OPTIMIZED PROBES AND PRIMERS AND METHODS OF USING SAME FOR THE DETECTION, SCREENING, ISOLATION AND SEQUENCING OF VANCOMYCIN RESISTANCE GENES AND VANCOMYCIN RESISTANT ENTEROCOCCI

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 15, 2011, is named 09108055.txt.

BACKGROUND

Enterococci are found within the normal intestinal flora and the female genital tract of humans, other mammals and birds. Enterococcus is intrinsically resistant (i.e., resistant to a low level) to β-lactam-based antibiotics (e.g., ampicillin, penicillin) and aminoglycosides (e.g., gentamicin, kanamycin, and neomycin). Enterococcus can acquire resistance to glycopeptides, such as vancomycin, and high concentrations of both β-lactam-based antibiotics and aminoglycosides, among others.

VRE is a threat to immunocompromised individuals, individuals recovering from surgical procedures and those generally in poor health. An individual can be colonized with VRE, which may or may not become a full-blown infection. Although colonized individuals can remain asymptomatic for months, even years, such persons are capable of transmitting VRE to others. VRE is rarely a concern for healthy adults, and is usually cleared from the host without intervention. Infections typically occur at sites such as wounds and urinary tract infections from in dwelling catheters. Infected patients can become septic. VRE is frequently transmitted person-to-person by healthcare workers (HCW) whose hands have become contaminated with VRE that is present in the feces, urine, or blood of an infected or colonized person. VRE can also be spread indirectly via hand contact with open wounds or contaminated environmental surfaces. Colonized individuals could also infect themselves through contact with feces, urine, blood or surfaces contaminated with their own feces, urine or blood. VRE can persist for weeks on environmental surfaces and medical instruments. Consequently, these surfaces are also potential modes of transmission and potential testing areas.

Culture-based diagnostic methods remain the definitive methods of choice for the determination of VRE. Bacterial colonies growing on Bile Esculin Azide plates supplemented with vancomycin (BEAV) could be presumed as VRE based on colonial morphology, but additional culture steps would be required for confirmation. This process can take 48 hours or longer. Culture can also have a high false negative rate. Adoption of nucleic acid-based tests (NAT) (such as polymerase chase reaction (PCR)) has led to diagnostic tests with significantly better turn-around time (2-5 hours), but many of the available tests lack sensitivity and specificity.

Detection of the vancomycin resistance genes from the genus Enterococcus, as well as other non-Enterococcal genera, would allow for improved treatments of bacterial infections. Furthermore, determination of whether the vancomycin resistance genes are from the Enterococcal genera would enable effective treatment decisions. A rapid and accurate diagnostic test panel for the detection of vancomycin-resistance genes and for detection of VRE would provide clinicians with an effective tool for diagnosis and supporting subsequent effective treatment regimens. A rapid screening panel for screening patients at risk for developing vancomycin resistance-associated and VRE-associated diseases would also provide clinicals with an efficient method to screen at-risk patients.

SUMMARY

Described herein are nucleic acid probes and primers for detecting, isolating and sequencing all known, characterized variants of the vanA, vanB, vanC1, vanC2/3, vanD, vanE, and vanG vancomycin resistance genes (particularly the vanA and vanB genes) from the genus Enterococcus, as well as other non-Enterococcal genera, with a high degree of sensitivity and specificity. Also described herein are nucleic acid probes and primers for determining whether the vancomycin resistance genes are from the Enterococcal genera. A diagnostic test that distinguishes multiple drug resistance genes simultaneously and also determines whether the organism is VRE is necessary because such detection is critical in patient and personnel screening and surveillance of inanimate objects to eliminate the transmission of potentially deadly healthcare-associated infections (HAIs).

Patient, personnel and inanimate object screening, combined with barrier isolation and contact precautions of VRE-carriers, has been shown to be effective in controlling VRE infections; in some cases reducing to undetectable levels the VRE in clinical facilities. The assays described herein are critical components of a resistance screening program to screen patients admitted to and personnel working in clinical settings for VRE and VRSA. The assays described herein are also used to screen environmental surfaces for evidence that vancomycin-resistant organisms are or were present in a hospital setting. Additionally, the assays described herein are used to identify or confirm the identification of an isolate as containing vancomycin resistance and whether the organism is from the genera Enterococcus.

Enterococci are common commensal bacteria located in the gut microflora. Enterococcus faecium (Efm) and Enterococcus faecalis (Efs) are two of the most common Enterococcal species that have been shown to have vancomycin resistance. One marker for Efm and Efs is the sodA gene, which encodes the enzyme superoxide dismutase A (Efm sodA and Efs sodA). The sodA gene is frequently used as a bacterial species-specific marker. Other markers, identified through in silico analysis, target novel genes from Efm and Efs (Efm novel and Efs novel). An additional marker, a dual marker, identified through in silico analysis, binds to novel genes found in both E. faecium and E. faecalis (Efm/Efs dual).

Many facilities utilize culture-based methods for the determination and detection of antibiotic resistance genes, which requires days to obtain the results. The methods of detection of the resistance markers described herein occurs within a minimal number of hours, allowing clinicians to rapidly determine the appropriate contact precautions or treatment for individuals harboring vancomycin-resistant organisms, avoiding needless precautions for resistance-negative individuals and avoiding the careless use of antibiotics that have no or little treatment efficacy.

One embodiment is directed to an isolated nucleic acid sequence comprising a sequence selected from the group consisting of: SEQ ID NOS: 1-846.

One embodiment is directed to a method of hybridizing one or more isolated nucleic acid sequences comprising a sequence selected from the group consisting of: SEQ ID NOS: 1-502 to a vancomycin-resistance gene sequence, comprising contacting one or more isolated nucleic acid sequences to a sample comprising the vancomycin-resistance gene under conditions suitable for hybridization. In a particular embodiment, the vancomycin-resistance gene sequence is a genomic sequence, a naturally occurring plasmid, a naturally occurring transposable element, a template sequence or a sequence derived from an artificial construct. In a particular embodiment, the method(s) further comprise isolating and/or sequencing the hybridized vancomycin-resistance gene sequence.

One embodiment is directed to a primer set comprising at least one forward primer selected from the group consisting of SEQ ID NOS: 1, 6, 19, 22, 23, 26, 28, 29, 33, 34, 37-42, 45, 48, 53, 59 (vanA); 61, 68, 70-72, 75, 81, 83, 89, 93, 94, 103-105, 107 and 111 (vanB); 123, 127, 130, 133, 138, 141, 144, 148, 151, 156, 158, 161, 162, 165, 168, 170, 171, 175, 178, 180, 183-185, 188, 191, 193, 194, 196, 198, and 200-204 (vanC1); 206, 210, 213, 217, 220, 221, 222, 224-226, 228-238, 240, 242 and 243 (vanC2/3); 388, 391, 394, 396, 399, 409, 415, 416, 425, 428, 435, 438, 440, 443, 445, 462, 465, 468, 471, 474, 477, 480, 483, 488, 491, 494-498 and 500-502 (vanD); 334, 337-380 and 382-387 (vanE); 244, 249, 252, 253, 255, 256, 260, 263, 264, 267, 270-272, 275-283, 285, 288, 291, 293, 295, 297-300, 302, 303, 305, 307, 309-311, 313-318, 321, 322, 325-333 (vanG); and at least one reverse primer selected from the group consisting of SEQ ID NOS: SEQ ID NOS: 3, 5, 8, 10, 21, 31, 32, 36, 44, 47, 51, 52, 55 and 60 (vanA); 63, 65, 66, 74, 77, 85-88, 90, 91, 95, and 97-102 (vanB); 125, 129, 132, 135, 137, 140, 143, 146, 147, 150, 153, 155, 157, 160, 164, 167, 169, 173, 174, 177, 179, 187, 190 and 192 (vanC1); 208, 209, 212, 215, 216, 219, 223, 227, 239 and 241 (vanC2/3); 390, 393, 395, 398, 401, 411, 417, 419, 421, 426, 430, 434, 437, 439, 442, 444, 447-455, 458-461, 464, 467, 470, 473, 476, 479, 482, 486, 490 and 493 (vanD); 336 and 381 (vanE); 246-248, 250, 251, 254, 258, 259, 262, 266, 269, 274, 284, 286, 287, 290, 301, 304, 306, 308, 312, 320 and 324 (vanG).

One embodiment is directed to a primer set comprising at least one forward primer selected from the group consisting of SEQ ID NOS: 517 (Efm sodA); 577, 586, 590, 598, 599, 600 (Efs sodA); and at least one reverse primer selected from the group consisting of SEQ ID NOS: 529 (Efm sodA); 617, 623, 624, 625, 637 and 640. (Efs sodA).

One embodiment is directed to a primer set comprising at least one forward primer selected from the group consisting of SEQ ID NOS: 683, 687, 692 (Efm novel); 758, 772, 773, 775 (Efs novel) and at least one reverse primer selected from the group consisting of SEQ ID NOS: 707, 720, 723 (Efm novel); 785, 791, 797, 799 and 803 (Efs novel).

One embodiment is directed to a primer set comprising at least one forward primer selected from the group consisting of SEQ ID NO: 843 (Efm/Efs dual); and at least one reverse primer selected from the group consisting of SEQ ID NOS: 845 and 846 (Efm/Efs dual).

One embodiment is directed to a primer set (at least one forward primer and at least one reverse primer) selected from the group consisting of: Groups 1-644 of Tables 5, 6, 8B, 9B, 10B, 11B, and 12.

One embodiment is directed to a method of producing a nucleic acid product, comprising contacting one or more isolated nucleic acid sequences selected from the group consisting of SEQ ID NOS: 1-846 to a sample comprising a vancomycin-resistance gene and/or an Efm and/or Efs sodA and/or Efm and/or Efs novel gene and/or dual marker genes under conditions suitable for nucleic acid polymerization. In a particular embodiment, the nucleic acid product is a vanA amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 1, 6, 19, 22, 23, 26, 28, 29, 33, 34, 37-42, 45, 48, 53 and 59 and at least one reverse primer selected from the group consisting of SEQ ID NOS: 3, 5, 8, 10, 21, 31, 32, 36, 44, 47, 51, 52, 55 and 60. In a particular embodiment, the nucleic acid product is a vanB amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 61, 68, 70-72, 75, 81, 83, 89, 93, 94, 103-105, 107 and 111 and at least one reverse primer selected from the group consisting of SEQ ID NOS: 63, 65, 66, 74, 77, 85-88, 90, 91, 95, and 97-102. In a particular embodiment, the nucleic acid product is a vanC1 amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 123, 127, 130, 133, 138, 141, 144, 148, 151, 156, 158, 161, 162, 165, 168, 170, 171, 175, 178, 180, 183-185, 188, 191, 193, 194, 196, 198, and 200-204 and at least one reverse primer selected from the group consisting of SEQ ID NOS: 125, 129, 132, 135, 137, 140, 143, 146, 147, 150, 153, 155, 157, 160, 164, 167, 169, 173, 174, 177, 179, 187, 190 and 192. In a particular embodiment, the nucleic acid product is a vanC2/3 amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 206, 210, 213, 217, 220, 221, 222, 224-226, 228-238, 240, 242 and 243 and at least one reverse primer selected from the group consisting of SEQ ID NOS: 208, 209, 212, 215, 216, 219, 223, 227, 239 and 241. In a particular embodiment, the nucleic acid product is a vanD amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 388, 391, 394, 396, 399, 409, 415, 416, 425, 428, 435, 438, 440, 443, 445, 462, 465, 468, 471, 474, 477, 480, 483, 488, 491, 494-498 and 500-502 and at least one reverse primer selected from the group consisting of SEQ ID NOS: 390, 393, 395, 398, 401, 411, 417, 419, 421, 426, 430, 434, 437, 439, 442, 444, 447-455, 458-461, 464, 467, 470, 473, 476, 479, 482, 486, 490 and 493. In a particular embodiment, the nucleic acid product is a vanE amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 334, 337-380 and 382-387 and at least one reverse primer selected from the group consisting of SEQ ID NOS: 336 and 381. In a particular embodiment, the nucleic acid product is a vanG amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 244, 249, 252, 253, 255, 256, 260, 263, 264, 267, 270-272, 275-283, 285, 288, 291, 293, 295, 297-300, 302, 303, 305, 307, 309-311, 313-318, 321, 322, 325-333 and at least one reverse primer selected from the group consisting of SEQ ID NOS: 246-248, 250, 251, 254, 258, 259, 262, 266, 269, 274, 284, 286, 287, 290, 301, 304, 306, 308, 312, 320 and 324. In a particular embodiment, the nucleic acid product is a Efm sodA amplicon produced using at least one forward primer consisting of SEQ ID NOS: 517 and at least one reverse primer consisting of SEQ ID NOS: 529. In a particular embodiment, the nucleic acid product is a Efs sodA amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 577, 586, 590, 598, 599, 600; and at least one reverse primer selected from the group consisting of SEQ ID NOS: 617, 623, 624, 625, 637 and 640. In a particular embodiment, the nucleic acid product is a Efm novel amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 683, 687, 692; and at least one reverse primer selected from the group consisting of SEQ ID NOS: 707, 720, 723. In a particular embodiment, the nucleic acid product is a Efs novel amplicon produced using at least one forward primer selected from the group consisting of SEQ ID NOS: 758, 772, 773, 775; and at least one reverse primer selected from the group consisting of SEQ ID NOS: 785, 791, 797, 799 and 803. In a particular embodiment, the nucleic acid product is a Efm dual and Efs dual amplicon produced using at least one forward primer consisting of SEQ ID NO: 843; and at least one reverse primer consisting of SEQ ID NOS: 845 and 846.

One embodiment is directed to a probe that hybridized to an amplicon produced as described herein, e.g., using the primers described herein. In a particular embodiment, the probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA); 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205 (vanC1); 207, 211 (vanC2/3); 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 475, 478, 481, 484, 485, 487, 489, 492, 499 (vanD); 335 (vanE); 245, 257, 261, 265, 273, 289, 292, 294, 296, 319, 323 (vanG). In a particular embodiment, the probe comprises a sequence selected from the group consisting of SEQ ID NOS: 555, 562, 571 (Efm sodA); 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); 728, 750 (Efm novel); 815, 832 (Efs novel), and 844 (Efm/Efs dual).

In a particular embodiment, the probe(s) is labeled with a detectable label selected from the group consisting of: a fluorescent label, a chemiluminescent label, a quencher, a radioactive label, biotin and gold.

One embodiment is directed to a set of probes that hybridize to an amplicon produced as described herein, e.g., using the primers described herein. In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA), and a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB). In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA), a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB), and a third probe comprises SEQ ID NO: 505. In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 555, 562, 571 (Efm sodA); a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 644, 650, 654, 659, 661-665, 667, 673, 675-677 (Efs sodA). In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 555, 562, 571 (Efm sodA); a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); a third probe comprises SEQ ID NO: 505. In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 728, 750 (Efm novel); a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 815, 832 (Efs novel). In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 728, 750 (Efm novel); a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 815, 832 (Efs novel); a third probe comprises SEQ ID NO: 505. In a particular embodiment, a probe comprises a sequence consisting of SEQ ID NO: 844 (Efm/Efs dual). In a particular embodiment, a probe comprises a sequence consisting of SEQ ID NO: 844 (Efm/Efs dual) and a second probe comprises SEQ ID NO: 505.

In a particular embodiment, the first probe is labeled with a first detectable label and the second probe is labeled with a second detectable label. In a particular embodiment, the first probe and the second probe are labeled with the same detectable label. In a particular embodiment, the first probe is labeled with a first detectable label, the second probe is labeled with a second detectable label and the third probe is labeled with a third detectable label. One embodiment is directed to a probe that hybridizes directly to the genomic sequences of the target without amplification. In a particular embodiment, the probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA); 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205 (vanC1); 207, 211 (vanC2/3); 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 475, 478, 481, 484, 485, 487, 489, 492, 499 (vanD); 335 (vanE); 245, 257, 261, 265, 273, 289, 292, 294, 296, 319, 323 (vanG). In a particular embodiment, the probe comprises a sequence selected from the group consisting of SEQ ID NOS: 555, 562, 571 (Efm sodA); 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); 728, 750 (Efm novel); 815, 832 (Efs novel); and 844 (Efm/Efs dual).

One embodiment is directed to a set of probes that hybridize directly to the genomic sequences of the target without amplification. In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA), and a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB). In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA), a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB), a third probe comprises SEQ ID NO: 505. In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA), a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB), a third probe comprises SEQ ID NOS: 555, 562, 571 (Efm sodA); and a fourth probe comprises a sequence selected from the group consisting of SEQ ID NOS: 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA). In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA), a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB), a third probe comprises SEQ ID NOS: 555, 562, 571 (Efm sodA); and a fourth probe comprises a sequence selected from the group consisting of SEQ ID NOS: 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); a fifth probe comprises SEQ ID NO: 505. In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA), a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB), a third probe comprises SEQ ID NOS: 728, 750 (Efm novel); a fourth probe comprises a sequence selected from the group consisting of SEQ ID NOS: 815, 832 (Efs novel) and a fifth probe comprises SEQ ID NO: 505. In a particular embodiment, a first probe comprises a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA), a second probe comprises a sequence selected from the group consisting of SEQ ID NOS: 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB), a third probe comprises SEQ ID NOS: 844 (Efs/Efm dual) and a fourth probe comprises SEQ ID: 505.

In one embodiment, the probe(s) is fluorescently labeled and the step of detecting the binding of the probe to the amplified product comprises measuring the fluorescence of the sample. In one embodiment, the probe comprises a fluorescent reporter moiety and a quencher of fluorescence-quenching moiety. Upon probe hybridization with the amplified product, the exonuclease activity of a DNA polymerase dissociates the probe's fluorescent reporter and the quencher, resulting in the unquenched emission of fluorescence, which is detected. An increase in the amplified product causes a proportional increase in fluorescence, due to cleavage of the probe and release of the reporter moiety of the probe. The amplified product is quantified in real time as it accumulates. In another embodiment, each probe in the multiplex reaction is labeled with a different distinguishable and detectable label.

In a particular embodiment, the probes are molecular beacons. Molecular beacons are single-stranded probes that form a stem-and-loop structure. A fluorophore is covalently linked to one end of the stem and a quencher is covalently linked to the other end of the stem forming a stem hybrid; fluorescence is quenched when the formation of the stem loop positions the fluorophore proximal to the quencher. When a molecular beacon hybridizes to a target nucleic acid sequence, the probe undergoes a conformational change that results in the dissociation of the stem hybrid and, thus the fluorophore and the quencher move away from each other, enabling the probe to fluoresce brightly. Molecular beacons can be labeled with differently colored fluorophores to detect different target sequences. Any of the probes described herein may be designed and utilized as molecular beacons.

One embodiment is directed a method for detecting a vancomycin-resistance gene(s) in a sample, comprising: (a) contacting the sample with at least one forward primer comprising a sequence selected from the group consisting of: SEQ ID NOS: 1, 6, 19, 22, 23, 26, 28, 29, 33, 34, 37-42, 45, 48, 53 and 59 (vanA); 61, 68, 70-72, 75, 81, 83, 89, 93, 94, 103-105, 107 and 111 (vanB); 123, 127, 130, 133, 138, 141, 144, 148, 151, 156, 158, 161, 162, 165, 168, 170, 171, 175, 178, 180, 183-185, 188, 191, 193, 194, 196, 198, and 200-204 (vanC1); 206, 210, 213, 217, 220, 221, 222, 224-226, 228-238, 240, 242 and 243 (vanC2/3); 388, 391, 394, 396, 399, 409, 415, 416, 425, 428, 435, 438, 440, 443, 445, 462, 465, 468, 471, 474, 477, 480, 483, 488, 491, 494-498 and 500-502 (vanD); 334, 337-380 and 382-387 (vanE); 244, 249, 252, 253, 255, 256, 260, 263, 264, 267, 270-272, 275-283, 285, 288, 291, 293, 295, 297-300, 302, 303, 305, 307, 309-311, 313-318, 321, 322, 325-333 (vanG), and at least one reverse primer comprising a sequence selected from the group consisting of: SEQ ID NOS: 3, 5, 8, 10, 21, 31, 32, 36, 44, 47, 51, 52, 55 and 60 (vanA); 63, 65, 66, 74, 77, 85-88, 90, 91, 95, and 97-102 (vanB); 125, 129, 132, 135, 137, 140, 143, 146, 147, 150, 153, 155, 157, 160, 164, 167, 169, 173, 174, 177, 179, 187, 190 and 192 (vanC1); 208, 209, 212, 215, 216, 219, 223, 227, 239 and 241 (vanC2/3); 390, 393, 395, 398, 401, 411, 417, 419, 421, 426, 430, 434, 437, 439, 442, 444, 447-455, 458-461, 464, 467, 470, 473, 476, 479, 482, 486, 490 and 493 (vanD); 336 and 381 (vanE); 246-248, 250, 251, 254, 258, 259, 262, 266, 269, 274, 284, 286, 287, 290, 301, 304, 306, 308, 312, 320 and 324 (vanG) under conditions such that nucleic acid amplification occurs to yield an amplicon; and (b) contacting the amplicon with one or more probes comprising one or more sequences selected from the group consisting of: SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA); 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205 (vanC1); 207, 211 (vanC2/3); 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 475, 478, 481, 484, 485, 487, 489, 492, 499 (vanD); 335 (vanE); 245, 257, 261, 265, 268, 273, 289, 292, 294, 296, 319, 323 (vanG) under conditions such that hybridization of the probe to the amplicon occurs, wherein hybridization of the probe is indicative of a vancomycin-resistance gene(s) in the sample.

One embodiment is directed a method for detecting an Enterococcal Efm sodA or Efs sodA gene(s) or novel gene or dual marker in a sample, comprising: (a) contacting the sample with at least one forward primer comprising a sequence selected from the group consisting of: SEQ ID NOS: 517 (Efm sodA); 577, 586, 590, 598, 599, 600 (Efs sodA); 683, 687, 692 (Efm novel); 758, 772, 773, 775 (Efs novel); and 843 (Efm/Efs dual); and at least one reverse primer comprising a sequence selected from the group consisting of SEQ ID NOS: 529 (Efm sodA); 617, 623, 624, 625, 637, 640 (Efs sodA); 707, 720, 723 (Efm novel); 785, 791, 797, 799, 803 (Efs novel); 845 and 846 (Efm/Efs dual) under conditions such that nucleic acid amplification occurs to yield an amplicon; and (b) contacting the amplicon with one or more probes comprising one or more sequences selected from the group consisting of: SEQ ID NOS: 555, 562, 571 (Efm sodA); 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); 728, 750 (Efm novel); 815, 832 (Efs novel), and 844 (Efm/Efs dual).

One embodiment is directed a method for detecting a vancomycin-resistance gene(s) or an Enterococcal marker gene in a sample, comprising: (a) contacting the sample with at least one forward primer comprising a sequence selected from the group consisting of: SEQ ID NOS: 1, 6, 19, 22, 23, 26, 28, 29, 33, 34, 37-42, 45, 48, 53 and 59 (vanA); 61, 68, 70-72, 75, 81, 83, 89, 93, 94, 103-105, 107 and 111 (vanB); 123, 127, 130, 133, 138, 141, 144, 148, 151, 156, 158, 161, 162, 165, 168, 170, 171, 175, 178, 180, 183-185, 188, 191, 193, 194, 196, 198, and 200-204 (vanC1); 206, 210, 213, 217, 220, 221, 222, 224-226, 228-238, 240, 242 and 243 (vanC2/3); 388, 391, 394, 396, 399, 409, 415, 416, 425, 428, 435, 438, 440, 443, 445, 462, 465, 468, 471, 474, 477, 480, 483, 488, 491, 494-498 and 500-502 (vanD); 334, 337-380 and 382-387 (vanE); 244, 249, 252, 253, 255, 256, 260, 263, 264, 267, 270-272, 275-283, 285, 288, 291, 293, 295, 297-300, 302, 303, 305, 307, 309-311, 313-318, 321, 322, 325-333 (vanG), 517 (Efm sodA); 577, 586, 590, 598, 599, 600 (Efs sodA); 683, 687, 692 (Efm novel); 758, 772, 773, 775 (Efs novel); and 843 (Efm/Efs dual), and at least one reverse primer comprising a sequence selected from the group consisting of: SEQ ID NOS: 3, 5, 8, 10, 21, 31, 32, 36, 44, 47, 51, 52, 55 and 60 (vanA); 63, 65, 66, 74, 77, 85-88, 90, 91, 95, and 97-102 (vanB); 125, 129, 132, 135, 137, 140, 143, 146, 147, 150, 153, 155, 157, 160, 164, 167, 169, 173, 174, 177, 179, 187, 190 and 192 (vanC1); 208, 209, 212, 215, 216, 219, 223, 227, 239 and 241 (vanC2/3); 390, 393, 395, 398, 401, 411, 417, 419, 421, 426, 430, 434, 437, 439, 442, 444, 447-455, 458-461, 464, 467, 470, 473, 476, 479, 482, 486, 490 and 493 (vanD); 336 and 381 (vanE); 246-248, 250, 251, 254, 258, 259, 262, 266, 269, 274, 284, 286, 287, 290, 301, 304, 306, 308, 312, 320 and 324 (vanG), 529 (Efm sodA); 617, 623, 624, 625, 637, 640 (Efs sodA); 707, 720, 723 (Efm novel); 785, 791, 797, 799, 803 (Efs novel); 845 and 846 (Efm/Efs dual) under conditions such that nucleic acid amplification occurs to yield an amplicon; and (b) contacting the amplicon with one or more probes comprising one or more sequences selected from the group consisting of: SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA); 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205 (vanC1); 207, 211 (vanC2/3); 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 475, 478, 481, 484, 485, 487, 489, 492, 499 (vanD); 335 (vanE); 245, 257, 261, 265, 268, 273, 289, 292, 294, 296, 319, 323 (vanG), 555, 562, 571 (Efm sodA); 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); 728, 750 (Efm novel); 815, 832 (Efs novel), and 844 (Efm/Efs dual) under conditions such that hybridization of the probe to the amplicon occurs, wherein hybridization of the probe is indicative of a vancomycin-resistance gene(s) in the sample.

In a particular embodiment, each of the one or more probes is labeled with a different detectable label. In a particular embodiment, the one or more probes are labeled with the same detectable label. In a particular embodiment, the sample is selected from the group consisting of: blood, serum, plasma, enriched peripheral blood mononuclear cells, neoplastic or other tissue obtained from biopsies, cerebrospinal fluid, saliva, fluids collected from the ear, eye, mouth, and respiratory airways, sputum, skin, tears, oropharyngeal swabs, nasopharyngeal swabs, throat swabs, urine, anal-rectal swabs, feces, skin swabs, nasal aspirates, nasal wash, fluids and cells obtained by the perfusion of tissues of both human and animal origin, and fluids and cells derived from the culturing of human cells, including human stem cells and human cartilage or fibroblasts. In one embodiment, the sample is from a human, is non-human in origin, or is derived from an inanimate object or environmental surfaces. In a particular embodiment, the at least one forward primer, the at least one reverse primer and the one or more probes are selected from the group consisting of: Groups 1-212 of Table 5, Groups 213-601 of Table 6, Groups 603-605 of Table 8B, Groups 606-627 of Table 9B, Groups 628-636 of Table 10B, Groups 637-643 of Table 11B, and Group 644 of Table 12. In a particular embodiment, the method(s) further comprise isolating and/or sequencing the vancomycin-resistance gene sequence(s) and/or Enterococcal sodA or novel gene or dual marker sequence(s) in a sample.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of a vancomycin-resistance gene (vanA gene), comprising a nucleotide sequence selected from the group consisting of: (1) SEQ ID NOS: 1 and 3; (2) SEQ ID NOS: 1 and 32; (3) SEQ ID NOS: 1 and 5; (4) SEQ ID NOS: 19 and 21; (5) SEQ ID NOS: 19 and 3; (6) SEQ ID NOS: 19 and 32; (7) SEQ ID NOS: 19 and 47; (8) SEQ ID NOS: 19 and 5; (9) SEQ ID NOS: 19 and 52; (10) SEQ ID NOS: 19 and 55; (11) SEQ ID NOS: 22 and 21; (12) SEQ ID NOS: 22 and 3; (13) SEQ ID NOS: 22 and 32; (14) SEQ ID NOS: 22 and 5; (15) SEQ ID NOS: 23 and 21; (16) SEQ ID NOS: 23 and 3; (17) SEQ ID NOS: 23 and 32; (18) SEQ ID NOS: 23 and 5; (19) SEQ ID NOS: 26 and 3; (20) SEQ ID NOS: 26 and 32; (21) SEQ ID NOS: 26 and 47; (22) SEQ ID NOS: 26 and 5; (23) SEQ ID NOS: 26 and 51; (24) SEQ ID NOS: 28 and 3; (25) SEQ ID NOS: 28 and 32; (26) SEQ ID NOS: 28 and 5; (27) SEQ ID NOS: 29 and 21; (28) SEQ ID NOS: 29 and 3; (29) SEQ ID NOS: 29 and 5; (30) SEQ ID NOS: 29 and 8; (31) SEQ ID NOS: 33 and 21; (32) SEQ ID NOS: 33 and 3; (33) SEQ ID NOS: 33 and 32; (34) SEQ ID NOS: 33 and 5; (35) SEQ ID NOS: 34 and 36; (36) SEQ ID NOS: 34 and 52; (37) SEQ ID NOS: 37 and 3; (38) SEQ ID NOS: 37 and 32; (39) SEQ ID NOS: 37 and 5; (40) SEQ ID NOS: 38 and 3; (41) SEQ ID NOS: 38 and 32; (42) SEQ ID NOS: 38 and 5; (43) SEQ ID NOS: 39 and 3; (44) SEQ ID NOS: 39 and 32; (45) SEQ ID NOS: 39 and 5; (46) SEQ ID NOS: 40 and 3; (47) SEQ ID NOS: 40 and 32; (48) SEQ ID NOS: 40 and 5; (49) SEQ ID NOS: 41 and 21; (50) SEQ ID NOS: 42 and 44; (51) SEQ ID NOS: 45 and 47; (52) SEQ ID NOS: 48 and 47; (53) SEQ ID NOS: 53 and 47; (54) SEQ ID NOS: 59 and 52; (55) SEQ ID NOS: 59 and 60; (56) SEQ ID NOS: 6 and 10; (57) SEQ ID NOS: 6 and 21; (58) SEQ ID NOS: 6 and 3; (59) SEQ ID NOS: 6 and 31; (60) SEQ ID NOS: 6 and 32; (61) SEQ ID NOS: 6 and 5; and (62) SEQ ID NOS: 6 and 8.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of a vancomycin-resistance gene (vanB gene), comprising a nucleotide sequence selected from the group consisting of: (1) SEQ ID NOS: 103 and 65; (2) SEQ ID NOS: 103 and 66; (3) SEQ ID NOS: 103 and 86; (4) SEQ ID NOS: 103 and 87; (5) SEQ ID NOS: 103 and 88; (6) SEQ ID NOS: 104 and 66; (7) SEQ ID NOS: 105 and 66; (8) SEQ ID NOS: 107 and 66; (9) SEQ ID NOS: 111 and 63; (10) SEQ ID NOS: 111 and 66; (11) SEQ ID NOS: 111 and 88; (12) SEQ ID NOS: 61 and 63; (13) SEQ ID NOS: 61 and 65; (14) SEQ ID NOS: 61 and 66; (15) SEQ ID NOS: 61 and 74; (16) SEQ ID NOS: 61 and 77; (17) SEQ ID NOS: 61 and 97; (18) SEQ ID NOS: 68 and 63; (19) SEQ ID NOS: 68 and 65; (20) SEQ ID NOS: 68 and 66; (21) SEQ ID NOS: 68 and 74; (22) SEQ ID NOS: 68 and 77; (23) SEQ ID NOS: 70 and 63; (24) SEQ ID NOS: 70 and 74; (25) SEQ ID NOS: 70 and 77; (26) SEQ ID NOS: 71 and 63; (27) SEQ ID NOS: 71 and 74; (28) SEQ ID NOS: 71 and 77; (29) SEQ ID NOS: 72 and 74; (30) SEQ ID NOS: 75 and 74; (31) SEQ ID NOS: 81 and 65; (32) SEQ ID NOS: 81 and 66; (33) SEQ ID NOS: 81 and 77; (34) SEQ ID NOS: 81 and 87; (35) SEQ ID NOS: 81 and 88; (36) SEQ ID NOS: 81 and 95; (37) SEQ ID NOS: 83 and 101; (38) SEQ ID NOS: 83 and 65; (39) SEQ ID NOS: 83 and 66; (40) SEQ ID NOS: 83 and 85; (41) SEQ ID NOS: 83 and 86; (42) SEQ ID NOS: 83 and 87; (43) SEQ ID NOS: 83 and 88; (44) SEQ ID NOS: 83 and 95; (45) SEQ ID NOS: 89 and 100; (46) SEQ ID NOS: 89 and 101; (47) SEQ ID NOS: 89 and 102; (48) SEQ ID NOS: 89 and 65; (49) SEQ ID NOS: 89 and 66; (50) SEQ ID NOS: 89 and 85; (51) SEQ ID NOS: 89 and 86; (52) SEQ ID NOS: 89 and 87; (53) SEQ ID NOS: 89 and 88; (54) SEQ ID NOS: 89 and 90; (55) SEQ ID NOS: 89 and 91; (56) SEQ ID NOS: 89 and 95; (57) SEQ ID NOS: 89 and 98; (58) SEQ ID NOS: 89 and 99; (59) SEQ ID NOS: 93 and 101; (60) SEQ ID NOS: 93 and 65; (61) SEQ ID NOS: 93 and 66; (62) SEQ ID NOS: 93 and 85; (63) SEQ ID NOS: 93 and 86; (64) SEQ ID NOS: 93 and 87; (65) SEQ ID NOS: 93 and 88; (66) SEQ ID NOS: 93 and 90; (67) SEQ ID NOS: 93 and 95; (68) SEQ ID NOS: 93 and 98; (69) SEQ ID NOS: 94 and 65; (70) SEQ ID NOS: 94 and 66; (71) SEQ ID NOS: 94 and 87; (72) SEQ ID NOS: 94 and 88; and (73) SEQ ID NOS: 94 and 95.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of a vancomycin-resistance gene (vanC1 gene), comprising a nucleotide sequence selected from the group consisting of: (1) SEQ ID NOS: 123 and 125; (2) SEQ ID NOS: 127 and 129; (3) SEQ ID NOS: 130 and 132; (4) SEQ ID NOS: 133 and 135; (5) SEQ ID NOS: 133 and 137; (6) SEQ ID NOS: 138 and 140; (7) SEQ ID NOS: 141 and 137; (8) SEQ ID NOS: 141 and 143; (9) SEQ ID NOS: 141 and 147; (10) SEQ ID NOS: 141 and 179; (11) SEQ ID NOS: 144 and 137; (12) SEQ ID NOS: 144 and 146; (13) SEQ ID NOS: 144 and 147; (14) SEQ ID NOS: 144 and 157; (15) SEQ ID NOS: 148 and 137; (16) SEQ ID NOS: 148 and 150; (17) SEQ ID NOS: 151 and 153; (18) SEQ ID NOS: 151 and 155; (19) SEQ ID NOS: 156 and 150; (20) SEQ ID NOS: 158 and 160; (21) SEQ ID NOS: 161 and 137; (22) SEQ ID NOS: 161 and 147; (23) SEQ ID NOS: 161 and 150; (24) SEQ ID NOS: 161 and 153; (25) SEQ ID NOS: 161 and 190; (26) SEQ ID NOS: 161 and 192; (27) SEQ ID NOS: 162 and 164; (28) SEQ ID NOS: 165 and 167; (29) SEQ ID NOS: 168 and 169; (30) SEQ ID NOS: 170 and 169; (31) SEQ ID NOS: 171 and 173; (32) SEQ ID NOS: 171 and 174; (33) SEQ ID NOS: 175 and 177; (34) SEQ ID NOS: 178 and 179; (35) SEQ ID NOS: 180 and 146; (36) SEQ ID NOS: 183 and 150; (37) SEQ ID NOS: 184 and 174; (38) SEQ ID NOS: 185 and 187; (39) SEQ ID NOS: 188 and 137; (40) SEQ ID NOS: 188 and 150; (41) SEQ ID NOS: 188 and 190; (42) SEQ ID NOS: 191 and 137; (43) SEQ ID NOS: 191 and 150; (44) SEQ ID NOS: 191 and 153; (45) SEQ ID NOS: 191 and 190; (46) SEQ ID NOS: 191 and 192; (47) SEQ ID NOS: 193 and 137; (48) SEQ ID NOS: 193 and 150; (49) SEQ ID NOS: 193 and 153; (50) SEQ ID NOS: 193 and 190; (51) SEQ ID NOS: 194 and 147; (52) SEQ ID NOS: 194 and 160; (53) SEQ ID NOS: 196 and 147; (54) SEQ ID NOS: 196 and 160; (55) SEQ ID NOS: 198 and 179; (56) SEQ ID NOS: 200 and 179; (57) SEQ ID NOS: 201 and 179; (58) SEQ ID NOS: 202 and 179; (59) SEQ ID NOS: 203 and 147; (60) SEQ ID NOS: 204 and 179; and (61) SEQ ID NOS: 204 and 187.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of a vancomycin-resistance gene (vanC2/C3 gene), comprising a nucleotide sequence selected from the group consisting of: (1) SEQ ID NOS: 206 and 208; (2) SEQ ID NOS: 206 and 209; (3) SEQ ID NOS: 206 and 216; (4) SEQ ID NOS: 206 and 219; (5) SEQ ID NOS: 206 and 227; (6) SEQ ID NOS: 210 and 209; (7) SEQ ID NOS: 210 and 212; (8) SEQ ID NOS: 210 and 215; (9) SEQ ID NOS: 210 and 216; (10) SEQ ID NOS: 210 and 219; (11) SEQ ID NOS: 210 and 223; (12) SEQ ID NOS: 210 and 227; (13) SEQ ID NOS: 213 and 215; (14) SEQ ID NOS: 217 and 209; (15) SEQ ID NOS: 217 and 216; (16) SEQ ID NOS: 217 and 219; (17) SEQ ID NOS: 217 and 223; (18) SEQ ID NOS: 217 and 227; (19) SEQ ID NOS: 220 and 209; (20) SEQ ID NOS: 220 and 219; (21) SEQ ID NOS: 220 and 223; (22) SEQ ID NOS: 220 and 227; (23) SEQ ID NOS: 221 and 209; (24) SEQ ID NOS: 221 and 216; (25) SEQ ID NOS: 221 and 219; (26) SEQ ID NOS: 221 and 227; (27) SEQ ID NOS: 222 and 209; (28) SEQ ID NOS: 222 and 216; (29) SEQ ID NOS: 222 and 219; (30) SEQ ID NOS: 222 and 223; (31) SEQ ID NOS: 222 and 227; (32) SEQ ID NOS: 224 and 212; (33) SEQ ID NOS: 224 and 215; (34) SEQ ID NOS: 224 and 216; (35) SEQ ID NOS: 225 and 209; (36) SEQ ID NOS: 225 and 212; (37) SEQ ID NOS: 225 and 216; (38) SEQ ID NOS: 226 and 209; (39) SEQ ID NOS: 226 and 212; (40) SEQ ID NOS: 226 and 216; (41) SEQ ID NOS: 228 and 215; (42) SEQ ID NOS: 229 and 209; (43) SEQ ID NOS: 229 and 215; (44) SEQ ID NOS: 230 and 219; (45) SEQ ID NOS: 231 and 212; (46) SEQ ID NOS: 231 and 215; (47) SEQ ID NOS: 232 and 216; (48) SEQ ID NOS: 233 and 212; (49) SEQ ID NOS: 234 and 215; (50) SEQ ID NOS: 235 and 215; (51) SEQ ID NOS: 235 and 239; (52) SEQ ID NOS: 235 and 241; (53) SEQ ID NOS: 236 and 216; (54) SEQ ID NOS: 237 and 209; (55) SEQ ID NOS: 237 and 215; (56) SEQ ID NOS: 238 and 215; (57) SEQ ID NOS: 240 and 216; (58) SEQ ID NOS: 242 and 216; and (59) SEQ ID NOS: 243 and 215.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of a vancomycin-resistance gene (vanD gene), comprising a nucleotide sequence selected from the group consisting of: (1) SEQ ID NOS: 388 and 390; (2) SEQ ID NOS: 391 and 393; (3) SEQ ID NOS: 391 and 434; (4) SEQ ID NOS: 394 and 393; (5) SEQ ID NOS: 396 and 398; (6) SEQ ID NOS: 396 and 419; (7) SEQ ID NOS: 396 and 419; (8) SEQ ID NOS: 399 and 401; (9) SEQ ID NOS: 399 and 401; (10) SEQ ID NOS: 399 and 401; (11) SEQ ID NOS: 399 and 401; (12) SEQ ID NOS: 399 and 444; (13) SEQ ID NOS: 399 and 444; (14) SEQ ID NOS: 415 and 401; (15) SEQ ID NOS: 416 and 417; (16) SEQ ID NOS: 435 and 437; (17) SEQ ID NOS: 438 and 439; (18) SEQ ID NOS: 440 and 442; (19) SEQ ID NOS: 443 and 434; (20) SEQ ID NOS: 445 and 447; (21) SEQ ID NOS: 445 and 448; (22) SEQ ID NOS: 445 and 449; (23) SEQ ID NOS: 445 and 450; (24) SEQ ID NOS: 445 and 451; (25) SEQ ID NOS: 445 and 452; (26) SEQ ID NOS: 445 and 453; (27) SEQ ID NOS: 445 and 454; (28) SEQ ID NOS: 445 and 455; (29) SEQ ID NOS: 445 and 459; (30) SEQ ID NOS: 445 and 460; (31) SEQ ID NOS: 445 and 461; (32) SEQ ID NOS: 456 and 458; (33) SEQ ID NOS: 462 and 464; (34) SEQ ID NOS: 465 and 467; (35) SEQ ID NOS: 468 and 470; (36) SEQ ID NOS: 471 and 473; (37) SEQ ID NOS: 474 and 476; (38) SEQ ID NOS: 477 and 479; (39) SEQ ID NOS: 480 and 482; (40) SEQ ID NOS: 483 and 479; (41) SEQ ID NOS: 483 and 486; (42) SEQ ID NOS: 488 and 490; (43) SEQ ID NOS: 491 and 493; (44) SEQ ID NOS: 494 and 486; (45) SEQ ID NOS: 495 and 493; (46) SEQ ID NOS: 496 and 486; (47) SEQ ID NOS: 497 and 486; (48) SEQ ID NOS: 498 and 486; (49) SEQ ID NOS: 500 and 486; (50) SEQ ID NOS: 501 and 486; and (51) SEQ ID NOS: 502 and 493.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of a vancomycin-resistance gene (vanE gene), comprising a nucleotide sequence selected from the group consisting of: (1) SEQ ID NOS: 334 and 336; (2) SEQ ID NOS: 337 and 336; (3) SEQ ID NOS: 338 and 336; (4) SEQ ID NOS: 338 and 381; (5) SEQ ID NOS: 339 and 336; (6) SEQ ID NOS: 340 and 336; (7) SEQ ID NOS: 341 and 336; (8) SEQ ID NOS: 341 and 381; (9) SEQ ID NOS: 342 and 336; (10) SEQ ID NOS: 342 and 381; (11) SEQ ID NOS: 343 and 336; (12) SEQ ID NOS: 344 and 336; (13) SEQ ID NOS: 344 and 381; (14) SEQ ID NOS: 345 and 336; (15) SEQ ID NOS: 346 and 336; (16) SEQ ID NOS: 347 and 336; (17) SEQ ID NOS: 347 and 381; (18) SEQ ID NOS: 348 and 336; (19) SEQ ID NOS: 348 and 381; (20) SEQ ID NOS: 349 and 336; (21) SEQ ID NOS: 349 and 381; (22) SEQ ID NOS: 350 and 336; (23) SEQ ID NOS: 350 and 381; (24) SEQ ID NOS: 351 and 336; (25) SEQ ID NOS: 352 and 336; (26) SEQ ID NOS: 353 and 336; (27) SEQ ID NOS: 354 and 336; (28) SEQ ID NOS: 355 and 336; (29) SEQ ID NOS: 355 and 381; (30) SEQ ID NOS: 356 and 336; (31) SEQ ID NOS: 357 and 336; (32) SEQ ID NOS: 358 and 336; (33) SEQ ID NOS: 359 and 336; (34) SEQ ID NOS: 359 and 381; (35) SEQ ID NOS: 360 and 336; (36) SEQ ID NOS: 360 and 381; (37) SEQ ID NOS: 361 and 336; (38) SEQ ID NOS: 362 and 336; (39) SEQ ID NOS: 363 and 336; (40) SEQ ID NOS: 364 and 336; (41) SEQ ID NOS: 365 and 336; (42) SEQ ID NOS: 366 and 336; (43) SEQ ID NOS: 367 and 336; (44) SEQ ID NOS: 368 and 336; (45) SEQ ID NOS: 369 and 336; (46) SEQ ID NOS: 370 and 336; (47) SEQ ID NOS: 371 and 336; (48) SEQ ID NOS: 372 and 336; (49) SEQ ID NOS: 373 and 336; (50) SEQ ID NOS: 374 and 336; (51) SEQ ID NOS: 375 and 336; (52) SEQ ID NOS: 376 and 336; (53) SEQ ID NOS: 377 and 336; (54) SEQ ID NOS: 378 and 336; (55) SEQ ID NOS: 379 and 336; (56) SEQ ID NOS: 380 and 336; (57) SEQ ID NOS: 382 and 381; (58) SEQ ID NOS: 383 and 381; (59) SEQ ID NOS: 384 and 381; (60) SEQ ID NOS: 385 and 381; (61) SEQ ID NOS: 386 and 381; and (62) SEQ ID NOS: 387 and 381.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of a vancomycin-resistance gene (vanG gene), comprising a nucleotide sequence selected from the group consisting of: (1) SEQ ID NOS: 244 and 246; (2) SEQ ID NOS: 244 and 247; (3) SEQ ID NOS: 244 and 248; (4) SEQ ID NOS: 244 and 250; (5) SEQ ID NOS: 244 and 251; (6) SEQ ID NOS: 244 and 254; (7) SEQ ID NOS: 244 and 258; (8) SEQ ID NOS: 244 and 259; (9) SEQ ID NOS: 244 and 284; (10) SEQ ID NOS: 244 and 286; (11) SEQ ID NOS: 244 and 287; (12) SEQ ID NOS: 249 and 246; (13) SEQ ID NOS: 249 and 248; (14) SEQ ID NOS: 249 and 286; (15) SEQ ID NOS: 249 and 301; (16) SEQ ID NOS: 249 and 306; (17) SEQ ID NOS: 249 and 308; (18) SEQ ID NOS: 249 and 312; (19) SEQ ID NOS: 252 and 246; (20) SEQ ID NOS: 252 and 262; (21) SEQ ID NOS: 253 and 246; (22) SEQ ID NOS: 255 and 246; (23) SEQ ID NOS: 256 and 246; (24) SEQ ID NOS: 260 and 258; (25) SEQ ID NOS: 263 and 258; (26) SEQ ID NOS: 264 and 266; (27) SEQ ID NOS: 267 and 269; (28) SEQ ID NOS: 270 and 266; (29) SEQ ID NOS: 270 and 269; (30) SEQ ID NOS: 271 and 266; (31) SEQ ID NOS: 272 and 274; (32) SEQ ID NOS: 275 and 269; (33) SEQ ID NOS: 276 and 269; (34) SEQ ID NOS: 277 and 269; (35) SEQ ID NOS: 278 and 266; (36) SEQ ID NOS: 279 and 269; (37) SEQ ID NOS: 280 and 266; (38) SEQ ID NOS: 280 and 269; (39) SEQ ID NOS: 281 and 269; (40) SEQ ID NOS: 282 and 266; (41) SEQ ID NOS: 282 and 269; (42) SEQ ID NOS: 283 and 269; (43) SEQ ID NOS: 285 and 259; (44) SEQ ID NOS: 288 and 290; (45) SEQ ID NOS: 288 and 304; (46) SEQ ID NOS: 291 and 290; (47) SEQ ID NOS: 293 and 290; (48) SEQ ID NOS: 293 and 304; (49) SEQ ID NOS: 295 and 258; (50) SEQ ID NOS: 297 and 290; (51) SEQ ID NOS: 298 and 290; (52) SEQ ID NOS: 298 and 304; (53) SEQ ID NOS: 299 and 290; (54) SEQ ID NOS: 300 and 290; (55) SEQ ID NOS: 302 and 274; (56) SEQ ID NOS: 303 and 290; (57) SEQ ID NOS: 303 and 304; (58) SEQ ID NOS: 305 and 274; (59) SEQ ID NOS: 305 and 290; (60) SEQ ID NOS: 307 and 274; (61) SEQ ID NOS: 307 and 290; (62) SEQ ID NOS: 309 and 290; (63) SEQ ID NOS: 310 and 290; (64) SEQ ID NOS: 311 and 258; (65) SEQ ID NOS: 313 and 290; (66) SEQ ID NOS: 314 and 290; (67) SEQ ID NOS: 314 and 320; (68) SEQ ID NOS: 315 and 290; (69) SEQ ID NOS: 316 and 290; (70) SEQ ID NOS: 317 and 290; (71) SEQ ID NOS: 318 and 290; (72) SEQ ID NOS: 321 and 258; (73) SEQ ID NOS: 322 and 324; (74) SEQ ID NOS: 325 and 266; (75) SEQ ID NOS: 325 and 274; (76) SEQ ID NOS: 326 and 274; (77) SEQ ID NOS: 327 and 266; (78) SEQ ID NOS: 328 and 274; (79) SEQ ID NOS: 329 and 274; (80) SEQ ID NOS: 330 and 274; (81) SEQ ID NOS: 331 and 274; (82) SEQ ID NOS: 332 and 266; and (83) SEQ ID NOS: 333 and 266.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of an Efm sodA gene, comprising a nucleotide sequence SEQ ID NOS: 610 and 622.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of an Efs sodA gene, comprising a nucleotide sequence selected from the group consisting of: 1) SEQ ID NOS: 577 and 617; (2) SEQ ID NOS: 577 and 623; (3) SEQ ID NOS: 577 and 624; (4) SEQ ID NOS: 577 and 625; (5) SEQ ID NOS: 577 and 637; (6) SEQ ID NOS: 577 and 640; (7) SEQ ID NOS: 586 and 617; (8) SEQ ID NOS: 586 and 623; (9) SEQ ID NOS: 586 and 624; (10) SEQ ID NOS: 586 and 625; (11) SEQ ID NOS: 586 and 637; (12) SEQ ID NOS: 586 and 640; (13) SEQ ID NOS: 590 and 617; (14) SEQ ID NOS: 590 and 623; (15) SEQ ID NOS: 590 and 624; (16) SEQ ID NOS: 590 and 625; (17) SEQ ID NOS: 590 and 637; (18) SEQ ID NOS: 590 and 640; (19) SEQ ID NOS: 598 and 617; (20) SEQ ID NOS: 598 and 623; (21) SEQ ID NOS: 598 and 624; (22) SEQ ID NOS: 598 and 625; (23) SEQ ID NOS: 598 and 637; (24) SEQ ID NOS: 598 and 640; (25) SEQ ID NOS: 599 and 617; (26) SEQ ID NOS: 599 and 623; (27) SEQ ID NOS: 599 and 624; (28) SEQ ID NOS: 599 and 625; (29) SEQ ID NOS: 599 and 637; (30) SEQ ID NOS: 599 and 640; (31) SEQ ID NOS: 600 and 617; (32) SEQ ID NOS: 600 and 623; (33) SEQ ID NOS: 600 and 624; (34) SEQ ID NOS: 600 and 625; (35) SEQ ID NOS: 600 and 637; (36) SEQ ID NOS: 600 and 640;

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of an Efm novel gene, comprising a nucleotide sequence selected from the from the group consisting of: SEQ ID NOS: 683 and 707; (2) SEQ ID NOS: 683 and 720; (3) SEQ ID NOS: 683 and 723; (4) SEQ ID NOS: 687 and 707; (5) SEQ ID NOS: 687 and 720; (6) SEQ ID NOS: 687 and 723; (7) SEQ ID NOS: 692 and 707; (8) SEQ ID NOS: 692 and 720; (9) SEQ ID NOS: 692 and 723.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of an Efs novel gene, comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 758 and 785; (2) SEQ ID NOS: 758 and 791; (3) SEQ ID NOS: 758 and 797; (4) SEQ ID NOS: 758 and 799; (5) SEQ ID NOS: 758 and 803; (6) SEQ ID NOS: 772 and 785; (7) SEQ ID NOS: 772 and 791; (8) SEQ ID NOS: 772 and 797; (9) SEQ ID NOS: 772 and 799; (10) SEQ ID NOS: 772 and 803; (11) SEQ ID NOS: 773 and 785; (12) SEQ ID NOS: 773 and 791; (13) SEQ ID NOS: 773 and 797; (14) SEQ ID NOS: 773 and 799; (15) SEQ ID NOS: 773 and 803; (16) SEQ ID NOS: 775 and 785; (17) SEQ ID NOS: 775 and 791; (18) SEQ ID NOS: 775 and 797; (19) SEQ ID NOS: 775 and 799; (20) SEQ ID NOS: 775 and 803.

One embodiment is directed to a primer set or collection of primer sets for amplifying DNA of Efm/Efs dual genes, comprising a nucleotide sequence consisting of: SEQ ID NOS: 843, 845 and 846.

A particular embodiment is directed to oligonucleotide probes for binding to DNA of a vancomycin-resistance gene(s), comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA); 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205 (vanC1); 207, 211 (vanC2/3); 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 475, 478, 481, 484, 485, 487, 489, 492, 499 (vanD); 335 (vanE); and 245, 257, 261, 265, 268, 273, 289, 292, 294, 296, 319, 323 (vanG).

A particular embodiment is directed to oligonucleotide probes for binding to DNA of an Efm sodA gene or Efs sodA gene or Efm novel gene or Efs novel gene or dual genes, comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 555, 562, 571 (Efm sodA); 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); 728, 750 (Efm novel); 815, 832 (Efs novel), and 844 (Efm/Efs dual).

One embodiment is directed to the simultaneous detection in a multiplex format of vancomycin resistance, specifically the resistance genes vanA, vanB, vanC, vanD, vanE and vanG.

One embodiment is directed to the simultaneous detection and differentiation in a multiplex format of the vanA and vanB resistance genes.

One embodiment is directed to the simultaneous detection in a multiplex format of VRE when an isolate is tested.

One embodiment is directed to primer sets for amplifying DNA of a vancomycin-resistance gene(s) simultaneously, comprising:

(a): (1) SEQ ID NOS: 1 and 3; (2) SEQ ID NOS: 1 and 32; (3) SEQ ID NOS: 1 and 5; (4) SEQ ID NOS: 19 and 21; (5) SEQ ID NOS: 19 and 3; (6) SEQ ID NOS: 19 and 32; (7) SEQ ID NOS: 19 and 47; (8) SEQ ID NOS: 19 and 5; (9) SEQ ID NOS: 19 and 52; (10) SEQ ID NOS: 19 and 55; (11) SEQ ID NOS: 22 and 21; (12) SEQ ID NOS: 22 and 3; (13) SEQ ID NOS: 22 and 32; (14) SEQ ID NOS: 22 and 5; (15) SEQ ID NOS: 23 and 21; (16) SEQ ID NOS: 23 and 3; (17) SEQ ID NOS: 23 and 32; (18) SEQ ID NOS: 23 and 5; (19) SEQ ID NOS: 26 and 3; (20) SEQ ID NOS: 26 and 32; (21) SEQ ID NOS: 26 and 47; (22) SEQ ID NOS: 26 and 5; (23) SEQ ID NOS: 26 and 51; (24) SEQ ID NOS: 28 and 3; (25) SEQ ID NOS: 28 and 32; (26) SEQ ID NOS: 28 and 5; (27) SEQ ID NOS: 29 and 21; (28) SEQ ID NOS: 29 and 3; (29) SEQ ID NOS: 29 and 5; (30) SEQ ID NOS: 29 and 8; (31) SEQ ID NOS: 33 and 21; (32) SEQ ID NOS: 33 and 3; (33) SEQ ID NOS: 33 and 32; (34) SEQ ID NOS: 33 and 5; (35) SEQ ID NOS: 34 and 36; (36) SEQ ID NOS: 34 and 52; (37) SEQ ID NOS: 37 and 3; (38) SEQ ID NOS: 37 and 32; (39) SEQ ID NOS: 37 and 5; (40) SEQ ID NOS: 38 and 3; (41) SEQ ID NOS: 38 and 32; (42) SEQ ID NOS: 38 and 5; (43) SEQ ID NOS: 39 and 3; (44) SEQ ID NOS: 39 and 32; (45) SEQ ID NOS: 39 and 5; (46) SEQ ID NOS: 40 and 3; (47) SEQ ID NOS: 40 and 32; (48) SEQ ID NOS: 40 and 5; (49) SEQ ID NOS: 41 and 21; (50) SEQ ID NOS: 42 and 44; (51) SEQ ID NOS: 45 and 47; (52) SEQ ID NOS: 48 and 47; (53) SEQ ID NOS: 53 and 47; (54) SEQ ID NOS: 59 and 52; (55) SEQ ID NOS: 59 and 60; (56) SEQ ID NOS: 6 and 10; (57) SEQ ID NOS: 6 and 21; (58) SEQ ID NOS: 6 and 3; (59) SEQ ID NOS: 6 and 31; (60) SEQ ID NOS: 6 and 32; (61) SEQ ID NOS: 6 and 5; and (62) SEQ ID NOS: 6 and 8 (forward and reverse primers for amplifying DNA of vanA, respectively); and

(b) (1) SEQ ID NOS: 103 and 65; (2) SEQ ID NOS: 103 and 66; (3) SEQ ID NOS: 103 and 86; (4) SEQ ID NOS: 103 and 87; (5) SEQ ID NOS: 103 and 88; (6) SEQ ID NOS: 104 and 66; (7) SEQ ID NOS: 105 and 66; (8) SEQ ID NOS: 107 and 66; (9) SEQ ID NOS: 111 and 63; (10) SEQ ID NOS: 111 and 66; (11) SEQ ID NOS: 111 and 88; (12) SEQ ID NOS: 61 and 63; (13) SEQ ID NOS: 61 and 65; (14) SEQ ID NOS: 61 and 66; (15) SEQ ID NOS: 61 and 74; (16) SEQ ID NOS: 61 and 77; (17) SEQ ID NOS: 61 and 97; (18) SEQ ID NOS: 68 and 63; (19) SEQ ID NOS: 68 and 65; (20) SEQ ID NOS: 68 and 66; (21) SEQ ID NOS: 68 and 74; (22) SEQ ID NOS: 68 and 77; (23) SEQ ID NOS: 70 and 63; (24) SEQ ID NOS: 70 and 74; (25) SEQ ID NOS: 70 and 77; (26) SEQ ID NOS: 71 and 63; (27) SEQ ID NOS: 71 and 74; (28) SEQ ID NOS: 71 and 77; (29) SEQ ID NOS: 72 and 74; (30) SEQ ID NOS: 75 and 74; (31) SEQ ID NOS: 81 and 65; (32) SEQ ID NOS: 81 and 66; (33) SEQ ID NOS: 81 and 77; (34) SEQ ID NOS: 81 and 87; (35) SEQ ID NOS: 81 and 88; (36) SEQ ID NOS: 81 and 95; (37) SEQ ID NOS: 83 and 101; (38) SEQ ID NOS: 83 and 65; (39) SEQ ID NOS: 83 and 66; (40) SEQ ID NOS: 83 and 85; (41) SEQ ID NOS: 83 and 86; (42) SEQ ID NOS: 83 and 87; (43) SEQ ID NOS: 83 and 88; (44) SEQ ID NOS: 83 and 95; (45) SEQ ID NOS: 89 and 100; (46) SEQ ID NOS: 89 and 101; (47) SEQ ID NOS: 89 and 102; (48) SEQ ID NOS: 89 and 65; (49) SEQ ID NOS: 89 and 66; (50) SEQ ID NOS: 89 and 85; (51) SEQ ID NOS: 89 and 86; (52) SEQ ID NOS: 89 and 87; (53) SEQ ID NOS: 89 and 88; (54) SEQ ID NOS: 89 and 90; (55) SEQ ID NOS: 89 and 91; (56) SEQ ID NOS: 89 and 95; (57) SEQ ID NOS: 89 and 98; (58) SEQ ID NOS: 89 and 99; (59) SEQ ID NOS: 93 and 101; (60) SEQ ID NOS: 93 and 65; (61) SEQ ID NOS: 93 and 66; (62) SEQ ID NOS: 93 and 85; (63) SEQ ID NOS: 93 and 86; (64) SEQ ID NOS: 93 and 87; (65) SEQ ID NOS: 93 and 88; (66) SEQ ID NOS: 93 and 90; (67) SEQ ID NOS: 93 and 95; (68) SEQ ID NOS: 93 and 98; (69) SEQ ID NOS: 94 and 65; (70) SEQ ID NOS: 94 and 66; (71) SEQ ID NOS: 94 and 87; (72) SEQ ID NOS: 94 and 88; and (73) SEQ ID NOS: 94 and 95 (forward and reverse primers for amplifying DNA of vanB, respectively).

A particular embodiment is directed to oligonucleotide probes for binding to DNA of vancomycin-resistance gene(s), comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA probes); and 62, 64, 67, 69, 73, 76, 78, 79, 80, 82, 84, 92, 96, 108-110, 112 (vanB probes).

One embodiment is directed to a kit for detecting DNA of a vancomycin-resistance gene(s) in a sample, comprising one or more probes comprising a sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA); 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205 (vanC1); 207, 211 (vanC2/3); 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 475, 478, 481, 484, 485, 487, 489, 492, 499 (vanD); 335 (vanE); 245, 257, 261, 265, 268, 273, 289, 292, 294, 296, 319, 323 (vanG) In a particular embodiment, the kit further comprises a) at least one forward primer comprising the sequence selected from the group consisting of: SEQ ID NOS: 1, 6, 19, 22, 23, 26, 28, 29, 33, 34, 37-42, 45, 48, 53 and 59 (vanA); 61, 68, 70-72, 75, 81, 83, 89, 93, 94, 103-105, 107 and 111 (vanB); 123, 127, 130, 133, 138, 141, 144, 148, 151, 156, 158, 161, 162, 165, 168, 170, 171, 175, 178, 180, 183-185, 188, 191, 193, 194, 196, 198, and 200-204 (vanC1); 206, 210, 213, 217, 220, 221, 222, 224-226, 228-238, 240, 242 and 243 (vanC2/3); 388, 391, 394, 396, 399, 409, 415, 416, 425, 428, 435, 438, 440, 443, 445, 462, 465, 468, 471, 474, 477, 480, 483, 488, 491, 494-498 and 500-502 (vanD); 334, 337-380 and 382-387 (vanE); 244, 249, 252, 253, 255, 256, 260, 263, 264, 267, 270-272, 275-283, 285, 288, 291, 293, 295, 297-300, 302, 303, 305, 307, 309-311, 313-318, 321, 322, 325-333 (vanG); and b) at least one reverse primer comprising the sequence selected from the group consisting of: SEQ ID NOS: SEQ ID NOS: SEQ ID NOS: 3, 5, 8, 10, 21, 31, 32, 36, 44, 47, 51, 52, 55 and 60 (vanA); 63, 65, 66, 74, 77, 85-88, 90, 91, 95, and 97-102 (vanB); 125, 129, 132, 135, 137, 140, 143, 146, 147, 150, 153, 155, 157, 160, 164, 167, 169, 173, 174, 177, 179, 187, 190 and 192 (vanC1); 208, 209, 212, 215, 216, 219, 223, 227, 239 and 241 (vanC2/3); 390, 393, 395, 398, 401, 411, 417, 419, 421, 426, 430, 434, 437, 439, 442, 444, 447-455, 458-461, 464, 467, 470, 473, 476, 479, 482, 486, 490 and 493 (vanD); 336 and 381 (vanE); 246-248, 250, 251, 254, 258, 259, 262, 266, 269, 274, 284, 286, 287, 290, 301, 304, 306, 308, 312, 320 and 324 (vanG).

One embodiment is directed to a kit for detecting DNA of a Efm sodA or Efs sodA gene or Efm novel gene or Efs novel gene or dual genes, in a sample, comprising one or more probes comprising a sequence selected from the group consisting of: SEQ ID NOS: 555, 562, 571 (Efm sodA); 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); 728, 750 (Efm novel); 815, 832 (Efs novel), and 844 (Efm/Efs dual). In a particular embodiment, the kit further comprises a) at least one forward primer comprising the sequence selected from the group consisting of: SEQ ID NOS: 517 (Efm sodA); 577, 586, 590, 598, 599, 600 (Efs sodA); 683, 687, 692 (Efm novel); 758, 772, 773, 775 (Efs novel); and 843 (Efm/Efs dual); and at least one reverse primer comprising a sequence selected from the group consisting of SEQ ID NOS: 529 (Efm sodA); 617, 623, 624, 625, 637, 640 (Efs sodA); 707, 720, 723 (Efm novel); 785, 791, 797, 799, 803 (Efs novel); 845 and 846 (Efm/Efs dual).

One embodiment is directed to a kit for detecting DNA of a vancomycin-resistance gene(s) or Enterococcal marker gene in a sample, comprising one or more probes comprising a sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA); 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205 (vanC1); 207, 211 (vanC2/3); 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 475, 478, 481, 484, 485, 487, 489, 492, 499 (vanD); 335 (vanE); 245, 257, 261, 265, 268, 273, 289, 292, 294, 296, 319, 323 (vanG), 555, 562, 571 (Efm sodA); 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); 728, 750 (Efm novel); 815, 832 (Efs novel), and 844 (Efm/Efs dual). In a particular embodiment, the kit further comprises a) at least one forward primer comprising the sequence selected from the group consisting of: SEQ ID NOS: 1, 6, 19, 22, 23, 26, 28, 29, 33, 34, 37-42, 45, 48, 53 and 59 (vanA); 61, 68, 70-72, 75, 81, 83, 89, 93, 94, 103-105, 107 and 111 (vanB); 123, 127, 130, 133, 138, 141, 144, 148, 151, 156, 158, 161, 162, 165, 168, 170, 171, 175, 178, 180, 183-185, 188, 191, 193, 194, 196, 198, and 200-204 (vanC1); 206, 210, 213, 217, 220, 221, 222, 224-226, 228-238, 240, 242 and 243 (vanC2/3); 388, 391, 394, 396, 399, 409, 415, 416, 425, 428, 435, 438, 440, 443, 445, 462, 465, 468, 471, 474, 477, 480, 483, 488, 491, 494-498 and 500-502 (vanD); 334, 337-380 and 382-387 (vanE); 244, 249, 252, 253, 255, 256, 260, 263, 264, 267, 270-272, 275-283, 285, 288, 291, 293, 295, 297-300, 302, 303, 305, 307, 309-311, 313-318, 321, 322, 325-333 (vanG); 517 (Efm sodA); 577, 586, 590, 598, 599, 600 (Efs sodA); 683, 687, 692 (Efm novel); 758, 772, 773, 775 (Efs novel); and 843 (Efm/Efs dual); and b) at least one reverse primer comprising the sequence selected from the group consisting of: SEQ ID NOS: SEQ ID NOS: SEQ ID NOS: 3, 5, 8, 10, 21, 31, 32, 36, 44, 47, 51, 52, 55 and 60 (vanA); 63, 65, 66, 74, 77, 85-88, 90, 91, 95, and 97-102 (vanB); 125, 129, 132, 135, 137, 140, 143, 146, 147, 150, 153, 155, 157, 160, 164, 167, 169, 173, 174, 177, 179, 187, 190 and 192 (vanC1); 208, 209, 212, 215, 216, 219, 223, 227, 239 and 241 (vanC2/3); 390, 393, 395, 398, 401, 411, 417, 419, 421, 426, 430, 434, 437, 439, 442, 444, 447-455, 458-461, 464, 467, 470, 473, 476, 479, 482, 486, 490 and 493 (vanD); 336 and 381 (vanE); 246-248, 250, 251, 254, 258, 259, 262, 266, 269, 274, 284, 286, 287, 290, 301, 304, 306, 308, 312, 320 and 324 (vanG); 529 (Efm sodA); 617, 623, 624, 625, 637, 640 (Efs sodA); 707, 720, 723 (Efm novel); 785, 791, 797, 799, 803 (Efs novel); 845 and 846 (Efm/Efs dual).

In a particular embodiment, the kit further comprises reagents for isolating and/or sequencing the vancomycin-resistance gene(s) in the sample. In a particular embodiment, the one or more probes are labeled with different detectable labels. In a particular embodiment, the one or more probes are labeled with the same detectable labels. In a particular embodiment, the at least one forward primer, the at least one reverse primer and the one or more probes are selected from the groups consisting of: Groups 1-212 of Table 5, Groups 213-601 of Table 6, Groups 603-605 of Table 8B, Groups 606-627 of Table 9B, Groups 628-636 of Table 10B, Groups 637-643 of Table 11B, and Group 644 of Table 12.

One embodiment is directed to a method for diagnosing a condition, syndrome or disease in a human associated with a vancomycin-resistant organism, comprising: a) contacting a sample with at least one forward and reverse primer set selected from the group consisting of: Groups 1-601 of Tables 5 and 6; b) conducting an amplification reaction, thereby producing an amplicon; and c) detecting the amplicon using one or more probes selected from the group consisting of: SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA); 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205 (vanC1); 207, 211 (vanC2/3); 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 475, 478, 481, 484, 485, 487, 489, 492, 499 (vanD); 335 (vanE); 245, 257, 261, 265, 268, 273, 289, 292, 294, 296, 319, 323 (vanG); wherein the generation of an amplicon is indicative of the presence of an organism resistant to vancomycin in the sample. In a particular embodiment, the sample is blood, serum, plasma, enriched peripheral blood mononuclear cells, neoplastic or other tissue obtained from biopsies, cerebrospinal fluid, saliva, fluids collected from the ear, eye, mouth, and respiratory airways, sputum, skin, tears, oropharyngeal swabs, nasopharyngeal swabs, throat swabs, urine, anal-rectal swabs, feces, skin swabs, nasal aspirates, nasal wash, fluids and cells obtained by the perfusion of tissues of both human and animal origin, and fluids and cells derived from the culturing of human cells, including human stem cells and human cartilage, fibroblasts or samples derived from inanimate objects. A sample may be collected from more than one collection site, e.g., blood and an anal-rectal swab. In a particular embodiment, the complications, conditions, syndromes or diseases in humans associated with a vancomycin-resistant organism are selected from the group consisting of: infections at indwelling sites and wounds, urinary tract infections, sepsis, infections from indwelling urinary or central venous catheters, and infections from abdominal or cardiothoracic surgery.

One embodiment is directed to a method for diagnosing a condition, syndrome or disease in a human associated with an Enterococcal organism, comprising: a) contacting a sample with at least one forward and reverse primer set selected from the group consisting of: Groups 603-644 of Tables 8B, 9B, 10B, 11B, and 12; b) conducting an amplification reaction, thereby producing an amplicon; and c) detecting the amplicon using one or more probes selected from the group consisting of: SEQ ID NOS: 555, 562, 571 (Efm sodA); 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); 728, 750 (Efm novel); 815, 832 (Efs novel), and 844 (Efm/Efs dual); wherein the generation of an amplicon is indicative of the presence of an Enterococcal organism in the sample. In a particular embodiment, the sample is blood, serum, plasma, enriched peripheral blood mononuclear cells, neoplastic or other tissue obtained from biopsies, cerebrospinal fluid, saliva, fluids collected from the ear, eye, mouth, and respiratory airways, sputum, skin, tears, oropharyngeal swabs, nasopharyngeal swabs, throat swabs, urine, anal-rectal swabs, feces, skin swabs, nasal aspirates, nasal wash, fluids and cells obtained by the perfusion of tissues of both human and animal origin, and fluids and cells derived from the culturing of human cells, including human stem cells and human cartilage, fibroblasts or samples derived from inanimate objects. A sample may be collected from more than one collection site, e.g., blood and an anal-rectal swab. In a particular embodiment, the complications, conditions, syndromes or diseases in humans associated with a vancomycin-resistant organism are selected from the group consisting of: infections at indwelling sites and wounds, urinary tract infections, sepsis, infections from indwelling urinary or central venous catheters, and infections from abdominal or cardiothoracic surgery.

One embodiment is directed to a kit for amplifying and sequencing DNA of a vancomycin-resistance gene(s) in a sample, comprising: a) at least one forward primer comprising the sequence selected from the group consisting of: SEQ ID NOS: 1, 6, 19, 22, 23, 26, 28, 29, 33, 34, 37-42, 45, 48, 53 and 59 (vanA); 61, 68, 70-72, 75, 81, 83, 89, 93, 94, 103-105, 107 and 111 (vanB); 123, 127, 130, 133, 138, 141, 144, 148, 151, 156, 158, 161, 162, 165, 168, 170, 171, 175, 178, 180, 183-185, 188, 191, 193, 194, 196, 198, and 200-204 (vanC1); 206, 210, 213, 217, 220, 221, 222, 224-226, 228-238, 240, 242 and 243 (vanC2/3); 388, 391, 394, 396, 399, 409, 415, 416, 425, 428, 435, 438, 440, 443, 445, 462, 465, 468, 471, 474, 477, 480, 483, 488, 491, 494-498 and 500-502 (vanD); 334, 337-380 and 382-387 (vanE); 244, 249, 252, 253, 255, 256, 260, 263, 264, 267, 270-272, 275-283, 285, 288, 291, 293, 295, 297-300, 302, 303, 305, 307, 309-311, 313-318, 321, 322, 325-333 (vanG); and b) at least one reverse primer comprising the sequence selected from the group consisting of: SEQ ID NOS: 3, 5, 8, 10, 21, 31, 32, 36, 44, 47, 51, 52, 55 and 60 (vanA); 63, 65, 66, 74, 77, 85-88, 90, 91, 95, and 97-102 (vanB); 125, 129, 132, 135, 137, 140, 143, 146, 147, 150, 153, 155, 157, 160, 164, 167, 169, 173, 174, 177, 179, 187, 190 and 192 (vanC1); 208, 209, 212, 215, 216, 219, 223, 227, 239 and 241 (vanC2/3); 390, 393, 395, 398, 401, 411, 417, 419, 421, 426, 430, 434, 437, 439, 442, 444, 447-455, 458-461, 464, 467, 470, 473, 476, 479, 482, 486, 490 and 493 (vanD); 336 and 381 (vanE); 246-248, 250, 251, 254, 258, 259, 262, 266, 269, 274, 284, 286, 287, 290, 301, 304, 306, 308, 312, 320 and 324 (vanG); and c) reagents for the sequencing of amplified DNA fragments.

One embodiment is directed to a kit for amplifying and sequencing DNA of an Enterococci specific gene in a sample, comprising: a) at least one forward primer comprising the sequence selected from the group consisting of: SEQ ID NOS: 517 (Efm sodA); 577, 586, 590, 598, 599, 600 (Efs sodA); 683, 687, 692 (Efm novel); 758, 772, 773, 775 (Efs novel); and 843 (Efm/Efs dual); and at least one reverse primer comprising a sequence selected from the group consisting of SEQ ID NOS: 529 (Efm sodA); 617, 623, 624, 625, 637, 640 (Efs sodA); 707, 720, 723 (Efm novel); 785, 791, 797, 799, 803 (Efs novel); 845 and 846 (Efm/Efs dual); and c) reagents for the sequencing of amplified DNA fragments.

In a particular embodiment, the sample is blood, serum, plasma, enriched peripheral blood mononuclear cells, neoplastic or other tissue obtained from biopsies, cerebrospinal fluid, saliva, fluids collected from the ear, eye, mouth, and respiratory airways, sputum, skin, tears, oropharyngeal swabs, nasopharyngeal swabs, throat swabs, urine, anal-rectal swabs, feces, skin swabs, nasal aspirates, nasal wash, fluids and cells obtained by the perfusion of tissues of both human and animal origin, and fluids and cells derived from the culturing of human cells, including human stem cells and human cartilage, fibroblasts or samples derived from inanimate objects. In a particular embodiment, the complications, conditions, syndromes or diseases in humans associated with a vancomycin-resistant organism are selected from the group consisting of: skin infections, such as boils, impetigo, cellulitis, and scalded skin syndrome; food poisoning, leading to abdominal cramps, nausea, vomiting, and diarrhea; bacteremia, resulting in a persistent fever and other signs of blood poisoning; toxic shock syndrome, resulting in high fever, nausea, vomiting, rash on palms and soles, confusion, muscle aches, seizures, headache; and septic arthritis, resulting in joint swelling, severe pain in the affected joint, fever, and shaking chills.

One embodiment is directed to an internal control plasmid and vancomycin-resistance positive control plasmids. The non-competitive internal control plasmid is a synthetic target that does not occur naturally in clinical sample types for which this assay is intended. The synthetic target sequence incorporates an artificial, random polynucleotide sequence with a known GC content. The synthetic target sequence is:

(SEQ ID NO: 503)

5′GCGAAGTGAGAATACGCCGTGTCGCAGTTTCCTTGAGCAGTGTCTCTA

AATGCCTCAAACCGTCGCATTTTTGGTTATAGCAGTAACTATATGGAGGT

CCGTAGGCGGCGTGCGTGGGGGCACCAAACTCATCCAACGGTCGACTGC

GCCTGTAGGGTCTTAAGAAGCGGCACCTCAGACCGATAGCATAGCACTT

AAAGAGGAATTGAATAATCAAGATGGGTATCCGACCGACGCGGAGTGAC

CGAGGAAGAGGACCCTGCATGTATCCTGAGAGTATAGTTGTCAGAGCAG

CAATTGATTCACCACCAAGGGACTTAGTCT 3′.

This internal control is detected by a forward primer (SEQ ID NO: 504), a reverse primer (SEQ ID NO: 506) and a probe (SEQ ID NO: 505). A plasmid vector containing the internal control target sequence (SEQ ID NO: 503) is included in the assay. The internal control plasmid is added directly to the reaction mix to monitor the integrity of the PCR reagents and the presence of PCR inhibitors.

The vancomycin-resistance positive control plasmid contain partial sequences for one or more of the vancomycin resistance targets (i.e. vanA, vanB, vanC, etc.), respectively. The positive control plasmids comprise forward primer, probe and reverse primer sequences for the given vancomycin resistance. An artificial polynucleotide sequence is inserted within the positive control sequence corresponding to the given target to allow the amplicon generated by the target primer pairs to be differentiated from the amplicon derived by the same primer pairs from a natural target by size, by a unique restriction digest profile, and by a probe directed against the artificial sequence. The positive control plasmids are intended to be used as a control to confirm that the assay is performing within specifications.

The oligonucleotides of the present invention and their resulting amplicons do not cross react and, thus, will work together without negatively impacting each other. The primers and probes of the present invention do not cross react with other potentially contaminating species that would be present in a sample matrix.

One embodiment is directed to a method of hybridizing one or more isolated nucleic acid sequences comprising a sequence selected from the group consisting of: SEQ ID NOS: 513-846 to a Enterococcus faecium specific gene and/or Enterococcus faecalis specific gene, comprising contacting one or more isolated nucleic acid sequences to a sample comprising the Enterococcus faecium specific gene and/or Enterococcus faecalis specific gene under conditions suitable for hybridization.

One embodiment is directed to a method of hybridizing one or more isolated nucleic acid sequences comprising a sequence selected from the group consisting of: SEQ ID NOS: 1-502 and 513-846 to a vancomycin-resistance gene and/or an Enterococcus faecium specific gene and/or an Enterococcus faecalis specific gene, comprising contacting one or more isolated nucleic acid sequences to a sample comprising the vancomycin-resistance gene and/or the Enterococcus faecium specific gene and/or Enterococcus faecalis specific gene under conditions suitable for hybridization.

One embodiment is directed to a kit for detecting an Enterococcus faecium specific gene and/or an Enterococcus faecalis specific gene in a sample, comprising one or more probes comprising a sequence selected from the group consisting of: SEQ ID NOS: 513-846.

One embodiment is directed to a kit for detecting a vancomycin-resistance gene and/or an Enterococcus faecium specific gene and/or Enterococcus faecalis specific gene in a sample, comprising one or more probes comprising a sequence selected from the group consisting of: SEQ ID NOS: 1-502 and 513-846.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot diagram of the amplification of the vanA synthetic construct.

FIG. 2 is a plot diagram of the amplification of the vanB synthetic construct.

FIG. 3 is an electropherogram and legend showing the migration of vanA and vanB PCR products during gel electrophoresis.

FIG. 4 is a plot diagram of the amplification of the E. faecium sodA oligonucleotide solution, E. faecalis sodA oligonucleotide solution, and both E. faecium and E. faecalis dual oligonucleotide solution.

FIG. 5 is an electropherogram and legend showing the migration of the E. faecium sodA, E. faecalis sodA, and both E. faecium and E. faecalis dual PCR products during gel electrophoresis.

DETAILED DESCRIPTION

A diagnostic or screening test that can detect multiple resistance genes simultaneously (the van genes), as well as determine whether a sample contains VRE, is necessary, as vancomycin resistant organisms are the major causative agents, for example, of HAIs.

Described herein are optimized probes and primers that, alone or in various combinations, allow for the amplification, detection, isolation, and sequencing of vancomycin genes that can be found in clinical isolates, including Enterococcal and Staphylococcal pathogens. Specific probes and primers, i.e., probes and primers that detect all known and characterized vancomycin have been discovered and are described herein. Nucleic acid primers and probes for detecting bacterial genetic material, especially the resistance genes vanA and vanB, and methods for designing and optimizing the respective primer and probe sequences, are described. The present invention also provides nucleic acid primers and probes for detecting the resistance genes van C, vanD, vanE and vanG. The present invention furthermore provides nucleic acid primers and probes for detecting the genus Enterococci.

The primers and probes described herein can be used, for example, to screen patients for the presence of the vanA, vanB, vanC, vanD, vanE and vanG resistance genes, Efs sodA, Efm sodA, Efs novel, Efm novel, Efm/Efs dual, e.g., in clinical isolates, including Enterococcal and Staphylococcal pathogens, in a multiplex format.

The primers and probes of the present invention can be used for the detection of the vancomycin-resistance genes in a multiplex format to allow detection of vancomycin resistant organisms (including VRE and vancomycin resistant Staphylococcus aureus (VRSA). Currently, the vancomycin-resistance genes are tested separately; however, the multiplex format option of the present invention allows relative comparisons to be made between these prevalent resistance genes.

Vancomycin Resistance

The importance of Enterococcus in vancomycin-resistant nosocomial infections or hospital acquired infections (HAIs) (vancomycin-resistant Enterococcus; VRE), has been the impetus for the development of therapeutic alternatives to vancomycin. One such alternative that is currently in use in the United States and European Union is linezolid. Linezolid is of the oxazolidinone class and is frequently used on multi-drug resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and VRE. Vancomycin resistance is classified according to six phenotypes: VanA, VanB, VanC (C1, C2, C3), VanD, VanE and VanG. VanA and VanB are inducible and transferable, while VanC, VanD, VanE and VanG are constitutive and non-transferable. In general, the VanA and VanB phenotypes are the most clinically important and found most often in E. faecium and E. faecalis. The VanC phenotype may also be clinically important as it is frequently associated with resistance infections caused by other Enterococcus species (such as E. gallinarum, E. casseliflavus, and E. flavescens). It is less clear if the VanD, VanE and VanG phenotypes are clinically important. Vancomycin-resistant E. faecium and E. faecalis corresponding to the VanD, VanE, or VanG phenotypes have been isolated, but the prevalence of these relative to the VanA and VanB phenotypes in these species is not known and may be much lower in clinical settings. (Cetinkaya et al., Clin. Microbiol. Rev. 13:686-707 (2000); McKessar et al., Antimicrob. Agents Chemother. 44:3224-3228 (2000); Perichon et al., Antimicrob. Agents Chemother. 41:2016-2018 (1997); Boyd et al., Antimicrob. Agents Chemother. 46:1977-1979 (2002); Domingo et al., Antimicrob. Agents Chemother. 49:4784-4786 (2005)).

The presence of non-enterococcal vancomycin resistance may not alter present treatment or control measures. However, since vanB genes may be transferred from intestinal flora to enterococcal species, knowledge of potential reservoirs of glycopeptide resistance genes is critical for maintaining VRE infection control over VRE and other vancomycin-resistant species. (Ballard et al., Antimicrob. Agents Chemother. 49:77-81 (2005); Ballard et al., Antimicrob. Agents Chemother. 49:1688-1694 (2005); Domingo et al., J. Antimicrob. Chemother. 55:466-474 (2005)).

The mechanism of vancomycin resistance involves substituting the D-alanine terminating residue of cell wall precursors to which vancomycin binds, with a D-lactate residue—VanA, VanB, or VanD phenotypes, or D-serine residue—VanC and VanE phenotypes, and presumably the VanG phenotype. The modified cell wall precursors have a lower affinity for vancomycin binding, neutralizing its effect. The VanA phenotype is highly resistant to vancomycin and another glycopeptide-class antibiotic, teicoplanin. The VanB phenotype is associated with moderate to high levels of vancomycin resistance, but sensitivity to teicoplanin. The VanC, VanE and VanG phenotypes are associated with lower levels of resistance to both vancomycin and teicoplanin, while the VanD phenotype is associated with moderate levels of resistance to both vancomycin and teicoplanin (de Lalla et al., Antimicrob Agents Chemother. 36:2192-2196 (1992); McKessar et al., Antimicrob. Agents Chemother. 44: 3224-3228 (2000); Perichon et al., Antimicrob. Agents Chemother. 41:2016-2018 (1997); Leclercq et al., Clin. Infect. Dis. 24:545-556 (1997); Yean et al., BMC Microbiology 7:112 (2007); Arthur et al., J. Bacteriol. 175:117-127 (1993)). Table 1 lists the minimal inhibitory concentration (MIC) for vancomycin and teicoplanin for each phenotype. (Cetinkaya et al., Clin. Microbiol. Rev. 13:686-707 (2000); McKessar et al., Antimicrob. Agents Chemother. 44: 3224-3228 (2000)).

TABLE 1

Minimal inhibitory concentration (MIC) for vancomycin

and teicoplanin for the resistance genes vanA,

vanB, vanC, vanD, vanE and vanG

MIC (μg/mL)
MIC (μg/mL)

Phenotype
Allele
vancomycin
teicoplanin

VanA
vanA
64->1000 (high)
16-512 (high)

VanB
vanB
4-1024 (can be high)
≦0.5 (sensitive)

VanC
vanC
2-32 (low)
≦0.5 (sensitive)

VanD
vanD
128 (moderate)
4 (moderate)

VanE
vanE
16 (low)
0.5 (sensitive)

VanG
vanG
12-16 (low)
0.5 (sensitive)

VRE infections can be treated with non-glycopeptide antibiotics such as cephalosporins and aminoglycosides; regardless of the phenotype, susceptibility testing is performed on isolates to determine the best course of treatment.

Diagnosis of VRE can be determined using bacteriological and molecular-based diagnostic tests to identify the type of vancomycin resistance (i.e. VanA, VanB, VanC phenotypes, etc.) and the infecting/colonizing Enterococcus species. Once VRE is identified, the isolate is subjected to further tests to predict its susceptibility to antibiotics (for treatment of infections) and, in some cases, is speciated to enable the infection to be tracked (for infection control).

Risk factors for VRE infection or colonization include indwelling urinary or central venous catheters; recent abdominal or cardiothoracic surgery; prolonged and/or frequent hospital stays; hospital stay on an ICU, oncology, or transplant ward; stay in a long-term care facility (LTCF); and prior treatment with vancomycin, cephalosporins, metronidazole or clindamycin, or multiple antibiotics.

A reduction or eradication of VRE can occur upon implementation of control measures. VRE incidence can be decreased in hospitals in which patient surveillance cultures are used in concert with barrier isolation of colonized patients. Active infection-control intervention, relying heavily on surveillance cultures to guide the isolation of colonized patients, is important to reducing and even eradicating VRE (Ostrowsky et al., N Engl J Med. 344:1427-1433 (2001)).

Active Surveillance Cultures (ACS), combined with contact precautions, has also been described as effective in VRE reduction and sustaining long-term control. Conversely, long-term VRE increases are observed in institutions not utilizing this approach. (Management of Multidrug-Resistant Organisms in Healthcare Settings, HIPAC/CDC (2006)).

Additional control methods include administrative controls, such as tracking and trending VRE infections/colonizations and establishing a system whereby VRE positive results trigger specific responses (i.e. administrative controls). Control methods will require screening. Testing has been shown to correlate with reduction in VRE occurrence or re-occurrence.

Culture-based methods are widely used to screen patients for the presence of VRE. Bacterial colonies growing on Bile Esculin Azide plates supplemented with vancomycin (BEAV) are preliminarily identified as VRE based on colonial morphology, but additional culture steps would be required for definitive confirmation. The Bile Esculin test differentiates enterococci and group D streptococci from non-group D viridans group streptococci. Bile Esculin positive colonies appear black and are preliminarily identified as enterococci before Gram-staining Gram-positive cocci (Enterococcus is Gram-positive) are then plated on a blood agar plate for isolation. The Gram status of the isolates is confirmed and they are subsequently checked for catalase and pyrrolinodyl peptidase activity. Catalase-negative and pyrrolinodyl peptidase positive isolates can be reported as Enterococcus spp. Positive isolates subjected to susceptibility testing can be classified as VRE if the minimal inhibitory concentration (MIC) of vancomycin is 32 μg/mL (Moellering, R., Clin Infect Dis. 14:1173-6 (1992)).

The use of automated systems allows one to speciate the Enterococcus isolate. Microscopic observation of motility can also be used to speciate Enterococcus. This entire process can take several days, with the first result suggesting Enterococcus obtained in 24-48 hours. These putative vancomycin-resistant isolates can be confirmed more quickly, perhaps within hours, by polymerase chain reaction (PCR) detection of any of the vancomycin-resistance markers. In addition, patient's clinical specimens can be screened rapidly using a PCR test designed to detect vancomycin-resistance genes. A positive result would suggest the need for barrier isolation, while a negative result may establish that barrier precautions are unnecessary.

In addition to VRE infections/colonizations, another form of vancomycin resistant bacteria has been observed. Vancomycin resistant Staphylococcus aureus (VRSA) are antimicrobial-resistant Staphylococci. Patients that develop VRSA infections usually have several underlying health conditions (such as diabetes), previous infections with MRSA, and recent hospitalizations. The spread of VRSA occurs through close physical contact with infected patients or contaminated material.

Assays

Tables 2 and 3 demonstrate possible diagnostic outcome scenarios using the probes and primers described herein in diagnostic methods.

TABLE 2

Possible diagnostic outcome scenarios using the

probes and primers of the present invention.

Cha.
Trg.
Results

1
vanA
+
−
+
−

2
vanB
−
+
+
−

3
IC
+
+
+
−

Interpretation
Type A
Type B
Type A
Invalid

van.
van.
and B van.
sample

resistance
resistance
resistance
result

Cha.., Fluorescence channel; Trg., target; +, target detected; −, target not detected; vanA, target corresponding to vancomycin resistance type A; vanB, target corresponding to vancomycin resistance type B; IC, internal control

TABLE 3

Possible diagnostic outcome scenarios using the

probes and primers of the present invention.

Chan.
Trg.
Results

1
vanA and/or
+
−

vanB

2
IC
+
−

Interpretation
Type A and/or B
Invalid sample result

vancomycin resistance

Chan., Fluorescence channel; Trg., target; +, target detected; -, target not detected; vanA, target corresponding to vancomycin resistance type A; vanB, target corresponding to vancomycin resistance type B; IC, internal control

Detection of the internal control (IC) indicates that the sample result is valid, where an absence of a signal corresponding to the IC indicates either an invalid result or that one or more of the specific targets is at a high starting concentration. A signal indicating a high starting concentration of specific target in the absence of an internal control signal is considered to be a valid sample result.

The advantages of a multiplex format are: (1) simplified and improved testing and analysis; (2) increased efficiency and cost-effectiveness; (3) decreased turnaround time (increased speed of reporting results); (4) increased productivity (less equipment time needed); and (5) coordination/standardization of results for patients for multiple organisms (reduces error from inter-assay variation).

Screening and diagnosis of the vancomycin resistance genes and VRE can lead to earlier and more effective treatment of a subject. The methods for diagnosing and detecting vancomycin resistance and VRE described herein can be coupled with effective treatment therapies (e.g., antibiotics). The antibiotic classes comprising non-glycopeptides such as cephalosporins and aminoglycosides are often prescribed for treatment of a vancomycin resistant infection. The treatments for such infections will depend upon the clinical disease state of the patient, as determinable by one of skill in the art.

The present invention therefore provides a method for specifically detecting the presence of antibiotic resistance genes in a given sample using the primers and probes provided herein. Of particular interest in this regard is the ability of the disclosed primers and probes, as well as those that can be designed according to the disclosed methods, to specifically detect all or a majority of presently characterized strains of known, characterized vancomycin-resistance genes. The optimized primers and probes are useful, therefore, for identifying and diagnosing the causative or contributing agents of disease caused by VRE, whereupon an appropriate treatment can then be administered to the individual to eradicate the bacteria.

The present invention provides one or more sets of primers that can anneal to all currently identified vancomycin-resistance genes and the genus Enterococci and thereby amplify a target from a biological sample. The present invention provides, for example, at least a first primer and at least a second primer for the vancomycin resistance genes vanA, vanB, vanC, vanD, van E and vanG, and the genus Enterococci, each of which comprises a nucleotide sequence designed according to the inventive principles disclosed herein, which are used together to amplify DNA from vancomycin-resistance genes and Enterococci in a mixed-flora sample in a multiplex assay.

Also provided herein are probes that hybridize to the vancomycin-resistance gene sequences and Enterococci sequences and/or amplified products derived from the vancomycin-resistance gene sequences and Enterococci sequences. A probe can be labeled, for example, such that when it binds to an amplified or unamplified target sequence, or after it has been cleaved after binding, a fluorescent signal is emitted that is detectable under various spectroscopy and light measuring apparatuses. The use of a labeled probe, therefore, can enhance the sensitivity of detection of a target in an amplification reaction of DNA of vancomycin-resistance genes because it permits the detection of bacterial-derived DNA at low template concentrations that might not be conducive to visual detection as a gel-stained amplification product.

Primers and probes are sequences that anneal to a bacterial genomic or bacterial genomic derived sequence, e.g., the antibiotic resistance genes of Enterococcus and/or Staphylococcus sequences, e.g., VRE and/or VRSA sequences (the “target” sequences). The target sequence can be, for example, an antibiotic resistance gene or a bacterial genome. In one embodiment, the entire gene sequence can be “scanned” for optimized primers and probes useful for detecting the antibiotic resistance genes. In other embodiments, particular regions of the gene can be scanned, e.g., regions that are documented in the literature as being useful for detecting multiple genes, regions that are conserved, or regions where sufficient information is available in, for example, a public database, with respect to the antibiotic resistance genes.

Sets or groups of primers and probes are generated based on the target to be detected. The set of all possible primers and probes can include, for example, sequences that include the variability at every site based on the known antibiotic resistance gene, or the primers and probes can be generated based on a consensus sequence of the target. The primers and probes are generated such that the primers and probes are able to anneal to a particular sequence under high stringency conditions. For example, one of skill in the art recognizes that for any particular sequence, it is possible to provide more than one oligonucleotide sequence that will anneal to the particular target sequence, even under high stringency conditions. The set of primers and probes to be sampled includes, for example, all such oligonucleotides for all known and characterized vancomycin resistance genes and for the genus Enterococci. Alternatively, the primers and probes include all such oligonucleotides for a given consensus sequence for a target.

Typically, stringent hybridization and washing conditions are used for nucleic acid molecules over about 500 bp. Stringent hybridization conditions include a solution comprising about 1 M Na⁺at 25° C. to 30 C below the Tm; e.g., 5×SSPE, 0.5% SDS, at 65 C; see, Ausubel, et al., Current Protocols in Molecular Biology, Greene Publishing, 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989). Tm is dependent on both the G+C content and the concentration of salt ions, e.g., Na⁺and K⁺. A formula to calculate the Tm of nucleic acid molecules greater than about 500 bp is Tm=81.5+0.41(% (G+C))−log₁₀[Na⁺]. Washing conditions are generally performed at least at equivalent stringency conditions as the hybridization. If the background levels are high, washing can be performed at higher stringency, such as around 15° C. below the Tm.

The set of primers and probes, once determined as described above, are optimized for hybridizing to a plurality of antibiotic resistance genes by employing scoring and/or ranking steps that provide a positive or negative preference or “weight” to certain nucleotides in a target nucleic acid strain sequence. If a consensus sequence is used to generate the full set of primers and probes, for example, then a particular primer sequence is scored for its ability to anneal to the corresponding sequence of every known native target sequence. Even if a probe were originally generated based on a consensus, the validation of the probe is in its ability to specifically anneal and detect every, or a large majority of, target sequences. The particular scoring or ranking steps performed depend upon the intended use for the primer and/or probe, the particular target nucleic acid sequence, and the number of resistance genes of that target nucleic acid sequence. The methods of the invention provide optimal primer and probe sequences because they hybridize to all or a subset of vancomycin resistance genes and the genus Enterococci. Once optimized oligonucleotides are identified that can anneal to such genes, the sequences can then further be optimized for use, for example, in conjunction with another optimized sequence as a “primer set” or for use as a probe. A “primer set” is defined as at least one forward primer and one reverse primer.

Described herein are methods for using the primers and probes for producing a nucleic acid product, for example, comprising contacting one or more nucleic acid sequences of SEQ ID NOS: 1-502 and 600-939 to a sample comprising the vancomycin-resistance genes and the Enterococci marker sequences under conditions suitable for nucleic acid polymerization. The primers and probes can additionally be used to sequence the DNA of the vancomycin-resistance genes and the Enterococci marker sequences, or used as diagnostics to, for example, detect vancomycin resistance genes in a clinical isolate sample, e.g., obtained from a subject, e.g., a mammalian subject. Particular combinations for amplifying DNA of vancomycin-resistance genes include, for example, using at least one forward primer selected from the group consisting of: SEQ ID NOS: 1, 6, 19, 22, 23, 26, 28, 29, 33, 34, 37-42, 45, 48, 53 and 59 (vanA); 61, 68, 70-72, 75, 81, 83, 89, 93, 94, 103-105, 107 and 111 (vanB); 123, 127, 130, 133, 138, 141, 144, 148, 151, 156, 158, 161, 162, 165, 168, 170, 171, 175, 178, 180, 183-185, 188, 191, 193, 194, 196, 198, and 200-204 (vanC1); 206, 210, 213, 217, 220, 221, 222, 224-226, 228-238, 240, 242 and 243 (vanC2/3); 388, 391, 394, 396, 399, 409, 415, 416, 425, 428, 435, 438, 440, 443, 445, 462, 465, 468, 471, 474, 477, 480, 483, 488, 491, 494-498 and 500-502 (vanD); 334, 337-380 and 382-387 (vanE); 244, 249, 252, 253, 255, 256, 260, 263, 264, 267, 270-272, 275-283, 285, 288, 291, 293, 295, 297-300, 302, 303, 305, 307, 309-311, 313-318, 321, 322, 325-333 (vanG), and using at least one reverse primer selected from the group consisting of: SEQ ID NOS: 3, 5, 8, 10, 21, 31, 32, 36, 44, 47, 51, 52, 55 and 60 (vanA); 63, 65, 66, 74, 77, 85-88, 90, 91, 95, and 97-102 (vanB); 125, 129, 132, 135, 137, 140, 143, 146, 147, 150, 153, 155, 157, 160, 164, 167, 169, 173, 174, 177, 179, 187, 190 and 192 (vanC1); 208, 209, 212, 215, 216, 219, 223, 227, 239 and 241 (vanC2/3); 390, 393, 395, 398, 401, 411, 417, 419, 421, 426, 430, 434, 437, 439, 442, 444, 447-455, 458-461, 464, 467, 470, 473, 476, 479, 482, 486, 490 and 493 (vanD); 336 and 381 (vanE); and 246-248, 250, 251, 254, 258, 259, 262, 266, 269, 274, 284, 286, 287, 290, 301, 304, 306, 308, 312, 320 and 324 (vanG). Particular combinations for amplifying DNA of Enterococci marker sequences include, for example, using at least one forward primer selected from the group consisting of: SEQ ID NOS: 517 (Efm sodA); 577, 586, 590, 598, 599, 600 (Efs sodA); 683, 687, 692 (Efm novel); 758, 772, 773, 775 (Efs novel); and 843 (Efm/Efs dual); and at least one reverse primer comprising a sequence selected from the group consisting of SEQ ID NOS: 529 (Efm sodA); 617, 623, 624, 625, 637, 640 (Efs sodA); 707, 720, 723 (Efm novel); 785, 791, 797, 799, 803 (Efs novel); 845 and 846 (Efm/Efs dual).

Methods are described for detecting vancomycin resistance genes in a sample, for example, comprising (1) contacting at least one forward and reverse primer set, e.g., SEQ ID NOS: SEQ ID NOS: SEQ ID NOS: 1, 6, 19, 22, 23, 26, 28, 29, 33, 34, 37-42, 45, 48, 53 and 59 (vanA); 61, 68, 70-72, 75, 81, 83, 89, 93, 94, 103-105, 107 and 111 (vanB); 123, 127, 130, 133, 138, 141, 144, 148, 151, 156, 158, 161, 162, 165, 168, 170, 171, 175, 178, 180, 183-185, 188, 191, 193, 194, 196, 198, and 200-204 (vanC1); 206, 210, 213, 217, 220, 221, 222, 224-226, 228-238, 240, 242 and 243 (vanC2/3); 388, 391, 394, 396, 399, 409, 415, 416, 425, 428, 435, 438, 440, 443, 445, 462, 465, 468, 471, 474, 477, 480, 483, 488, 491, 494-498 and 500-502 (vanD); 334, 337-380 and 382-387 (vanE); 244, 249, 252, 253, 255, 256, 260, 263, 264, 267, 270-272, 275-283, 285, 288, 291, 293, 295, 297-300, 302, 303, 305, 307, 309-311, 313-318, 321, 322, 325-333 (vanG) (forward primers) and SEQ ID NOS: 3, 5, 8, 10, 21, 31, 32, 36, 44, 47, 51, 52, 55 and 60 (vanA); 63, 65, 66, 74, 77, 85-88, 90, 91, 95, and 97-102 (vanB); 125, 129, 132, 135, 137, 140, 143, 146, 147, 150, 153, 155, 157, 160, 164, 167, 169, 173, 174, 177, 179, 187, 190 and 192 (vanC1); 208, 209, 212, 215, 216, 219, 223, 227, 239 and 241 (vanC2/3); 390, 393, 395, 398, 401, 411, 417, 419, 421, 426, 430, 434, 437, 439, 442, 444, 447-455, 458-461, 464, 467, 470, 473, 476, 479, 482, 486, 490 and 493 (vanD); 336 and 381 (vanE); 246-248, 250, 251, 254, 258, 259, 262, 266, 269, 274, 284, 286, 287, 290, 301, 304, 306, 308, 312, 320 and 324 (vanG) (reverse primers) to a sample; (2) conducting an amplification; and (3) detecting the generation of an amplified product, wherein the generation of an amplified product indicates the presence of vancomycin genes from Enterococcus and/or Staphylococcus pathogens in a clinical isolate sample.

Methods are described for detecting the Enterococci marker sequences in a sample, for example, comprising (1) contacting at least one forward and reverse primer set, e.g., SEQ ID NOS: 517 (Efm sodA); 577, 586, 590, 598, 599, 600 (Efs sodA); 683, 687, 692 (Efm novel); 758, 772, 773, 775 (Efs novel); and 843 (Efm/Efs dual) (forward primers); and SEQ ID NOS: 529 (Efm sodA); 617, 623, 624, 625, 637, 640 (Efs sodA); 707, 720, 723 (Efm novel); 785, 791, 797, 799, 803 (Efs novel); 845 and 846 (Efm/Efs dual) (reverse primers) to a sample; (2) conducting an amplification; and (3) detecting the generation of an amplified product, wherein the generation of an amplified product indicates the presence of Enterococcus in a clinical isolate sample.

The detection of amplicons using probes described herein can be performed, for example, using a labeled probe, e.g., the probe comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56-58 (vanA); 62, 64, 67, 69, 73, 76, 78-80, 82, 84, 92, 96, 108-110, 112 (vanB); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205 (vanC1); 207, 211 (vanC2/3); 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 475, 478, 481, 484, 485, 487, 489, 492, 499 (vanD); 335 (vanE); 245, 257, 261, 265, 268, 273, 289, 292, 294, 296, 319, 323 (vanG); 555, 562, 571 (Efm sodA); 644, 650, 654, 659, 661, 662, 663, 664, 665, 667, 673, 675, 676, 677 (Efs sodA); 728, 750 (Efm novel); 815, 832 (Efs novel), and 844 (Efm/Efs dual) that hybridizes to one of the strands of the amplicon generated by at least one forward and reverse primer set. The probe(s) can be, for example, fluorescently labeled, thereby indicating that the detection of the probe involves measuring the fluorescence of the sample of the bound probe, e.g., after bound probes have been isolated. Probes can also be fluorescently labeled in such a way, for example, such that they only fluoresce upon hybridizing to their target, thereby eliminating the need to isolate hybridized probes. The probe can also comprise a fluorescent reporter moiety and a quencher of fluorescence moiety. Upon probe hybridization with the amplified product, the exonuclease activity of a DNA polymerase can be used to dissociate the probe's reporter and quencher, resulting in the unquenched emission of fluorescence, which is detected. An increase in the amplified product causes a proportional increase in fluorescence, due to cleavage of the probe and release of the reporter moiety of the probe. The amplified product is quantified in real time as it accumulates. For multiplex reactions involving more than one distinct probe, each of the probes can be labeled with a different distinguishable and detectable label.

The probes can be molecular beacons. Molecular beacons are single-stranded probes that form a stem-loop structure. A fluorophore can be, for example, covalently linked to one end of the stem and a quencher can be covalently linked to the other end of the stem forming a stem hybrid. When a molecular beacon hybridizes to a target nucleic acid sequence, the probe undergoes a conformational change that results in the dissociation of the stem hybrid and, thus the fluorophore and the quencher move away from each other, enabling the probe to fluoresce brightly. Molecular beacons can be labeled with differently colored fluorophores to detect different target sequences. Any of the probes described herein can be modified and utilized as molecular beacons.

Primer or probe sequences can be ranked according to specific hybridization parameters or metrics that assign a score value indicating their ability to anneal to bacterial strains under highly stringent conditions. Where a primer set is being scored, a “first” or “forward” primer is scored and the “second” or “reverse”-oriented primer sequences can be optimized similarly but with potentially additional parameters, followed by an optional evaluation for primer dimmers, for example, between the forward and reverse primers.

The scoring or ranking steps that are used in the methods of determining the primers and probes include, for example, the following parameters: a target sequence score for the target nucleic acid sequence(s), e.g., the PriMD® score; a mean conservation score for the target nucleic acid sequence(s); a mean coverage score for the target nucleic acid sequence(s); 100% conservation score of a portion (e.g., 5′ end, center, 3′ end) of the target nucleic acid sequence(s); a species score; a strain score; a subtype score; a serotype score; an associated disease score; a year score; a country of origin score; a duplicate score; a patent score; and a minimum qualifying score. Other parameters that are used include, for example, the number of mismatches, the number of critical mismatches (e.g., mismatches that result in the predicted failure of the sequence to anneal to a target sequence), the number of native strain sequences that contain critical mismatches, and predicted Tm values. The term “Tm” refers to the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are known in the art (Berger and Kimmel (1987) Meth. Enzymol., Vol. 152: Guide To Molecular Cloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.) Vols. 1-3, Cold Spring Harbor Laboratory).

The resultant scores represent steps in determining nucleotide or whole target nucleic acid sequence preference, while tailoring the primer and/or probe sequences so that they hybridize to a plurality of target nucleic acid sequences. The methods of determining the primers and probes also can comprise the step of allowing for one or more nucleotide changes when determining identity between the candidate primer and probe sequences and the target nucleic acid sequences, or their complements.

In another embodiment, the methods of determining the primers and probes comprise the steps of comparing the candidate primer and probe nucleic acid sequences to “exclusion nucleic acid sequences” and then rejecting those candidate nucleic acid sequences that share identity with the exclusion nucleic acid sequences. In another embodiment, the methods comprise the steps of comparing the candidate primer and probe nucleic acid sequences to “inclusion nucleic acid sequences” and then rejecting those candidate nucleic acid sequences that do not share identity with the inclusion nucleic acid sequences.

In other embodiments of the methods of determining the primers and probes, optimizing primers and probes comprises using a polymerase chain reaction (PCR) penalty score formula comprising at least one of a weighted sum of: primer Tm−optimal Tm; difference between primer Tms; amplicon length−minimum amplicon length; and distance between the primer and a TaqMan® probe. The optimizing step also can comprise determining the ability of the candidate sequence to hybridize with the most target nucleic acid strain sequences (e.g., the most target organisms or genes). In another embodiment, the selecting or optimizing step comprises determining which sequences have mean conservation scores closest to 1, wherein a standard of deviation on the mean conservation scores is also compared.

In other embodiments, the methods further comprise the step of evaluating which target nucleic acid sequences are hybridized by an optimal forward primer and an optimal reverse primer, for example, by determining the number of base pair differences between target nucleic acid sequences in a database. For example, the evaluating step can comprise performing an in silico polymerase chain reaction, involving (1) rejecting the forward primer and/or reverse primer if it does not meet inclusion or exclusion criteria; (2) rejecting the forward primer and/or reverse primer if it does not amplify a medically valuable nucleic acid; (3) conducting a BLAST analysis to identify forward primer sequences and/or reverse primer sequences that overlap with a published and/or patented sequence; (4) and/or determining the secondary structure of the forward primer, reverse primer, and/or target. In an embodiment, the evaluating step includes evaluating whether the forward primer sequence, reverse primer sequence, and/or probe sequence hybridizes to sequences in the database other than the nucleic acid sequences that are representative of the target strains.

The present invention provides oligonucleotides that have preferred primer and probe qualities. These qualities are specific to the sequences of the optimized probes, however, one of skill in the art would recognize that other molecules with similar sequences could also be used. The oligonucleotides provided herein comprise a sequence that shares at least about 60-70% identity with a sequence described in Tables 5, 6, 8A, 8B, 9A, 9B, 10A, 10B, 11A, 11B, and 12. In addition, the sequences can be incorporated into longer sequences, provided they function to specifically anneal to and identify bacterial strains. In another embodiment, the invention provides a nucleic acid comprising a sequence that shares at least about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with the sequences of Tables 5, 6, 8A, 8B, 9A, 9B, 10A, 10B, 11A, 11B, and 12 or complement thereof. The terms “homology” or “identity” or “similarity” refer to sequence relationships between two nucleic acid molecules and can be determined by comparing a nucleotide position in each sequence when aligned for purposes of comparison. The term “homology” refers to the relatedness of two nucleic acid or protein sequences. The term “identity” refers to the degree to which nucleic acids are the same between two sequences. The term “similarity” refers to the degree to which nucleic acids are the same, but includes neutral degenerate nucleotides that can be substituted within a codon without changing the amino acid identity of the codon, as is well known in the art. The primer and/or probe nucleic acid sequences of the invention are complementary to the target nucleic acid sequence. The probe and/or primer nucleic acid sequences of the invention are optimal for identifying numerous strains of a target nucleic acid, e.g., vancomycin-resistance genes and the Enterococci marker sequences. In an embodiment, the nucleic acids of the invention are primers for the synthesis (e.g., amplification) of target nucleic acid sequences and/or probes for identification, isolation, detection, or analysis of target nucleic acid sequences, e.g., an amplified target nucleic acid that is amplified using the primers of the invention.

The present oligonucleotides hybridize with more than one antibiotic resistance gene (gene as determined by differences in its sequence). The probes and primers provided herein can, for example, allow for the detection of currently identified vancomycin resistance genes or a subset thereof. In addition, the primers and probes of the present invention, depending on the vancomycin resistance gene sequence(s), can allow for the detection of previously unidentified antibiotic resistance genes and VRE. The methods of the invention provide for optimal primers and probes, and sets thereof, and combinations of sets thereof, which can hybridize with a larger number of targets than available primers and probes.

In other aspects, the invention also provides vectors (e.g., plasmid, phage, expression), cell lines (e.g., mammalian, insect, yeast, bacterial), and kits comprising any of the sequences of the invention described herein. The invention further provides known or previously unknown target nucleic acid strain sequences that are identified, for example, using the methods of the invention. In an embodiment, the target nucleic acid sequence is an amplification product. In another embodiment, the target nucleic acid sequence is a native or synthetic nucleic acid. The primers, probes, and target nucleic acid sequences, vectors, cell lines, and kits can have any number of uses, such as diagnostic, investigative, confirmatory, monitoring, predictive or prognostic.

Diagnostic kits that comprise one or more of the oligonucleotides described herein, which are useful for screening for and/or detecting the presence of vancomycin resistance and VRE in an individual and/or from a sample, are provided herein. An individual can be a human male, human female, human adult, human child, or human fetus. An individual can also be any mammal, reptile, avian, fish, or amphibian. Hence, an individual can be a primate, pig, horse, cattle, sheep, dog, rabbit, guinea pig, rodent, bird or fish. A sample includes any item, surface, material, clothing, or environment, for example, sewage or water treatment plants, in which it may be desirable to test for the presence of vancomycin resistance genes and VRE. Thus, for instance, the present invention includes testing door handles, faucets, table surfaces, elevator buttons, chairs, toilet seats, sinks, kitchen surfaces, children's cribs, bed linen, pillows, keyboards, and so on, for the presence of vancomycin resistance genes and VRE.

A probe of the present invention can comprise a label such as, for example, a fluorescent label, a chemiluminescent label, a radioactive label, biotin, gold, dendrimers, aptamer, enzymes, proteins, quenchers and molecular motors. In an embodiment, the probe is a hydrolysis probe, such as, for example, a TaqMan® probe. In other embodiments, the probes of the invention are molecular beacons, any fluorescent probes, and probes that are replaced by any double stranded DNA binding dyes (e.g., SYBR Green®1).

Oligonucleotides of the present invention do not only include primers that are useful for conducting the aforementioned amplification reactions, but also include oligonucleotides that are attached to a solid support, such as, for example, a microarray, multiwell plate, column, bead, glass slide, polymeric membrane, glass microfiber, plastic tubes, cellulose, and carbon nanostructures. Hence, detection of vancomycin resistance genes and VRE can be performed by exposing such an oligonucleotide-covered surface to a sample such that the binding of a complementary strain DNA sequence to a surface-attached oligonucleotide elicits a detectable signal or reaction.

Oligonucleotides of the present invention also include primers for isolating and sequencing nucleic acid sequences derived from any identified or yet to be isolated and identified vancomycin-resistance gene and VRE.

One embodiment of the invention uses solid support-based oligonucleotide hybridization methods to detect gene expression. Solid support-based methods suitable for practicing the present invention are widely known and are described (PCT application WO 95/11755; Huber et al., Anal. Biochem., 299:24, 2001; Meiyanto et al., Biotechniques, 31:406, 2001; Relogio et al., Nucleic Acids Res., 30:e51, 2002; the contents of which are incorporated herein by reference in their entirety). Any solid surface to which oligonucleotides can be bound, covalently or non-covalently, can be used. Such solid supports include, but are not limited to, filters, polyvinyl chloride dishes, silicon or glass based chips.

In certain embodiments, the nucleic acid molecule can be directly bound to the solid support or bound through a linker arm, which is typically positioned between the nucleic acid sequence and the solid support. A linker arm that increases the distance between the nucleic acid molecule and the substrate can increase hybridization efficiency. There are a number of ways to position a linker arm. In one common approach, the solid support is coated with a polymeric layer that provides linker arms with a plurality of reactive ends/sites. A common example of this type is glass slides coated with polylysine (U.S. Pat. No. 5,667,976, the contents of which are incorporated herein by reference in its entirety), which are commercially available. Alternatively, the linker arm can be synthesized as part of or conjugated to the nucleic acid molecule, and then this complex is bonded to the solid support. One approach, for example, takes advantage of the extremely high affinity biotin-streptavidin interaction. The streptavidin-biotinylated reaction is stable enough to withstand stringent washing conditions and is sufficiently stable that it is not cleaved by laser pulses used in some detection systems, such as matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Therefore, streptavidin can be covalently attached to a solid support, and a biotinylated nucleic acid molecule will bind to the streptavidin-coated surface. In one version of this method, an amino-coated silicon wafer is reacted with the n-hydroxysuccinimido-ester of biotin and complexed with streptavidin. Biotinylated oligonucleotides are bound to the surface at a concentration of about 20 fmol DNA per mm².

One can alternatively directly bind DNA to the support using carbodiimides, for example. In one such method, the support is coated with hydrazide groups, and then treated with carbodiimide. Carboxy-modified nucleic acid molecules are then coupled to the treated support. Epoxide-based chemistries are also being employed with amine modified oligonucleotides. Other chemistries for coupling nucleic acid molecules to solid substrates are known to those of skill in the art.

The nucleic acid molecules, e.g., the primers and probes of the present invention, must be delivered to the substrate material, which is suspected of containing or is being tested for the presence of vancomycin resistance genes and VRE. Because of the miniaturization of the arrays, delivery techniques must be capable of positioning very small amounts of liquids in very small regions, very close to one another and amenable to automation. Several techniques and devices are available to achieve such delivery. Among these are mechanical mechanisms (e.g., arrayers from GeneticMicroSystems, MA, USA) and ink jet technology. Very fine pipets can also be used.

Other formats are also suitable within the context of this invention. For example, a 96-well format with fixation of the nucleic acids to a nitrocellulose or nylon membrane can also be employed.

After the nucleic acid molecules have been bound to the solid support, it is often useful to block reactive sites on the solid support that are not consumed in binding to the nucleic acid molecule. In the absence of the blocking step, excess primers and/or probes can, to some extent, bind directly to the solid support itself, giving rise to non-specific binding. Non-specific binding can sometimes hinder the ability to detect low levels of specific binding. A variety of effective blocking agents (e.g., milk powder, serum albumin or other proteins with free amine groups, polyvinylpyrrolidine) can be used and others are known to those skilled in the art (U.S. Pat. No. 5,994,065, the contents of which are incorporated herein by reference in their entirety). The choice depends at least in part upon the binding chemistry.

One embodiment uses oligonucleotide arrays, e.g., microarrays, that can be used to simultaneously observe the expression of a number of vancomycin resistance genes and VRE. Oligonucleotide arrays comprise two or more oligonucleotide probes provided on a solid support, wherein each probe occupies a unique location on the support. The location of each probe can be predetermined, such that detection of a detectable signal at a given location is indicative of hybridization to an oligonucleotide probe of a known identity. Each predetermined location can contain more than one molecule of a probe, but each molecule within the predetermined location has an identical sequence. Such predetermined locations are termed features. There can be, for example, from 2, 10, 100, 1,000, 2,000 or 5,000 or more of such features on a single solid support. In one embodiment, each oligonucleotide is located at a unique position on an array at least 2, at least 3, at least 4, at least 5, at least 6, or at least 10 times.

Oligonucleotide probe arrays for detecting gene expression can be made and used according to conventional techniques described (Lockhart et al., Nat. Biotech., 14:1675-1680, 1996; McGall et al., Proc. Natl. Acad. Sci. USA, 93:13555, 1996; Hughes et al., Nat. Biotechnol., 19:342, 2001). A variety of oligonucleotide array designs are suitable for the practice of this invention.

Generally, a detectable molecule, also referred to herein as a label, can be incorporated or added to an array's probe nucleic acid sequences. Many types of molecules can be used within the context of this invention. Such molecules include, but are not limited to, fluorochromes, chemiluminescent molecules, chromogenic molecules, radioactive molecules, mass spectrometry tags, proteins, and the like. Other labels will be readily apparent to one skilled in the art.

Oligonucleotide probes used in the methods of the present invention, including microarray techniques, can be generated using PCR. PCR primers used in generating the probes are chosen, for example, based on the sequences of Tables 6-8. In one embodiment, oligonucleotide control probes also are used. Exemplary control probes can fall into at least one of three categories referred to herein as (1) normalization controls, (2) expression level controls and (3) negative controls. In microarray methods, one or more of these control probes can be provided on the array with the inventive cell cycle gene-related oligonucleotides.

Normalization controls correct for dye biases, tissue biases, dust, slide irregularities, malformed slide spots, etc. Normalization controls are oligonucleotide or other nucleic acid probes that are complementary to labeled reference oligonucleotides or other nucleic acid sequences that are added to the nucleic acid sample to be screened. The signals obtained from the normalization controls, after hybridization, provide a control for variations in hybridization conditions, label intensity, reading efficiency and other factors that can cause the signal of a perfect hybridization to vary between arrays. The normalization controls also allow for the semi-quantification of the signals from other features on the microarray. In one embodiment, signals (e.g., fluorescence intensity or radioactivity) read from all other probes used in the method are divided by the signal from the control probes, thereby normalizing the measurements.

Virtually any probe can serve as a normalization control. Hybridization efficiency varies, however, with base composition and probe length. Preferred normalization probes are selected to reflect the average length of the other probes being used, but they also can be selected to cover a range of lengths. Further, the normalization control(s) can be selected to reflect the average base composition of the other probe(s) being used. In one embodiment, only one or a few normalization probes are used, and they are selected such that they hybridize well (i.e., without forming secondary structures) and do not match any test probes. In one embodiment, the normalization controls are mammalian genes.

“Negative control” probes are not complementary to any of the test oligonucleotides (i.e., the inventive cell cycle gene-related oligonucleotides), normalization controls, or expression controls. In one embodiment, the negative control is a mammalian gene that is not complementary to any other sequence in the sample.

The terms “background” and “background signal intensity” refer to hybridization signals resulting from non-specific binding or other interactions between the labeled target nucleic acids (e.g., mRNA present in the biological sample) and components of the oligonucleotide array. Background signals also can be produced by intrinsic fluorescence of the array components themselves. A single background signal can be calculated for the entire array, or a different background signal can be calculated for each target nucleic acid. In one embodiment, background is calculated as the average hybridization signal intensity for the lowest 5 to 10 percent of the oligonucleotide probes being used, or, where a different background signal is calculated for each target gene, for the lowest 5 to 10 percent of the probes for each gene. Where the oligonucleotide probes corresponding to a particular target hybridize well and, hence, appear to bind specifically to a target sequence, they should not be used in a background signal calculation. Alternatively, background can be calculated as the average hybridization signal intensity produced by hybridization to probes that are not complementary to any sequence found in the sample (e.g., probes directed to nucleic acids of the opposite sense or to genes not found in the sample). In microarray methods, background can be calculated as the average signal intensity produced by regions of the array that lack any oligonucleotides probes at all.

In an alternative embodiment, the nucleic acid molecules are directly or indirectly coupled to an enzyme. Following hybridization, a chromogenic substrate is applied and the colored product is detected by a camera, such as a charge-coupled camera. Examples of such enzymes include alkaline phosphatase, horseradish peroxidase and the like. A probe can be labeled with an enzyme or, alternatively, the probe is labeled with a moiety that is capable of binding to another moiety that is linked to the enzyme. For example, in the biotin-streptavidin interaction, the streptavidin is conjugated to an enzyme such as horseradish peroxidase (HRP). A chromogenic substrate is added to the reaction and is processed/cleaved by the enzyme. The product of the cleavage forms a color, either in the UV or visible spectrum. In another embodiment, streptavidin alkaline phosphatase can be used in a labeled streptavidin-biotin immunoenzymatic antigen detection system.

The invention also provides methods of labeling nucleic acid molecules with cleavable mass spectrometry tags (CMST; U.S. Patent Application No. 60/279,890). After an assay is complete, and the uniquely CMST-labeled probes are distributed across the array, a laser beam is sequentially directed to each member of the array. The light from the laser beam both cleaves the unique tag from the tag-nucleic acid molecule conjugate and volatilizes it. The volatilized tag is directed into a mass spectrometer. Based on the mass spectrum of the tag and knowledge of how the tagged nucleotides were prepared, one can unambiguously identify the nucleic acid molecules to which the tag was attached (WO 9905319).

The nucleic acids, primers and probes of the present invention can be labeled readily by any of a variety of techniques. When the diversity panel is generated by amplification, the nucleic acids can be labeled during the reaction by incorporation of a labeled dNTP or use of labeled amplification primer. If the amplification primers include a promoter for an RNA polymerase, a post-reaction labeling can be achieved by synthesizing RNA in the presence of labeled NTPs. Amplified fragments that were unlabeled during amplification or unamplified nucleic acid molecules can be labeled by one of a number of end labeling techniques or by a transcription method, such as nick-translation, random-primed DNA synthesis. Details of these methods are known to one of skill in the art and are set out in methodology books. Other types of labeling reactions are performed by denaturation of the nucleic acid molecules in the presence of a DNA-binding molecule, such as RecA, and subsequent hybridization under conditions that favor the formation of a stable RecA-incorporated DNA complex.

In another embodiment, PCR-based methods are used to detect gene expression. These methods include reverse-transcriptase-mediated polymerase chain reaction (RT-PCR) including real-time and endpoint quantitative reverse-transcriptase-mediated polymerase chain reaction (Q-RTPCR). These methods are well known in the art. For example, methods of quantitative PCR can be carried out using kits and methods that are commercially available from, for example, Applied BioSystems and Stratagene®. See also Kochanowski, Quantitative PCR Protocols (Humana Press, 1999); Innis et al., supra.; Vandesompele et al., Genome Biol., 3: RESEARCH0034, 2002; Stein, Cell Mol. Life Sci. 59:1235, 2002.

The forward and reverse amplification primers and internal hybridization probe is designed to hybridize specifically and uniquely with one nucleotide sequence derived from the transcript of a target gene. In one embodiment, the selection criteria for primer and probe sequences incorporates constraints regarding nucleotide content and size to accommodate TaqMan® requirements. SYBR Green® can be used as a probe-less Q-RTPCR alternative to the TaqMan®-type assay, discussed above (ABI Prism® 7900 Sequence Detection System User Guide Applied Biosystems, chap. 1-8, App. A-F. (2002)). A device measures changes in fluorescence emission intensity during PCR amplification. The measurement is done in “real time,” that is, as the amplification product accumulates in the reaction. Other methods can be used to measure changes in fluorescence resulting from probe digestion. For example, fluorescence polarization can distinguish between large and small molecules based on molecular tumbling (U.S. Pat. No. 5,593,867).

The primers and probes of the present invention may anneal to or hybridize to various Enterococcus and/or Staphylococcus genetic material or genetic material derived therefrom, or other genetic material derived therefrom, such as RNA, DNA, cDNA, or a PCR product.

A “sample” that is tested for the presence of vancomycin resistance genes and VRE includes, but is not limited to a tissue sample, such as, for example, blood, serum, plasma, enriched peripheral blood mononuclear cells, neoplastic or other tissue obtained from biopsies, cerebrospinal fluid, saliva, fluids collected from the ear, eye, mouth, and respiratory airways, sputum, skin, tears, oropharyngeal swabs, nasopharyngeal swabs, throat swabs, urine, anal-rectal swabs, feces, skin swabs, nasal aspirates, nasal wash, fluids and cells obtained by the perfusion of tissues of both human and animal origin, and fluids and cells derived from the culturing of human cells, including human stem cells and human cartilage or fibroblasts. The tissue sample may be fresh, fixed, preserved, or frozen. A sample also includes any item, surface, material, or clothing, or environment, for example, sewage or water treatment plants, in which it may be desirable to test for the presence of vancomycin resistance genes and VRE. Thus, for instance, the present invention includes testing door handles, faucets, table surfaces, elevator buttons, chairs, toilet seats, sinks, kitchen surfaces, children's cribs, bed linen, pillows, keyboards, and so on, for the presence of vancomycin resistance genes and VRE.

The target nucleic acid strain that is amplified may be RNA or DNA or a modification thereof. Thus, the amplifying step can comprise isothermal or non-isothermal reactions, such as polymerase chain reaction, Scorpion® primers, molecular beacons, SimpleProbes®, HyBeacons®, cycling probe technology, Invader Assay, self-sustained sequence replication, nucleic acid sequence-based amplification, ramification amplifying method, hybridization signal amplification method, rolling circle amplification, multiple displacement amplification, thermophilic strand displacement amplification, transcription-mediated amplification, ligase chain reaction, signal mediated amplification of RNA, split promoter amplification, Q-Beta replicase, isothermal chain reaction, one cut event amplification, loop-mediated isothermal amplification, molecular inversion probes, ampliprobe, headloop DNA amplification, and ligation activated transcription. The amplifying step can be conducted on a solid support, such as a multiwell plate, array, column, bead, glass slide, polymeric membrane, glass microfiber, plastic tubes, cellulose, and carbon nanostructures. The amplifying step also comprises in situ hybridization. The detecting step can comprise gel electrophoresis, fluorescence resonant energy transfer, or hybridization to a labeled probe, such as a probe labeled with biotin, at least one fluorescent moiety, an antigen, a molecular weight tag, and a modifier of probe Tm. The detection step can also comprise the incorporation of a label (e.g., fluorescent or radioactive) during an extension reaction. The detecting step comprises measuring fluorescence, mass, charge, and/or chemiluminescence.

The target nucleic acid strain may not need amplification and may be RNA or DNA or a modification thereof. If amplification is not necessary, the target nucleic acid strain can be denatured to enable hybridization of a probe to the target nucleic acid sequence.

Hybridization may be detected in a variety of ways and with a variety of equipment. In general, the methods can be categorized as those that rely upon detectable molecules incorporated into the diversity panels and those that rely upon measurable properties of double-stranded nucleic acids (e.g., hybridized nucleic acids) that distinguish them from single-stranded nucleic acids (e.g., unhybridized nucleic acids). The latter category of methods includes intercalation of dyes, such as, for example, ethidium bromide, into double-stranded nucleic acids, differential absorbance properties of double and single stranded nucleic acids, binding of proteins that preferentially bind double-stranded nucleic acids, and the like.

EXEMPLIFICATION
Example 1
Scoring a Set of Predicted Annealing Oligonucleotides

Each of the sets of primers and probes selected is ranked by a combination of methods as individual primers and probes and as a primer/probe set. This involves one or more methods of ranking (e.g., joint ranking, hierarchical ranking, and serial ranking) where sets of primers and probes are eliminated or included based on any combination of the following criteria, and a weighted ranking again based on any combination of the following criteria, for example: (A) Percentage Identity to Target Strains; (B) Conservation Score; (C) Coverage Score; (D) Strain/Subtype/Serotype Score; (E) Associated Disease Score; (F) Duplicates Sequences Score; (G) Year and Country of Origin Score; (H) Patent Score, and (I) Epidemiology Score.

(A) Percentage Identity

A percentage identity score is based upon the number of target nucleic acid strain (e.g., native) sequences that can hybridize with perfect conservation (the sequences are perfectly complimentary) to each primer or probe of a primer set and probe set. If the score is less than 100%, the program ranks additional primer set and probe sets that are not perfectly conserved. This is a hierarchical scale for percent identity starting with perfect complimentarity, then one base degeneracy through to the number of degenerate bases that would provide the score closest to 100%. The position of these degenerate bases would then be ranked. The methods for calculating the conservation is described under section B.

(i) Individual Base Conservation Score

A set of conservation scores is generated for each nucleotide base in the consensus sequence and these scores represent how many of the target nucleic acid strains sequences have a particular base at this position. For example, a score of 0.95 for a nucleotide with an adenosine, and 0.05 for a nucleotide with a cytidine means that 95% of the native sequences have an A at that position and 5% have a C at that position. A perfectly conserved base position is one where all the target nucleic acid strain sequences have the same base (either an A, C, G, or T/U) at that position. If there is an equal number of bases (e.g., 50% A & 50% T) at a position, it is identified with an N.

(ii) Candidate Primer/Probe Sequence Conservation

An overall conservation score is generated for each candidate primer or probe sequence that represents how many of the target nucleic acid strain sequences will hybridize to the primers or probes. A candidate sequence that is perfectly complimentary to all the target nucleic acid strain sequences will have a score of 1.0 and rank the highest. For example, illustrated below in Table 4 are three different 10-base candidate probe sequences that are targeted to different regions of a consensus target nucleic acid strain sequence. Each candidate probe sequence is compared to a total of 10 native sequences.

TABLE 4

#1.
A
A
A
C
A
C
G
T
G
C

0.7
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0

(SEQ ID NO: 847)

→Number of target nucleic acid strain sequences that are perfectly

complimentary - 7. Three out of the ten sequences do not have an

A at position 1.

#2.
C
C
T
T
G
T
T
C
C
A

1.0
0.9
1.0
0.9
0.9
1.0
1.0
1.0
1.0
1.0

(SEQ ID NO: 848)

→Number of target nucleic acid strain sequences that are perfectly

complimentary - 7, 8, or 9. At least one target nucleic acid strain does

not have a C at position 2, T at position 4, or G at position 5. These

differences may all be on one target nucleic acid strain molecule or

may be on two or three separate molecules.

#3.
C
A
G
G
G
A
C
G
A
T

1.0
1.0
1.0
1.0
1.0
0.9
0.8
1.0
1.0
1.0

(SEQ ID NO: 849)

→Number of target nucleic acid strain sequences that are perfectly

complimentary - 7 or 8. At least one target nucleic acid strain does

not have an A at position 6 and at least two target nucleic acid strain

do not have a C at position 7. These differences may all be on one

target nucleic acid strain molecule or may be on two separate molecules.

A simple arithmetic mean for each candidate sequence would generate the same value of 0.97. The number of target nucleic acid strain sequences identified by each candidate probe sequence, however, can be very different. Sequence #1 can only identify 7 native sequences because of the 0.7 (out of 1.0) score by the first base—A. Sequence #2 has three bases each with a score of 0.9; each of these could represent a different or shared target nucleic acid strain sequence. Consequently, Sequence #2 can identify 7, 8 or 9 target nucleic acid strain sequences. Similarly, Sequence #3 can identify 7 or 8 of the target nucleic acid strain sequences. Sequence #2 would, therefore, be the best choice if all the three bases with a score of 0.9 represented the same 9 target nucleic acid strain sequences.

(iii) Overall Conservation Score of the Primer and Probe Set—Percent Identity

The same method described in (ii) when applied to the complete primer set and probe set will generate the percent identity for the set (see A above). For example, using the same sequences illustrated above, if Sequences #1 and #2 are primers and Sequence #3 is a probe, then the percent identity for the target can be calculated from how many of the target nucleic acid sequences are identified with perfect complementarity to all three primer/probe sequences. The percent identity could be no better than 0.7 (7 out of 10 target nucleic acid strain sequences) but as little as 0.1 if each of the degenerate bases reflects a different target nucleic acid strain sequence. Again, an arithmetic mean of these three sequences would be 0.97. As none of the above examples were able to capture all the target nucleic acid strain sequences because of the degeneracy (scores of less than 1.0), the ranking system takes into account that a certain amount of degeneracy can be tolerated under normal hybridization conditions, for example, during a polymerase chain reaction. The ranking of these degeneracies is described in (iv) below.

An in silico evaluation determines how many native sequences (e.g., original sequences submitted to public databases) are identified by a given candidate primer/probe set. The ideal candidate primer/probe set is one that can perform PCR and the sequences are perfectly complementary to all the known native sequences that were used to generate the consensus sequence. If there is no such candidate, then the sets are ranked according to how many degenerate bases can be accepted and still hybridize to just the target sequence during the PCR and yet identify all the native sequences.

The hybridization conditions, for TaqMan® as an example, are: 10-50 mM Tris-HCl pH 8.3, 50 mM KCl, 0.1-0.2% Triton® X-100 or 0.1% Tween®, 1-5 mM MgCl₂. The hybridization is performed at 58-60° C. for the primers and 68-70° C. for the probe. The in silico PCR identifies native sequences that are not amplifiable using the candidate primers and probe set. The rules can be as simple as counting the number of degenerate bases to more sophisticated approaches based on exploiting the PCR criteria used by the PriMD® software. Each target nucleic acid strain sequence has a value or weight (see Score assignment above). If the failed target nucleic acid strain sequence is medically valuable, the primer/probe set is rejected. This in silico analysis provides a degree of confidence for a given genotype and is important when new sequences are added to the databases. New target nucleic acid strain sequences are automatically entered into both the “include” and “exclude” categories. Published primer and probes will also be ranked by the PriMD software.

(iv) Position (5′ to 3′) of the Base Conservation Score

In an embodiment, primers do not have bases in the terminal five positions at the 3′ end with a score less than 1. This is one of the last parameters to be relaxed if the method fails to select any candidate sequences. The next best candidate having a perfectly conserved primer would be one where the poorer conserved positions are limited to the terminal bases at the 5′ end. The closer the poorer conserved position is to the 5′ end, the better the score. For probes, the position criteria are different. For example, with a TaqMan® probe, the most destabilizing effect occurs in the center of the probe. The 5′ end of the probe is also important as this contains the reporter molecule that must be cleaved, following hybridization to the target, by the polymerase to generate a sequence-specific signal. The 3′ end is less critical. Therefore, a sequence with a perfectly conserved middle region will have the higher score. The remaining ends of the probe are ranked in a similar fashion to the 5′ end of the primer. Thus, the next best candidate to a perfectly conserved TaqMan® probe would be one where the poorer conserved positions are limited to the terminal bases at either the 5′ or 3′ ends. The hierarchical scoring will select primers with only one degeneracy first, then primers with two degeneracies next and so on. The relative position of each degeneracy will then be ranked favoring those that are closest to the 5′ end of the primers and those closest to the 3′ end of the TaqMan® probe. If there are two or more degenerate bases in a primer and probe set the ranking will initially select the sets where the degeneracies occur on different sequences.

B. Coverage Score

The total number of aligned sequences is considered under a coverage score. A value is assigned to each position based on how many times that position has been reported or sequenced. Alternatively, coverage can be defined as how representative the sequences are of the known strains, subtypes etc., or their relevance to a certain diseases. For example, the target nucleic acid strain sequences for a particular gene may be very well conserved and show complete coverage but certain strains are not represented in those sequences.

A sequence is included if it aligns with any part of the consensus sequence, which is usually a whole gene or a functional unit, or has been described as being a representative of this gene. Even though a base position is perfectly conserved it may only represent a fraction of the total number of sequences (for example, if there are very few sequences). For example, region A of a gene shows a 100% conservation from 20 sequence entries while region B in the same gene shows a 98% conservation but from 200 sequence entries. There is a relationship between conservation and coverage if the sequence shows some persistent variability. As more sequences are aligned, the conservation score falls, but this effect is lessened as the number of sequences gets larger. Unless the number of sequences is very small (e.g., under 10) the value of the coverage score is small compared to that of the conservation score. To obtain the best consensus sequence, artificial spaces are allowed to be introduced. Such spaces are not considered in the coverage score.

C. Strain/Subtype/Serotype Score

A value is assigned to each strain or subtype or serotype based upon its relevance to a disease. For example, bacterial strains and/or species that are linked to high frequencies of infection will have a higher score than strains that are generally regarded as benign. The score is based upon sufficient evidence to automatically associate a particular strain with a disease. For example, certain strains of adenovirus are not associated with diseases of the upper respiratory system. Accordingly, there will be sequences included in the consensus sequence that are not associated with diseases of the upper respiratory system.

D. Associated Disease Score

The associated disease score pertains to strains that are not known to be associated with a particular disease (to differentiate from D above). Here, a value is assigned only if the submitted sequence is directly linked to the disease and that disease is pertinent to the assay.

E. Duplicate Sequences Score

If a particular sequence has been sequenced more than once it will have an effect on representation, for example, a strain that is represented by 12 entries in GenBank of which six are identical and the other six are unique. Unless the identical sequences can be assigned to different strains/subtypes (usually by sequencing other gene or by immunology methods) they will be excluded from the scoring.

F. Year and Country of Origin Score

The year and country of origin scores are important in terms of the age of the human population and the need to provide a product for a global market. For example, strains identified or collected many years ago may not be relevant today. Furthermore, it is probably difficult to obtain samples that contain these older strains. Certain divergent strains from more obscure countries or sources may also be less relevant to the locations that will likely perform clinical tests, or may be more important for certain countries (e.g., North America, Europe, or Asia).

G. Patent Score

Candidate target strain sequences published in patents are searched electronically and annotated such that patented regions are excluded. Alternatively, candidate sequences are checked against a patented sequence database.

H. Minimum Qualifying Score

The minimum qualifying score is determined by expanding the number of allowed mismatches in each set of candidate primers and probes until all possible native sequences are represented (e.g., has a qualifying hit).

I. Other

A score is given to based on other parameters, such as relevance to certain patients (e.g., pediatrics, immunocompromised) or certain therapies (e.g., target those strains that respond to treatment) or epidemiology. The prevalence of an organism/strain and the number of times it has been tested for in the community can add value to the selection of the candidate sequences. If a particular strain is more commonly tested then selection of it would be more likely. Strain identification can be used to select better vaccines.

Example 2
Primer/Probe Evaluation

Once the candidate primers and probes have received their scores and have been ranked, they are evaluated using any of a number of methods of the invention, such as BLAST analysis and secondary structure analysis.

A. BLAST Analysis

The candidate primer/probe sets are submitted to BLAST analysis to check for possible overlap with any published sequences that might be missed by the Include/Exclude function. It also provides a useful summary.

B. Secondary Structure

The methods of the present invention include analysis of nucleic acid secondary structure. This includes the structures of the primers and/or probes, as well as their intended target strain sequences. The methods and software of the invention predict the optimal temperatures for annealing, but assumes that the target (e.g., RNA or DNA) does not have any significant secondary structure. For example, if the starting material is RNA, the first stage is the creation of a complimentary strand of DNA (cDNA) using a specific primer. This is usually performed at temperatures where the RNA template can have significant secondary structure thereby preventing the annealing of the primer. Similarly, after denaturation of a double stranded DNA target (for example, an amplicon after PCR), the binding of the probe is dependent on there being no major secondary structure in the amplicon.

The methods of the invention can either use this information as a criteria for selecting primers and probes or evaluate any secondary structure of a selected sequence, for example, by cutting and pasting candidate primer or probe sequences into a commercial internet link that uses software dedicated to analyzing secondary structure, such as, for example, MFOLD (Zuker et al. (1999) Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide in RNA Biochemistry and Biotechnology, J. Barciszewski and B. F. C. Clark, eds., NATO ASI Series, Kluwer Academic Publishers).

C. Evaluating the Primer and Probe Sequences

The methods and software of the invention may also analyze any nucleic acid sequence to determine its suitability in a nucleic acid amplification-based assay. For example, it can accept a competitor's primer set and determine the following information: (1) How it compares to the primers of the invention (e.g., overall rank, PCR and conservation ranking, etc.); (2) How it aligns to the exclude libraries (e.g., assessing cross-hybridization)—also used to compare primer and probe sets to newly published sequences; and (3) If the sequence has been previously published. This step requires keeping a database of sequences published in scientific journals, posters, and other presentations.

Example 3
Multiplexing

The Exclude/Include capability is ideally suited for designing multiplex reactions. The parameters for designing multiple primer and probe sets adhere to a more stringent set of parameters than those used for the initial Exclude/Include function. Each set of primers and probe, together with the resulting amplicon, is screened against the other sets that constitute the multiplex reaction. As new targets are accepted, their sequences are automatically added to the Exclude category.

The database is designed to interrogate the online databases to determine and acquire, if necessary, any new sequences relevant to the targets. These sequences are evaluated against the optimal primer/probe set. If they represent a new genotype or strain, then a multiple sequence alignment may be required.

Example 4
Sequences Identified for Detecting the Antibiotic Resistance Genes vanA, vanB, vanC, vanD, vanE and vanG Gene Variants

The set of primers and probes were then scored according to the methods described herein to identify the optimized primers and probes of Table 5 (vanA and vanB), and Table 6 (vanC, vanD, vanE and vanG). It should be noted that the primers, as they are sequences that anneal to a plurality of all identified or unidentified vancomycin-resistance genes, can also be used as probes either in the presence or absence of amplification of a sample.

TABLE 5

Optimized Primers and Probes for the Detection of vanA and vanB Resistance Genes.

Group No.
Forward Primer
Probe
Reverse Primer

vanA Sets

1
SEQ ID NO: 1
SEQ ID NO: 2
SEQ ID NO: 3

TTGTGCGGTATTGGGAAACAGT
TGATTTGGTCCACCTCGCCAACAACTAACGC
CGACTTCCTGATGAATACGAAAGATTCC

2
SEQ ID NO: 1
SEQ ID NO: 4
SEQ ID NO: 3

TTGTGCGGTATTGGGAAACAGT
CCTGATTTGGTCCACCTCGCCAACAACTAACG
CGACTTCCTGATGAATACGAAAGATTCC

3
SEQ ID NO: 1
SEQ ID NO: 2
SEQ ID NO: 5

TTGTGCGGTATTGGGAAACAGT
TGATTTGGTCCACCTCGCCAACAACTAACGC
CTCGACTTCCTGATGAATACGAAAGATTC

4
SEQ ID NO: 1
SEQ ID NO: 4
SEQ ID NO: 5

TTGTGCGGTATTGGGAAACAGT
CCTGATTTGGTCCACCTCGCCAACAACTAACG
CTCGACTTCCTGATGAATACGAAAGATTC

5
SEQ ID NO: 6
SEQ ID NO: 7
SEQ ID NO: 8

CGCGTTCAGGCTCATCCTT
TCACAGCCCGAAACAGCCTGCTCAATTAAGAT
ACTGTTTCCCAATACCGCACAAC

TTTGC

6
SEQ ID NO: 6
SEQ ID NO: 9
SEQ ID NO: 10

CGCGTTCAGGCTCATCCTT
CTCACAGCCCGAAACAGCCTGCTCAATTAAGA
ACTGTTTCCCAATACCGCACAA

TTT

7
SEQ ID NO: 6
SEQ ID NO: 11
SEQ ID NO: 10

CGCGTTCAGGCTCATCCTT
CTCACAGCCCGAAACAGCCTGCTCAATTAAGA
ACTGTTTCCCAATACCGCACAA

TT

8
SEQ ID NO: 6
SEQ ID NO: 12
SEQ ID NO: 10

CGCGTTCAGGCTCATCCTT
CTCACAGCCCGAAACAGCCTGCTCAATTAAGA
ACTGTTTCCCAATACCGCACAA

TTTTG

9
SEQ ID NO: 6
SEQ ID NO: 13
SEQ ID NO: 10

CGCGTTCAGGCTCATCCTT
CTCACAGCCCGAAACAGCCTGCTCAATTAAGAT
ACTGTTTCCCAATACCGCACAA

10
SEQ ID NO: 6
SEQ ID NO: 14
SEQ ID NO: 8

CGCGTTCAGGCTCATCCTT
ACAGCCCGAAACAGCCTGCTCAATTAAGATTTT
ACTGTTTCCCAATACCGCACAAC

GCT

11
SEQ ID NO: 6
SEQ ID NO: 15
SEQ ID NO: 10

CGCGTTCAGGCTCATCCTT
TCACAGCCCGAAACAGCCTGCTCAATTAAGAT
ACTGTTTCCCAATACCGCACAA

12
SEQ ID NO: 6
SEQ ID NO: 16
SEQ ID NO: 10

CGCGTTCAGGCTCATCCTT
TCACAGCCCGAAACAGCCTGCTCAATTAAGATT
ACTGTTTCCCAATACCGCACAA

13
SEQ ID NO: 6
SEQ ID NO: 17
SEQ ID NO: 10

CGCGTTCAGGCTCATCCTT
TCACAGCCCGAAACAGCCTGCTCAATTAAGAT
ACTGTTTCCCAATACCGCACAA

TTTG

14
SEQ ID NO: 6
SEQ ID NO: 14
SEQ ID NO: 10

CGCGTTCAGGCTCATCCTT
ACAGCCCGAAACAGCCTGCTCAATTAAGATTTT
ACTGTTTCCCAATACCGCACAA

GCT

15
SEQ ID NO: 6
SEQ ID NO: 18
SEQ ID NO: 10

CGCGTTCAGGCTCATCCTT
CAGCCCGAAACAGCCTGCTCAATTAAGATTTTG
ACTGTTTCCCAATACCGCACAA

CT

16
SEQ ID NO: 19
SEQ ID NO: 20
SEQ ID NO: 21

AGCAAAATCTTAATTGAGCAGGC
CCCAATACCGCACAACCGACCTCACAG
GGTCCACCTCGCCAACA

TGTTT

17
SEQ ID NO: 22
SEQ ID NO: 20
SEQ ID NO: 21

CAGCAAAATCTTAATTGAGCAGG
CCCAATACCGCACAACCGACCTCACAG
GGTCCACCTCGCCAACA

CTGTTT

18
SEQ ID NO: 6
SEQ ID NO: 20
SEQ ID NO: 21

CGCGTTCAGGCTCATCCTT
CCCAATACCGCACAACCGACCTCACAG
GGTCCACCTCGCCAACA

19
SEQ ID NO: 23
SEQ ID NO: 20
SEQ ID NO: 21

GCAAAATCTTAATTGAGCAGGCT
CCCAATACCGCACAACCGACCTCACAG
GGTCCACCTCGCCAACA

GTTT

20
SEQ ID NO: 6
SEQ ID NO: 24
SEQ ID NO: 21

CGCGTTCAGGCTCATCCTT
CAATACCGCACAACCGACCTCACAGCC
GGTCCACCTCGCCAACA

21
SEQ ID NO: 6
SEQ ID NO: 25
SEQ ID NO: 3

CGCGTTCAGGCTCATCCTT
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

22
SEQ ID NO: 22
SEQ ID NO: 25
SEQ ID NO: 3

CAGCAAAATCTTAATTGAGCAGG
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

CTGTTT

23
SEQ ID NO: 19
SEQ ID NO: 25
SEQ ID NO: 3

AGCAAAATCTTAATTGAGCAGGC
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

TGTTT

24
SEQ ID NO: 26
SEQ ID NO: 27
SEQ ID NO: 3

GGCTGTGAGGTCGGTTGTG
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

25
SEQ ID NO: 22
SEQ ID NO: 27
SEQ ID NO: 3

CAGCAAAATCTTAATTGAGCAGG
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

CTGTTT

26
SEQ ID NO: 19
SEQ ID NO: 27
SEQ ID NO: 3

AGCAAAATCTTAATTGAGCAGGC
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

TGTTT

27
SEQ ID NO: 23
SEQ ID NO: 25
SEQ ID NO: 3

GCAAAATCTTAATTGAGCAGGCT
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

GTTT

28
SEQ ID NO: 22
SEQ ID NO: 25
SEQ ID NO: 5

CAGCAAAATCTTAATTGAGCAGG
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

CTGTTT

29
SEQ ID NO: 19
SEQ ID NO: 25
SEQ ID NO: 5

AGCAAAATCTTAATTGAGCAGGC
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

TGTTT

30
SEQ ID NO: 6
SEQ ID NO: 25
SEQ ID NO: 5

CGCGTTCAGGCTCATCCTT
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

31
SEQ ID NO: 28
SEQ ID NO: 27
SEQ ID NO: 3

GGCTGTGAGGTCGGTTGT
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

32
SEQ ID NO: 29
SEQ ID NO: 7
SEQ ID NO: 8

GGCGCGTTCAGGCTCATC
TCACAGCCCGAAACAGCCTGCTCAATTAAGAT
ACTGTTTCCCAATACCGCACAAC

TTTGC

33
SEQ ID NO: 23
SEQ ID NO: 25
SEQ ID NO: 5

GCAAAATCTTAATTGAGCAGGCT
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

GTTT

34
SEQ ID NO: 23
SEQ ID NO: 27
SEQ ID NO: 3

GCAAAATCTTAATTGAGCAGGCT
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

GTTT

35
SEQ ID NO: 19
SEQ ID NO: 27
SEQ ID NO: 5

AGCAAAATCTTAATTGAGCAGGC
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

TGTTT

36
SEQ ID NO: 26
SEQ ID NO: 27
SEQ ID NO: 5

GGCTGTGAGGTCGGTTGTG
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

37
SEQ ID NO: 22
SEQ ID NO: 27
SEQ ID NO: 5

CAGCAAAATCTTAATTGAGCAGG
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

CTGTTT

38
SEQ ID NO: 28
SEQ ID NO: 27
SEQ ID NO: 5

GGCTGTGAGGTCGGTTGT
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

39
SEQ ID NO: 19
SEQ ID NO: 30
SEQ ID NO: 3

AGCAAAATCTTAATTGAGCAGGC
CAGCCTGATTTGGTCCACCTCGCCA
CGACTTCCTGATGAATACGAAAGATTCC

TGTTT

40
SEQ ID NO: 23
SEQ ID NO: 27
SEQ ID NO: 5

GCAAAATCTTAATTGAGCAGGCT
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

GTTT

41
SEQ ID NO: 6
SEQ ID NO: 14
SEQ ID NO: 31

CGCGTTCAGGCTCATCCTT
ACAGCCCGAAACAGCCTGCTCAATTAAGATTTT
CACTGTTTCCCAATACCGCACAA

GCT

42
SEQ ID NO: 6
SEQ ID NO: 25
SEQ ID NO: 32

CGCGTTCAGGCTCATCCTT
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

43
SEQ ID NO: 19
SEQ ID NO: 25
SEQ ID NO: 32

AGCAAAATCTTAATTGAGCAGGC
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

TGTTT

44
SEQ ID NO: 22
SEQ ID NO: 25
SEQ ID NO: 32

CAGCAAAATCTTAATTGAGCAGG
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

CTGTTT

45
SEQ ID NO: 23
SEQ ID NO: 25
SEQ ID NO: 32

GCAAAATCTTAATTGAGCAGGCT
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

GTTT

46
SEQ ID NO: 22
SEQ ID NO: 27
SEQ ID NO: 32

CAGCAAAATCTTAATTGAGCAGG
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

CTGTTT

47
SEQ ID NO: 26
SEQ ID NO: 27
SEQ ID NO: 32

GGCTGTGAGGTCGGTTGTG
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

48
SEQ ID NO: 28
SEQ ID NO: 27
SEQ ID NO: 32

GGCTGTGAGGTCGGTTGT
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

49
SEQ ID NO: 19
SEQ ID NO: 27
SEQ ID NO: 32

AGCAAAATCTTAATTGAGCAGGC
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

TGTTT

50
SEQ ID NO: 23
SEQ ID NO: 27
SEQ ID NO: 32

GCAAAATCTTAATTGAGCAGGCT
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

GTTT

51
SEQ ID NO: 33
SEQ ID NO: 20
SEQ ID NO: 21

GGCGCGTTCAGGCTCAT
CCCAATACCGCACAACCGACCTCACAG
GGTCCACCTCGCCAACA

52
SEQ ID NO: 29
SEQ ID NO: 20
SEQ ID NO: 21

GGCGCGTTCAGGCTCATC
CCCAATACCGCACAACCGACCTCACAG
GGTCCACCTCGCCAACA

53
SEQ ID NO: 33
SEQ ID NO: 24
SEQ ID NO: 21

GGCGCGTTCAGGCTCAT
CAATACCGCACAACCGACCTCACAGCC
GGTCCACCTCGCCAACA

54
SEQ ID NO: 34
SEQ ID NO: 35
SEQ ID NO: 36

GGGCTGTGAGGTCGGTTGT
CGTACTGCAGCCTGATTTGGTCCACCTCG
GCTCGACTTCCTGATGAATACGAAAGAT

55
SEQ ID NO: 37
SEQ ID NO: 25
SEQ ID NO: 3

GAGGTCGGTTGTGCGGTATTG
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

56
SEQ ID NO: 33
SEQ ID NO: 25
SEQ ID NO: 3

GGCGCGTTCAGGCTCAT
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

57
SEQ ID NO: 38
SEQ ID NO: 25
SEQ ID NO: 3

AGGTCGGTTGTGCGGTATTG
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

58
SEQ ID NO: 39
SEQ ID NO: 25
SEQ ID NO: 3

TGAGGTCGGTTGTGCGGTATT
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

59
SEQ ID NO: 40
SEQ ID NO: 25
SEQ ID NO: 3

GAGGTCGGTTGTGCGGTATT
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

60
SEQ ID NO: 1
SEQ ID NO: 25
SEQ ID NO: 3

TTGTGCGGTATTGGGAAACAGT
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

61
SEQ ID NO: 29
SEQ ID NO: 25
SEQ ID NO: 3

GGCGCGTTCAGGCTCATC
CTGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

62
SEQ ID NO: 37
SEQ ID NO: 27
SEQ ID NO: 3

GAGGTCGGTTGTGCGGTATTG
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

63
SEQ ID NO: 38
SEQ ID NO: 27
SEQ ID NO: 3

AGGTCGGTTGTGCGGTATTG
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

64
SEQ ID NO: 39
SEQ ID NO: 27
SEQ ID NO: 3

TGAGGTCGGTTGTGCGGTATT
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

65
SEQ ID NO: 40
SEQ ID NO: 27
SEQ ID NO: 3

GAGGTCGGTTGTGCGGTATT
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

66
SEQ ID NO: 33
SEQ ID NO: 25
SEQ ID NO: 5

GGCGCGTTCAGGCTCAT
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

67
SEQ ID NO: 40
SEQ ID NO: 25
SEQ ID NO: 5

GAGGTCGGTTGTGCGGTATT
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

68
SEQ ID NO: 37
SEQ ID NO: 25
SEQ ID NO: 5

GAGGTCGGTTGTGCGGTATTG
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

69
SEQ ID NO: 38
SEQ ID NO: 25
SEQ ID NO: 5

AGGTCGGTTGTGCGGTATTG
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

70
SEQ ID NO: 39
SEQ ID NO: 25
SEQ ID NO: 5

TGAGGTCGGTTGTGCGGTATT
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

71
SEQ ID NO: 1
SEQ ID NO: 25
SEQ ID NO: 5

TTGTGCGGTATTGGGAAACAGT
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

72
SEQ ID NO: 1
SEQ ID NO: 27
SEQ ID NO: 3

TTGTGCGGTATTGGGAAACAGT
TGCAGCCTGATTTGGTCCACCTCGC
CGACTTCCTGATGAATACGAAAGATTCC

73
SEQ ID NO: 29
SEQ ID NO: 25
SEQ ID NO: 5

GGCGCGTTCAGGCTCATC
CTGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

74
SEQ ID NO: 38
SEQ ID NO: 27
SEQ ID NO: 5

AGGTCGGTTGTGCGGTATTG
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

75
SEQ ID NO: 40
SEQ ID NO: 27
SEQ ID NO: 5

GAGGTCGGTTGTGCGGTATT
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

76
SEQ ID NO: 37
SEQ ID NO: 27
SEQ ID NO: 5

GAGGTCGGTTGTGCGGTATTG
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

77
SEQ ID NO: 39
SEQ ID NO: 27
SEQ ID NO: 5

TGAGGTCGGTTGTGCGGTATT
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

78
SEQ ID NO: 1
SEQ ID NO: 27
SEQ ID NO: 5

TTGTGCGGTATTGGGAAACAGT
TGCAGCCTGATTTGGTCCACCTCGC
CTCGACTTCCTGATGAATACGAAAGATTC

79
SEQ ID NO: 1
SEQ ID NO: 25
SEQ ID NO: 32

TTGTGCGGTATTGGGAAACAGT
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

80
SEQ ID NO: 38
SEQ ID NO: 25
SEQ ID NO: 32

AGGTCGGTTGTGCGGTATTG
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

81
SEQ ID NO: 40
SEQ ID NO: 25
SEQ ID NO: 32

GAGGTCGGTTGTGCGGTATT
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

82
SEQ ID NO: 33
SEQ ID NO: 25
SEQ ID NO: 32

GGCGCGTTCAGGCTCAT
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

83
SEQ ID NO: 37
SEQ ID NO: 25
SEQ ID NO: 32

GAGGTCGGTTGTGCGGTATTG
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

84
SEQ ID NO: 39
SEQ ID NO: 25
SEQ ID NO: 32

TGAGGTCGGTTGTGCGGTATT
CTGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

85
SEQ ID NO: 40
SEQ ID NO: 27
SEQ ID NO: 32

GAGGTCGGTTGTGCGGTATT
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

86
SEQ ID NO: 37
SEQ ID NO: 27
SEQ ID NO: 32

GAGGTCGGTTGTGCGGTATTG
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

87
SEQ ID NO: 1
SEQ ID NO: 27
SEQ ID NO: 32

TTGTGCGGTATTGGGAAACAGT
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

88
SEQ ID NO: 38
SEQ ID NO: 27
SEQ ID NO: 32

AGGTCGGTTGTGCGGTATTG
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

89
SEQ ID NO: 39
SEQ ID NO: 27
SEQ ID NO: 32

TGAGGTCGGTTGTGCGGTATT
TGCAGCCTGATTTGGTCCACCTCGC
GCTCGACTTCCTGATGAATACGAAA

90
SEQ ID NO: 41
SEQ ID NO: 20
SEQ ID NO: 21

GGCAAGACAATATGACAGCAAA
CCCAATACCGCACAACCGACCTCACAG
GGTCCACCTCGCCAACA

ATCTTAATTG

91
SEQ ID NO: 42
SEQ ID NO: 43
SEQ ID NO: 44

CAGCTACGTTTACCTATCCTGTTT
CCGGCGCGTTCAGGCTCATCCTT
GCCTGCTCAATTAAGATTTTGCTGTCAT

TTGTTAAG

92
SEQ ID NO: 45
SEQ ID NO: 46
SEQ ID NO: 47

TGGCGAGGTGGACCAAATCA
ACTGCGTTTTCAGAGCCTTTTTCCGGCTC
GGTCTGCGGGAACGGTTAT

93
SEQ ID NO: 48
SEQ ID NO: 49
SEQ ID NO: 47

GTTGGCGAGGTGGACCAAAT
AGAGCCTTTTTCCGGCTCGACTTCCTGAT
GGTCTGCGGGAACGGTTAT

94
SEQ ID NO: 26
SEQ ID NO: 50
SEQ ID NO: 47

GGCTGTGAGGTCGGTTGTG
ACTGCGTTTTCAGAGCCTTTTTCCGGCTCG
GGTCTGCGGGAACGGTTAT

95
SEQ ID NO: 26
SEQ ID NO: 27
SEQ ID NO: 51

GGCTGTGAGGTCGGTTGTG
TGCAGCCTGATTTGGTCCACCTCGC
CTGCGTTTTCAGAGCCTTTTTCC

96
SEQ ID NO: 34
SEQ ID NO: 50
SEQ ID NO: 52

GGGCTGTGAGGTCGGTTGT
ACTGCGTTTTCAGAGCCTTTTTCCGGCTCG
AGGTCTGCGGGAACGGTTAT

97
SEQ ID NO: 53
SEQ ID NO: 54
SEQ ID NO: 47

AATCAGGCTGCAGTACGGAATCT
TGCGTTTTCAGAGCCTTTTTCCGGCTCG
GGTCTGCGGGAACGGTTAT

TT

98
SEQ ID NO: 19
SEQ ID 50
SEQ ID 55

AGCAAAATCTTAATTGAGCAGGC
ACTGCGTTTTCAGAGCCTTTTTCCGGCTCG
AAGGTCTGCGGGAACGGTTA

TGTTT

99
SEQ ID NO: 19
SEQ ID NO: 56
SEQ ID NO: 52

AGCAAAATCTTAATTGAGCAGGC
TGCGTTTTCAGAGCCTTTTTCCGGCTCGAC
AGGTCTGCGGGAACGGTTAT

TGTTT

100
SEQ ID NO: 19
SEQ ID NO: 50
SEQ ID NO: 52

AGCAAAATCTTAATTGAGCAGGC
ACTGCGTTTTCAGAGCCTTTTTCCGGCTCG
AGGTCTGCGGGAACGGTTAT

TGTTT

101
SEQ ID NO: 19
SEQ ID NO: 57
SEQ ID NO: 52

AGCAAAATCTTAATTGAGCAGGC
AACTGCGTTTTCAGAGCCTTTTTCCGGCTCG
AGGTCTGCGGGAACGGTTAT

TGTTT

102
SEQ ID NO: 19
SEQ ID NO: 54
SEQ ID NO: 47

AGCAAAATCTTAATTGAGCAGGC
TGCGTTTTCAGAGCCTTTTTCCGGCTCG
GGTCTGCGGGAACGGTTAT

TGTTT

103
SEQ ID NO: 19
SEQ ID NO: 46
SEQ ID NO: 47

AGCAAAATCTTAATTGAGCAGGC
ACTGCGTTTTCAGAGCCTTTTTCCGGCTC
GGTCTGCGGGAACGGTTAT

TGTTT

104
SEQ ID NO: 19
SEQ ID NO: 58
SEQ ID NO: 3

AGCAAAATCTTAATTGAGCAGGC
TGTTTCCCAATACCGCACAACCGACCTCAC
CGACTTCCTGATGAATACGAAAGATTCC

TGTTT

105
SEQ ID NO: 59
SEQ ID NO: 56
SEQ ID NO: 52

GCAGTACGGAATCTTTCGTATTC
TGCGTTTTCAGAGCCTTTTTCCGGCTCGAC
AGGTCTGCGGGAACGGTTAT

ATCAG

106
SEQ ID NO: 59
SEQ ID NO: 54
SEQ ID NO: 60

GCAGTACGGAATCTTTCGTATTC
TGCGTTTTCAGAGCCTTTTTCCGGCTCG
GGTCTGCGGGAACGGTTA

ATCAG

vanB Sets

107
SEQ ID NO: 61
SEQ ID NO: 62
SEQ ID NO: 63

AAATCACTGGCCTACATTCTTACA
TTCTATCGCAGCGTTAAGTTCTTCCGTACC
CGAAATCGCTTGCTCAATTAAGAT

108
SEQ ID NO: 61
SEQ ID NO: 64
SEQ ID NO: 65

AAATCACTGGCCTACATTCTTACA
TATCGCAGCGTTAAGTTCTTCCGTACCGTTTA
GCTTGCTCAATTAAGATTTTTCCATCA

109
SEQ ID NO: 61
SEQ ID NO: 64
SEQ ID NO: 66

AAATCACTGGCCTACATTCTTACA
TATCGCAGCGTTAAGTTCTTCCGTACCGTTTA
GCTCAATTAAGATTTTTCCATCATATTGTC

110
SEQ ID NO: 61
SEQ ID NO: 62
SEQ ID NO: 66

AAATCACTGGCCTACATTCTTACA
TTCTATCGCAGCGTTAAGTTCTTCCGTACC
GCTCAATTAAGATTTTTCCATCATATTGTC

111
SEQ ID NO: 61
SEQ ID NO: 67
SEQ ID NO: 66

AAATCACTGGCCTACATTCTTACA
CTTCTATCGCAGCGTTAAGTTCTTCCGTACC
GCTCAATTAAGATTTTTCCATCATATTGTC

112
SEQ ID NO: 68
SEQ ID NO: 64
SEQ ID NO: 65

AAATCACTGGCCTACATTCTTAC
TATCGCAGCGTTAAGTTCTTCCGTACCGTTTA
GCTTGCTCAATTAAGATTTTTCCATCA

AAAA

113
SEQ ID NO: 68
SEQ ID NO: 64
SEQ ID NO: 66

AAATCACTGGCCTACATTCTTAC
TATCGCAGCGTTAAGTTCTTCCGTACCGTTTA
GCTCAATTAAGATTTTTCCATCATATTGTC

AAAA

114
SEQ ID NO: 68
SEQ ID NO: 62
SEQ ID NO: 66

AAATCACTGGCCTACATTCTTAC
TTCTATCGCAGCGTTAAGTTCTTCCGTACC
GCTCAATTAAGATTTTTCCATCATATTGTC

AAAA

115
SEQ ID NO: 68
SEQ ID NO: 67
SEQ ID NO: 66

AAATCACTGGCCTACATTCTTAC
CTTCTATCGCAGCGTTAAGTTCTTCCGTACC
GCTCAATTAAGATTTTTCCATCATATTGTC

AAAA

116
SEQ ID NO: 61
SEQ ID NO: 69
SEQ ID NO: 63

AAATCACTGGCCTACATTCTTACA
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CGAAATCGCTTGCTCAATTAAGAT

117
SEQ ID NO: 70
SEQ ID NO: 69
SEQ ID NO: 63

AAATCACTGGCCTACATTCTTAC
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CGAAATCGCTTGCTCAATTAAGAT

AA

118
SEQ ID NO: 71
SEQ ID NO: 69
SEQ ID NO: 63

AAATCACTGGCCTACATTCTTAC
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CGAAATCGCTTGCTCAATTAAGAT

AAA

119
SEQ ID NO: 68
SEQ ID NO: 69
SEQ ID NO: 63

AAATCACTGGCCTACATTCTTAC
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CGAAATCGCTTGCTCAATTAAGAT

AAAA

120
SEQ ID NO: 72
SEQ ID NO: 73
SEQ ID NO: 74

AATCACTGGCCTACATTCTTACAA
AGATTTTTCCATCATATTGTCCTGCCGCTTCTAT
CCGAAATCGCTTGCTCAATTA

121
SEQ ID NO: 75
SEQ ID NO: 73
SEQ ID NO: 74

AATCACTGGCCTACATTCTTACA
AGATTTTTCCATCATATTGTCCTGCCGCTTCTAT
CCGAAATCGCTTGCTCAATTA

AA

122
SEQ ID NO: 70
SEQ ID NO: 76
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
TAAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AA

123
SEQ ID NO: 71
SEQ ID NO: 76
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
TAAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AAA

124
SEQ ID NO: 61
SEQ ID NO: 76
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTACA
TAAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

125
SEQ ID NO: 68
SEQ ID NO: 76
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
TAAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AAAA

126
SEQ ID NO: 71
SEQ ID NO: 78
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
AAGATTTTTCCATCATATTGTCCTGCCGCTTCTA
CCGAAATCGCTTGCTCAAT

AAA

127
SEQ ID NO: 61
SEQ ID NO: 79
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTACA
AAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

128
SEQ ID NO: 70
SEQ ID NO: 80
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
AAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AA
AT

129
SEQ ID NO: 70
SEQ ID NO: 79
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
AAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AA

130
SEQ ID NO: 71
SEQ ID NO: 80
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
AAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AAA
AT

131
SEQ ID NO: 61
SEQ ID NO: 78
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTACA
AAGATTTTTCCATCATATTGTCCTGCCGCTTCTA
CCGAAATCGCTTGCTCAAT

132
SEQ ID NO: 71
SEQ ID NO: 79
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
AAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AAA

133
SEQ ID NO: 70
SEQ ID NO: 78
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
AAGATTTTTCCATCATATTGTCCTGCCGCTTCTA
CCGAAATCGCTTGCTCAAT

AA

134
SEQ ID NO: 61
SEQ ID NO: 80
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTACA
AAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AT

135
SEQ ID NO: 68
SEQ ID NO: 80
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
AAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AAAA
AT

136
SEQ ID NO: 68
SEQ ID NO: 79
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
AAGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

AAAA

137
SEQ ID NO: 68
SEQ ID NO: 78
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
AAGATTTTTCCATCATATTGTCCTGCCGCTTCTA
CCGAAATCGCTTGCTCAAT

AAAA

138
SEQ ID NO: 81
SEQ ID NO: 82
SEQ ID NO: 77

CTTACCTACCCTGTCTTTGTGA
AGATTTTTCCATCATATTGTCCTGCCGCTTCT
CCGAAATCGCTTGCTCAAT

139
SEQ ID NO: 83
SEQ ID NO: 84
SEQ ID NO: 85

TTACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCCA

140
SEQ ID NO: 83
SEQ ID NO: 84
SEQ ID NO: 86

TTACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCCAT

141
SEQ ID NO: 83
SEQ ID NO: 84
SEQ ID NO: 65

TTACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCA

142
SEQ ID NO: 83
SEQ ID NO: 84
SEQ ID NO: 87

TTACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCAT

143
SEQ ID NO: 83
SEQ ID NO: 84
SEQ ID NO: 88

TTACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCATA

144
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 90

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
TCGCTTGCTCAATTAAGATTTTTCC

145
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 85

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCCA

146
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 86

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCCAT

147
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 65

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCA

148
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 87

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCAT

149
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 91

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
ATCGCTTGCTCAATTAAGATTTTTCC

150
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 88

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCATA

151
SEQ ID NO: 61
SEQ ID NO: 92
SEQ ID NO: 74

AAATCACTGGCCTACATTCTTACA
TTTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAATTA

152
SEQ ID NO: 70
SEQ ID NO: 92
SEQ ID NO: 74

AAATCACTGGCCTACATTCTTAC
TTTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAATTA

AA

153
SEQ ID NO: 71
SEQ ID NO: 92
SEQ ID NO: 74

AAATCACTGGCCTACATTCTTAC
TTTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAATTA

AAA

154
SEQ ID NO: 68
SEQ ID NO: 92
SEQ ID NO: 74

AAATCACTGGCCTACATTCTTAC
TTTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAATTA

AAAA

155
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 65

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCA

156
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 87

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCAT

157
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 88

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCATA

158
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 90

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
TCGCTTGCTCAATTAAGATTTTTCC

159
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 85

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCCA

160
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 86

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCCAT

161
SEQ ID NO: 94
SEQ ID NO: 84
SEQ ID NO: 65

CTTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCA

162
SEQ ID NO: 94
SEQ ID NO: 84
SEQ ID NO: 87

CTTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCAT

163
SEQ ID NO: 94
SEQ ID NO: 84
SEQ ID NO: 88

CTTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCATA

164
SEQ ID NO: 83
SEQ ID NO: 84
SEQ ID NO: 95

TTACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CTTGCTCAATTAAGATTTTTCCATCATATTGTC

165
SEQ ID NO: 61
SEQ ID NO: 96
SEQ ID NO: 97

AAATCACTGGCCTACATTCTTACA
CGCCTCCGGCTTGTCACCTTT
ACAAAGACAGGGTAGGTAAGC

166
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 95

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CTTGCTCAATTAAGATTTTTCCATCATATTGTC

167
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 66

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTCAATTAAGATTTTTCCATCATATTGTC

168
SEQ ID NO: 83
SEQ ID NO: 84
SEQ ID NO: 66

TTACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTCAATTAAGATTTTTCCATCATATTGTC

169
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 66

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
GCTCAATTAAGATTTTTCCATCATATTGTC

170
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 98

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
TCGCTTGCTCAATTAAGATTTTTCCA

171
SEQ ID NO: 61
SEQ ID NO: 69
SEQ ID NO: 74

AAATCACTGGCCTACATTCTTACA
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAATTA

172
SEQ ID NO: 70
SEQ ID NO: 69
SEQ ID NO: 74

AAATCACTGGCCTACATTCTTAC
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAATTA

AA

173
SEQ ID NO: 71
SEQ ID NO: 69
SEQ ID NO: 74

AAATCACTGGCCTACATTCTTAC
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAATTA

AAA

174
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 99

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
ATCGCTTGCTCAATTAAGATTTTTCCA

175
SEQ ID NO: 68
SEQ ID NO: 69
SEQ ID NO: 74

AAATCACTGGCCTACATTCTTAC
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAATTA

AAAA

176
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 100

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
TCGCTTGCTCAATTAAGATTTTTCCAT

177
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 95

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CTTGCTCAATTAAGATTTTTCCATCATATTGTC

178
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 101

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCC

179
SEQ ID NO: 83
SEQ ID NO: 84
SEQ ID NO: 101

TTACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCC

180
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 101

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCC

181
SEQ ID NO: 94
SEQ ID NO: 84
SEQ ID NO: 95

CTTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
CTTGCTCAATTAAGATTTTTCCATCATATTGTC

182
SEQ ID NO: 89
SEQ ID NO: 84
SEQ ID NO: 102

ACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
ATCGCTTGCTCAATTAAGATTTTTCCAT

183
SEQ ID NO: 93
SEQ ID NO: 84
SEQ ID NO: 98

TACCTACCCTGTCTTTGTGAAG
CACGGTCAGGTTCGTCCTTTGGC
TCGCTTGCTCAATTAAGATTTTTCCA

184
SEQ ID NO: 94
SEQ ID NO: 84
SEQ ID NO: 66

CTTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
GCTCAATTAAGATTTTTCCATCATATTGTC

185
SEQ ID NO: 61
SEQ ID NO: 92
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTACA
TTTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAAT

186
SEQ ID NO: 70
SEQ ID NO: 92
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
TTTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAAT

AA

187
SEQ ID NO: 71
SEQ ID NO: 92
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
TTTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAAT

AAA

188
SEQ ID NO: 81
SEQ ID NO: 84
SEQ ID NO: 88

CTTACCTACCCTGTCTTTGTGA
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCATA

189
SEQ ID NO: 81
SEQ ID NO: 84
SEQ ID NO: 65

CTTACCTACCCTGTCTTTGTGA
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCA

190
SEQ ID NO: 81
SEQ ID NO: 84
SEQ ID NO: 87

CTTACCTACCCTGTCTTTGTGA
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCAT

191
SEQ ID NO: 68
SEQ ID NO: 92
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
TTTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAAT

AAAA

192
SEQ ID NO: 103
SEQ ID NO: 84
SEQ ID NO: 86

TTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
CGCTTGCTCAATTAAGATTTTTCCAT

193
SEQ ID NO: 103
SEQ ID NO: 84
SEQ ID NO: 66

TTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
GCTCAATTAAGATTTTTCCATCATATTGTC

194
SEQ ID NO: 103
SEQ ID NO: 84
SEQ ID NO: 65

TTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCA

195
SEQ ID NO: 103
SEQ ID NO: 84
SEQ ID NO: 87

TTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCAT

196
SEQ ID NO: 103
SEQ ID NO: 84
SEQ ID NO: 88

TTACCTACCCTGTCTTTGTGAA
CACGGTCAGGTTCGTCCTTTGGC
GCTTGCTCAATTAAGATTTTTCCATCATA

197
SEQ ID NO: 81
SEQ ID NO: 84
SEQ ID NO: 66

CTTACCTACCCTGTCTTTGTGA
CACGGTCAGGTTCGTCCTTTGGC
GCTCAATTAAGATTTTTCCATCATATTGTC

198
SEQ ID NO: 71
SEQ ID NO: 69
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAAT

AAA

199
SEQ ID NO: 61
SEQ ID NO: 69
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTACA
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAAT

200
SEQ ID NO: 70
SEQ ID NO: 69
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAAT

AA

201
SEQ ID NO: 81
SEQ ID NO: 84
SEQ ID NO: 95

CTTACCTACCCTGTCTTTGTGA
CACGGTCAGGTTCGTCCTTTGGC
CTTGCTCAATTAAGATTTTTCCATCATATTGTC

202
SEQ ID NO: 68
SEQ ID NO: 69
SEQ ID NO: 77

AAATCACTGGCCTACATTCTTAC
TTTTCCATCATATTGTCCTGCCGCTTCTATCG
CCGAAATCGCTTGCTCAAT

AAAA

203
SEQ ID NO: 104
SEQ ID NO: 84
SEQ ID NO: 66

GCTTACCTACCCTGTCTTTGT
CACGGTCAGGTTCGTCCTTTGGC
GCTCAATTAAGATTTTTCCATCATATTGTC

204
SEQ ID NO: 105
SEQ ID NO: 84
SEQ ID NO: 66

CGCTTACCTACCCTGTCTTT
CACGGTCAGGTTCGTCCTTTGGC
GCTCAATTAAGATTTTTCCATCATATTGTC

205
SEQ ID NO: 89
SEQ ID NO: 106
SEQ ID NO: 66

ACCTACCCTGTCTTTGTGAAG
AGGTTCGTCCTTTGGCGTAACCAAAGTAAAC
GCTCAATTAAGATTTTTCCATCATATTGTC

206
SEQ ID NO: 107
SEQ ID NO: 84
SEQ ID NO: 66

CGCTTACCTACCCTGTCTT
CACGGTCAGGTTCGTCCTTTGGC
GCTCAATTAAGATTTTTCCATCATATTGTC

207
SEQ ID NO: 103
SEQ ID NO: 108
SEQ ID NO: 66

TTACCTACCCTGTCTTTGTGAA
CAGGTTCGTCCTTTGGCGTAACCA
GCTCAATTAAGATTTTTCCATCATATTGTC

208
SEQ ID NO: 93
SEQ ID NO: 109
SEQ ID NO: 66

TACCTACCCTGTCTTTGTGAAG
CCAAAGGACGAACCTGACCGTGC
GCTCAATTAAGATTTTTCCATCATATTGTC

209
SEQ ID NO: 89
SEQ ID NO: 110
SEQ ID NO: 66

ACCTACCCTGTCTTTGTGAAG
AGGACGAACCTGACCGTGCC
GCTCAATTAAGATTTTTCCATCATATTGTC

210
SEQ ID NO: 111
SEQ ID NO: 112
SEQ ID NO: 63

ATCACTGGCCTACATTCTTACA
CGCTTACCTACCCTGTCTTTGTGAAGC
CGAAATCGCTTGCTCAATTAAGAT

211
SEQ ID NO: 111
SEQ ID NO: 112
SEQ ID NO: 66

ATCACTGGCCTACATTCTTACA
CGCTTACCTACCCTGTCTTTGTGAAGC
GCTCAATTAAGATTTTTCCATCATATTGTC

212
SEQ ID NO: 111
SEQ ID NO: 112
SEQ ID NO: 88

ATCACTGGCCTACATTCTTACA
CGCTTACCTACCCTGTCTTTGTGAAGC
GCTTGCTCAATTAAGATTTTTCCATCATA

645
SEQ ID NO: 113
SEQ ID NO: 114
SEQ ID NO: 115

0000
0000
0000

646
SEQ ID NO: 116
SEQ ID NO: 117
SEQ ID NO: 118

0000
0000
0000

647
SEQ ID NO: 119
SEQ ID NO: 120
SEQ ID NO: 121

0000
0000
0000

648
SEQ ID NO: 122
SEQ ID NO: 113
SEQ ID NO: 114

0000
0000
0000

A PCR primer set for amplifying a vanA gene comprises at least one of the following sets of primer sequences: (1) SEQ ID NOS: 1 and 3; (2) SEQ ID NOS: 1 and 32; (3) SEQ ID NOS: 1 and 5; (4) SEQ ID NOS: 19 and 21; (5) SEQ ID NOS: 19 and 3; (6) SEQ ID NOS: 19 and 32; (7) SEQ ID NOS: 19 and 47; (8) SEQ ID NOS: 19 and 5; (9) SEQ ID NOS: 19 and 52; (10) SEQ ID NOS: 19 and 55; (11) SEQ ID NOS: 22 and 21; (12) SEQ ID NOS: 22 and 3; (13) SEQ ID NOS: 22 and 32; (14) SEQ ID NOS: 22 and 5; (15) SEQ ID NOS: 23 and 21; (16) SEQ ID NOS: 23 and 3; (17) SEQ ID NOS: 23 and 32; (18) SEQ ID NOS: 23 and 5; (19) SEQ ID NOS: 26 and 3; (20) SEQ ID NOS: 26 and 32; (21) SEQ ID NOS: 26 and 47; (22) SEQ ID NOS: 26 and 5; (23) SEQ ID NOS: 26 and 51; (24) SEQ ID NOS: 28 and 3; (25) SEQ ID NOS: 28 and 32; (26) SEQ ID NOS: 28 and 5; (27) SEQ ID NOS: 29 and 21; (28) SEQ ID NOS: 29 and 3; (29) SEQ ID NOS: 29 and 5; (30) SEQ ID NOS: 29 and 8; (31) SEQ ID NOS: 33 and 21; (32) SEQ ID NOS: 33 and 3; (33) SEQ ID NOS: 33 and 32; (34) SEQ ID NOS: 33 and 5; (35) SEQ ID NOS: 34 and 36; (36) SEQ ID NOS: 34 and 52; (37) SEQ ID NOS: 37 and 3; (38) SEQ ID NOS: 37 and 32; (39) SEQ ID NOS: 37 and 5; (40) SEQ ID NOS: 38 and 3; (41) SEQ ID NOS: 38 and 32; (42) SEQ ID NOS: 38 and 5; (43) SEQ ID NOS: 39 and 3; (44) SEQ ID NOS: 39 and 32; (45) SEQ ID NOS: 39 and 5; (46) SEQ ID NOS: 40 and 3; (47) SEQ ID NOS: 40 and 32; (48) SEQ ID NOS: 40 and 5; (49) SEQ ID NOS: 41 and 21; (50) SEQ ID NOS: 42 and 44; (51) SEQ ID NOS: 45 and 47; (52) SEQ ID NOS: 48 and 47; (53) SEQ ID NOS: 53 and 47; (54) SEQ ID NOS: 59 and 52; (55) SEQ ID NOS: 59 and 60; (56) SEQ ID NOS: 6 and 10; (57) SEQ ID NOS: 6 and 21; (58) SEQ ID NOS: 6 and 3; (59) SEQ ID NOS: 6 and 31; (60) SEQ ID NOS: 6 and 32; (61) SEQ ID NOS: 6 and 5; and (62) SEQ ID NOS: 6 and 8.

The preceding numbering of the 62 sets of primers does not correspond exactly to the “Group” numbering scheme in Table 5 because certain groups use the same primer set, but different internal probes. For example, Groups 1 and 2 of Table 5 each employ the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 3, but different internal probes in each instance, e.g., SEQ ID NOS: 2 and 4. Accordingly, primer set “(1)” of the preceding passage implies any one of Groups 1 or 2 of Table 5.

A probe for binding to an amplicon(s) of a vanA gene, or to a vanA gene target, comprises at least one of the following probe sequences: SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56, 57, 58 (vanA probes).

A PCR primer set for amplifying a vanB gene comprises at least one of the following sets of primer sequences: (1) SEQ ID NOS: 103 and 65; (2) SEQ ID NOS: 103 and 66; (3) SEQ ID NOS: 103 and 86; (4) SEQ ID NOS: 103 and 87; (5) SEQ ID NOS: 103 and 88; (6) SEQ ID NOS: 104 and 66; (7) SEQ ID NOS: 105 and 66; (8) SEQ ID NOS: 107 and 66; (9) SEQ ID NOS: 111 and 63; (10) SEQ ID NOS: 111 and 66; (11) SEQ ID NOS: 111 and 88; (12) SEQ ID NOS: 61 and 63; (13) SEQ ID NOS: 61 and 65; (14) SEQ ID NOS: 61 and 66; (15) SEQ ID NOS: 61 and 74; (16) SEQ ID NOS: 61 and 77; (17) SEQ ID NOS: 61 and 97; (18) SEQ ID NOS: 68 and 63; (19) SEQ ID NOS: 68 and 65; (20) SEQ ID NOS: 68 and 66; (21) SEQ ID NOS: 68 and 74; (22) SEQ ID NOS: 68 and 77; (23) SEQ ID NOS: 70 and 63; (24) SEQ ID NOS: 70 and 74; (25) SEQ ID NOS: 70 and 77; (26) SEQ ID NOS: 71 and 63; (27) SEQ ID NOS: 71 and 74; (28) SEQ ID NOS: 71 and 77; (29) SEQ ID NOS: 72 and 74; (30) SEQ ID NOS: 75 and 74; (31) SEQ ID NOS: 81 and 65; (32) SEQ ID NOS: 81 and 66; (33) SEQ ID NOS: 81 and 77; (34) SEQ ID NOS: 81 and 87; (35) SEQ ID NOS: 81 and 88; (36) SEQ ID NOS: 81 and 95; (37) SEQ ID NOS: 83 and 101; (38) SEQ ID NOS: 83 and 65; (39) SEQ ID NOS: 83 and 66; (40) SEQ ID NOS: 83 and 85; (41) SEQ ID NOS: 83 and 86; (42) SEQ ID NOS: 83 and 87; (43) SEQ ID NOS: 83 and 88; (44) SEQ ID NOS: 83 and 95; (45) SEQ ID NOS: 89 and 100; (46) SEQ ID NOS: 89 and 101; (47) SEQ ID NOS: 89 and 102; (48) SEQ ID NOS: 89 and 65; (49) SEQ ID NOS: 89 and 66; (50) SEQ ID NOS: 89 and 85; (51) SEQ ID NOS: 89 and 86; (52) SEQ ID NOS: 89 and 87; (53) SEQ ID NOS: 89 and 88; (54) SEQ ID NOS: 89 and 90; (55) SEQ ID NOS: 89 and 91; (56) SEQ ID NOS: 89 and 95; (57) SEQ ID NOS: 89 and 98; (58) SEQ ID NOS: 89 and 99; (59) SEQ ID NOS: 93 and 101; (60) SEQ ID NOS: 93 and 65; (61) SEQ ID NOS: 93 and 66; (62) SEQ ID NOS: 93 and 85; (63) SEQ ID NOS: 93 and 86; (64) SEQ ID NOS: 93 and 87; (65) SEQ ID NOS: 93 and 88; (66) SEQ ID NOS: 93 and 90; (67) SEQ ID NOS: 93 and 95; (68) SEQ ID NOS: 93 and 98; (69) SEQ ID NOS: 94 and 65; (70) SEQ ID NOS: 94 and 66; (71) SEQ ID NOS: 94 and 87; (72) SEQ ID NOS: 94 and 88; and (73) SEQ ID NOS: 94 and 95.

The preceding numbering of the 73 sets of primers does not correspond exactly to the “Group” numbering scheme in Table 5 because certain groups use the same primer set, but different internal probes. For example, Groups 109-111 of Table 5 each employ the forward primer of SEQ ID NO: 61 and the reverse primer of SEQ ID NO: 66, but different internal probes in each instance, e.g., SEQ ID NOS: 64, 62, and 67, respectively. Accordingly, primer set “(14)” of the preceding passage implies any one of Groups 109-111 of Table 5.

A probe for binding to an amplicon(s) of a vanB gene, or to a vanB gene target, comprises at least one of the following probe sequences: SEQ ID NOS: 62, 64, 67, 69, 73, 76, 78, 79, 80, 82, 84, 92, 96, 108, 109, 110 and 112 (vanB probes).

Any set of primers can be used simultaneously in a multiplex reaction with one or more other primer sets, so that multiple amplicons are amplified simultaneously.

TABLE 6

Optimized Primers and Probes for the Detection of vanC, vanD, vanE and vanG

Resistance Genes.

Group

No.
Forward Primer
Probe
Reverse Primer

vanC1 Sets

213
SEQ ID NO: 123
SEQ ID NO: 124
SEQ ID NO: 125

ATGTATGAACAAATGGCTCTTGCATCA
CGCTAGTGCTCCCACTTTGCTTTTATCCCGCTA
GATCAACACAGTAGAACCGTAAGCAAAAG

AC

214
SEQ ID NO: 123
SEQ ID NO: 126
SEQ ID NO: 125

ATGTATGAACAAATGGCTCTTGCATCA
CGCTAGTGCTCCCACTTTGCTTTTATCCCGC
GATCAACACAGTAGAACCGTAAGCAAAAG

AC

215
SEQ ID NO: 127
SEQ ID NO: 128
SEQ ID NO: 129

AAAAGGGATCACAAAAGTAACTGACA
CGCAGCCAATTTCAATACCCGCTATCGCC
CCAATCGTCAATTGCTCATTTCCTAAGAT

AAACAG

216
SEQ ID NO: 130
SEQ ID NO: 131
SEQ ID NO: 132

TCGATCGTTTTATTCAAGACCATGGAT
CCCTTTTGAAGAACCGGCTTCATTCGGCT
GATCAACACAGTAGAACCGTAAGCA

TC

217
SEQ ID NO: 133
SEQ ID NO: 134
SEQ ID NO: 135

AGGGATCACAAAAGTAACTGACAAAA
TGCCGCAGCCAATTTCAATACCCGCTA
ACCAATCGTCAATTGCTCATTTCCTA

CAG

218
SEQ ID NO: 133
SEQ ID NO: 136
SEQ ID NO: 137

AGGGATCACAAAAGTAACTGACAAAA
AAGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

CAG

219
SEQ ID NO: 138
SEQ ID NO: 139
SEQ ID NO: 140

TCGATCGTTTTATTCAAGACCATGGAT
CCCTTTTGAAGAACCGGCTTCATTCGGCT
ACCGTAAGCAAAAGCAGTCGTTA

TC

220
SEQ ID NO: 141
SEQ ID NO: 142
SEQ ID NO: 143

TTAACGACTGCTTTTGCTTACGGTTCT
ACCCGCTATCGCCTTTTGGATCAACACAGT
GTCGACAAGAGAAATCGCATCACA

221
SEQ ID NO: 144
SEQ ID NO: 145
SEQ ID NO: 146

CTTTTGCTTACGGTTCTACTGTGTTGA
CGCAGCCAATTTCAATACCCGCTATCGCC
ACAAGAGAAATCGCATCACAAGCA

222
SEQ ID NO: 141
SEQ ID NO: 142
SEQ ID NO: 147

TTAACGACTGCTTTTGCTTACGGTTCT
ACCCGCTATCGCCTTTTGGATCAACACAGT
CGCATCACAAGCACCAATCG

223
SEQ ID NO: 148
SEQ ID NO: 136
SEQ ID NO: 137

TCTGCATTAACGACTGCTTTTGCT
AAGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

224
SEQ ID NO: 148
SEQ ID NO: 149
SEQ ID NO: 150

TCTGCATTAACGACTGCTTTTGCT
TGCCGCAGCCAATTTCAATACCCGCTA
ACCAATCGTCAATTGCTCATTTCCTA

225
SEQ ID NO: 144
SEQ ID NO: 145
SEQ ID NO: 147

CTTTTGCTTACGGTTCTACTGTGTTGA
CGCAGCCAATTTCAATACCCGCTATCGCC
CGCATCACAAGCACCAATCG

226
SEQ ID NO: 141
SEQ ID NO: 136
SEQ ID NO: 137

TTAACGACTGCTTTTGCTTACGGTTCT
AAGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

227
SEQ ID NO: 151
SEQ ID NO: 152
SEQ ID NO: 153

GCTTTTGCTTACGGTTCTACTGTGTT
AGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCCTA

228
SEQ ID NO: 151
SEQ ID NO: 154
SEQ ID NO: 155

GCTTTTGCTTACGGTTCTACTGTGTT
CGCAGCCAATTTCAATACCCGCTATCGCCTTT
CACCAATCGTCAATTGCTCATTTCCTAA

229
SEQ ID NO: 156
SEQ ID NO: 149
SEQ ID NO: 150

GCTTTTGCTTACGGTTCTACTGTGTTG
TGCCGCAGCCAATTTCAATACCCGCTA
ACCAATCGTCAATTGCTCATTTCCTA

230
SEQ ID NO: 144
SEQ ID NO: 136
SEQ ID NO: 137

CTTTTGCTTACGGTTCTACTGTGTTGA
AAGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

231
SEQ ID NO: 144
SEQ ID NO: 145
SEQ ID NO: 157

CTTTTGCTTACGGTTCTACTGTGTTGA
CGCAGCCAATTTCAATACCCGCTATCGCC
ACCAATCGTCAATTGCTCATTTCCTAAG

232
SEQ ID NO: 158
SEQ ID NO: 159
SEQ ID NO: 160

ACTGTGTTGATCCAAAAGGCGATAG
TTCCTAAGATGCCGCAGCCAATTTCAATACC
GAAATCGCATCACAAGCACCAATC

CG

233
SEQ ID NO: 161
SEQ ID NO: 159
SEQ ID NO: 147

CTACTGTGTTGATCCAAAAGGCGATA
TTCCTAAGATGCCGCAGCCAATTTCAATACC
CGCATCACAAGCACCAATCG

CG

234
SEQ ID NO: 162
SEQ ID NO: 163
SEQ ID NO: 164

TTTATTCAAGACCATGGATTCCCGATC
TCCCTTTTGAAGAACCGGCTTCATTCGGCT
GCGCTGTTTTGTCAGTTACTTTTGTG

TTTATC

235
SEQ ID NO: 165
SEQ ID NO: 166
SEQ ID NO: 167

TTCAAGACCATGGATTCCCGATCTTTA
TGATCCCTTTTGAAGAACCGGCTTCATTCGGC
TGGAGCGCTGTTTTGTCAGTTAC

TCA

236
SEQ ID NO: 168
SEQ ID NO: 166
SEQ ID NO: 169

ATTCAAGACCATGGATTCCCGATCTTT
TGATCCCTTTTGAAGAACCGGCTTCATTCGGC
GGAGCGCTGTTTTGTCAGTTACTT

ATCA

237
SEQ ID NO: 170
SEQ ID NO: 166
SEQ ID NO: 169

CAAGACCATGGATTCCCGATCTTTAT
TGATCCCTTTTGAAGAACCGGCTTCATTCGGC
GGAGCGCTGTTTTGTCAGTTACTT

238
SEQ ID NO: 171
SEQ ID NO: 172
SEQ ID NO: 173

AAGACCATGGATTCCCGATCTTTATCA
TCCCTTTTGAAGAACCGGCTTCATTCGGC
TGGAGCGCTGTTTTGTCAGTTA

239
SEQ ID NO: 171
SEQ ID NO: 166
SEQ ID NO: 174

AAGACCATGGATTCCCGATCTTTATCA
TGATCCCTTTTGAAGAACCGGCTTCATTCGGC
TGGAGCGCTGTTTTGTCAGTT

240
SEQ ID NO: 175
SEQ ID NO: 176
SEQ ID NO: 177

ACGGTTCTACTGTGTTGATCCAAAAGG
AGATGCCGCAGCCAATTTCAATACCCGCT
CACCAATCGTCAATTGCTCATTTCCT

241
SEQ ID NO: 178
SEQ ID NO: 142
SEQ ID NO: 179

TCTGCATTAACGACTGCTTTTGCTTA
ACCCGCTATCGCCTTTTGGATCAACACAGT
TAAGATGCCGCAGCCAATTTCA

242
SEQ ID NO: 180
SEQ ID NO: 181
SEQ ID NO: 146

AAAGGCGATAGCGGGTATTGAAATTG
CCAATCGTCAATTGCTCATTTCCTAAGATGCC
ACAAGAGAAATCGCATCACAAGCA

GCAG

243
SEQ ID NO: 175
SEQ ID NO: 182
SEQ ID NO: 177

ACGGTTCTACTGTGTTGATCCAAAAGG
AAGATGCCGCAGCCAATTTCAATACCCGCT
CACCAATCGTCAATTGCTCATTTCCT

244
SEQ ID NO: 183
SEQ ID NO: 149
SEQ ID NO: 150

TACGGTTCTACTGTGTTGATCCAAAAGG
TGCCGCAGCCAATTTCAATACCCGCTA
ACCAATCGTCAATTGCTCATTTCCTA

245
SEQ ID NO: 184
SEQ ID NO: 166
SEQ ID NO: 174

AGACCATGGATTCCCGATCTTTATCA
TGATCCCTTTTGAAGAACCGGCTTCATTCGGC
TGGAGCGCTGTTTTGTCAGTT

246
SEQ ID NO: 185
SEQ ID NO: 186
SEQ ID NO: 187

CTGCATTAACGACTGCTTTTGCTTAC
ACCCGCTATCGCCTTTTGGATCAACACAGTAG
CTAAGATGCCGCAGCCAATTTC

247
SEQ ID NO: 188
SEQ ID NO: 189
SEQ ID NO: 190

CGGTTCTACTGTGTTGATCCAAAAGG
TGCCGCAGCCAATTTCAATACCCGCT
CACCAATCGTCAATTGCTCATTTCC

248
SEQ ID NO: 188
SEQ ID NO: 152
SEQ ID NO: 137

CGGTTCTACTGTGTTGATCCAAAAGG
AGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

249
SEQ ID NO: 188
SEQ ID NO: 149
SEQ ID NO: 150

CGGTTCTACTGTGTTGATCCAAAAGG
TGCCGCAGCCAATTTCAATACCCGCTA
ACCAATCGTCAATTGCTCATTTCCTA

250
SEQ ID NO: 191
SEQ ID NO: 136
SEQ ID NO: 192

TCTACTGTGTTGATCCAAAAGGCGATA
AAGATGCCGCAGCCAATTTCAATACCCGC
GCACCAATCGTCAATTGCTCATTTC

251
SEQ ID NO: 193
SEQ ID NO: 152
SEQ ID NO: 153

TTCTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCCTA

252
SEQ ID NO: 193
SEQ ID NO: 136
SEQ ID NO: 190

TTCTACTGTGTTGATCCAAAAGGCGATA
AAGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCC

253
SEQ ID NO: 191
SEQ ID NO: 152
SEQ ID NO: 192

TCTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
GCACCAATCGTCAATTGCTCATTTC

254
SEQ ID NO: 194
SEQ ID NO: 195
SEQ ID NO: 160

CAAAAGGCGATAGCGGGTATTGAAA
TCAATTGCTCATTTCCTAAGATGCCGCAGCCA
GAAATCGCATCACAAGCACCAATC

255
SEQ ID NO: 193
SEQ ID NO: 152
SEQ ID NO: 190

TTCTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCC

256
SEQ ID NO: 191
SEQ ID NO: 152
SEQ ID NO: 190

TCTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCC

257
SEQ ID NO: 193
SEQ ID NO: 136
SEQ ID NO: 137

TTCTACTGTGTTGATCCAAAAGGCGATA
AAGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

258
SEQ ID NO: 196
SEQ ID NO: 197
SEQ ID NO: 160

AAAAGGCGATAGCGGGTATTGAAA
TCAATTGCTCATTTCCTAAGATGCCGCAGCCAA
GAAATCGCATCACAAGCACCAATC

259
SEQ ID NO: 198
SEQ ID NO: 142
SEQ ID NO: 179

ATTAACGACTGCTTTTGCTTACGGTTCT
ACCCGCTATCGCCTTTTGGATCAACACAGT
TAAGATGCCGCAGCCAATTTCA

260
SEQ ID NO: 193
SEQ ID NO: 152
SEQ ID NO: 137

TTCTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

261
SEQ ID NO: 191
SEQ ID NO: 152
SEQ ID NO: 153

TCTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCCTA

262
SEQ ID NO: 193
SEQ ID NO: 152
SEQ ID NO: 150

TTCTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCTA

263
SEQ ID NO: 191
SEQ ID NO: 136
SEQ ID NO: 190

TCTACTGTGTTGATCCAAAAGGCGATA
AAGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCC

264
SEQ ID NO: 196
SEQ ID NO: 195
SEQ ID NO: 160

AAAAGGCGATAGCGGGTATTGAAA
TCAATTGCTCATTTCCTAAGATGCCGCAGCCA
GAAATCGCATCACAAGCACCAATC

265
SEQ ID NO: 161
SEQ ID NO: 136
SEQ ID NO: 192

CTACTGTGTTGATCCAAAAGGCGATA
AAGATGCCGCAGCCAATTTCAATACCCGC
GCACCAATCGTCAATTGCTCATTTC

266
SEQ ID NO: 193
SEQ ID NO: 199
SEQ ID NO: 150

TTCTACTGTGTTGATCCAAAAGGCGATA
TGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCTA

267
SEQ ID NO: 141
SEQ ID NO: 142
SEQ ID NO: 179

TTAACGACTGCTTTTGCTTACGGTTCT
ACCCGCTATCGCCTTTTGGATCAACACAGT
TAAGATGCCGCAGCCAATTTCA

268
SEQ ID NO: 161
SEQ ID NO: 152
SEQ ID NO: 153

CTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCCTA

269
SEQ ID NO: 191
SEQ ID NO: 199
SEQ ID NO: 150

TCTACTGTGTTGATCCAAAAGGCGATA
TGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCTA

270
SEQ ID NO: 161
SEQ ID NO: 136
SEQ ID NO: 190

CTACTGTGTTGATCCAAAAGGCGATA
AAGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCC

271
SEQ ID NO: 191
SEQ ID NO: 136
SEQ ID NO: 137

TCTACTGTGTTGATCCAAAAGGCGATA
AAGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

272
SEQ ID NO: 161
SEQ ID NO: 152
SEQ ID NO: 190

CTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
CACCAATCGTCAATTGCTCATTTCC

273
SEQ ID NO: 191
SEQ ID NO: 152
SEQ ID NO: 137

TCTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

274
SEQ ID NO: 191
SEQ ID NO: 152
SEQ ID NO: 150

TCTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCTA

275
SEQ ID NO: 161
SEQ ID NO: 136
SEQ ID NO: 137

CTACTGTGTTGATCCAAAAGGCGATA
AAGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

276
SEQ ID NO: 200
SEQ ID NO: 142
SEQ ID NO: 179

TAACGACTGCTTTTGCTTACGGTTCT
ACCCGCTATCGCCTTTTGGATCAACACAGT
TAAGATGCCGCAGCCAATTTCA

277
SEQ ID NO: 161
SEQ ID NO: 152
SEQ ID NO: 137

CTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCT

278
SEQ ID NO: 161
SEQ ID NO: 152
SEQ ID NO: 150

CTACTGTGTTGATCCAAAAGGCGATA
AGATGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCTA

279
SEQ ID NO: 161
SEQ ID NO: 199
SEQ ID NO: 150

CTACTGTGTTGATCCAAAAGGCGATA
TGCCGCAGCCAATTTCAATACCCGC
ACCAATCGTCAATTGCTCATTTCCTA

280
SEQ ID NO: 201
SEQ ID NO: 142
SEQ ID NO: 179

AACGACTGCTTTTGCTTACGGTTCT
ACCCGCTATCGCCTTTTGGATCAACACAGT
TAAGATGCCGCAGCCAATTTCA

281
SEQ ID NO: 194
SEQ ID NO: 195
SEQ ID NO: 147

CAAAAGGCGATAGCGGGTATTGAAA
TCAATTGCTCATTTCCTAAGATGCCGCAGCCA
CGCATCACAAGCACCAATCG

282
SEQ ID NO: 202
SEQ ID NO: 142
SEQ ID NO: 179

ACGACTGCTTTTGCTTACGGTTCT
ACCCGCTATCGCCTTTTGGATCAACACAGT
TAAGATGCCGCAGCCAATTTCA

283
SEQ ID NO: 203
SEQ ID NO: 195
SEQ ID NO: 147

CAAAAGGCGATAGCGGGTATTGAA
TCAATTGCTCATTTCCTAAGATGCCGCAGCCA
CGCATCACAAGCACCAATCG

284
SEQ ID NO: 194
SEQ ID NO: 197
SEQ ID NO: 147

CAAAAGGCGATAGCGGGTATTGAAA
TCAATTGCTCATTTCCTAAGATGCCGCAGCCAA
CGCATCACAAGCACCAATCG

285
SEQ ID NO: 204
SEQ ID NO: 205
SEQ ID NO: 187

CGACTGCTTTTGCTTACGGTTCT
TACCCGCTATCGCCTTTTGGATCAACACAGT
CTAAGATGCCGCAGCCAATTTC

286
SEQ ID NO: 203
SEQ ID NO: 197
SEQ ID NO: 147

CAAAAGGCGATAGCGGGTATTGAA
TCAATTGCTCATTTCCTAAGATGCCGCAGCCAA
CGCATCACAAGCACCAATCG

287
SEQ ID NO: 204
SEQ ID NO: 142
SEQ ID NO: 187

CGACTGCTTTTGCTTACGGTTCT
ACCCGCTATCGCCTTTTGGATCAACACAGT
CTAAGATGCCGCAGCCAATTTC

288
SEQ ID NO: 204
SEQ ID NO: 142
SEQ ID NO: 179

CGACTGCTTTTGCTTACGGTTCT
ACCCGCTATCGCCTTTTGGATCAACACAGT
TAAGATGCCGCAGCCAATTTCA

289
SEQ ID NO: 196
SEQ ID NO: 195
SEQ ID NO: 147

AAAAGGCGATAGCGGGTATTGAAA
TCAATTGCTCATTTCCTAAGATGCCGCAGCCA
CGCATCACAAGCACCAATCG

290
SEQ ID NO: 196
SEQ ID NO: 197
SEQ ID NO: 147

AAAAGGCGATAGCGGGTATTGAAA
TCAATTGCTCATTTCCTAAGATGCCGCAGCCAA
CGCATCACAAGCACCAATCG

vanC2/3 Sets

291
SEQ ID NO: 206
SEQ ID NO: 207
SEQ ID NO: 208

CGCCATTGCCTGAAACGATTG
AGTCTTGGTCTTAAAGGTCTTGCTCGCATCGA
TCAAGTATAGTTCTCCTTGATCCGTGACA

CT

292
SEQ ID NO: 206
SEQ ID NO: 207
SEQ ID NO: 209

CGCCATTGCCTGAAACGATTG
AGTCTTGGTCTTAAAGGTCTTGCTCGCATCGA
AGTATAGTTCTCCTTGATCCGTGACA

CT

293
SEQ ID NO: 210
SEQ ID NO: 211
SEQ ID NO: 212

CCTGAAACGATTGAAACCAAGGTCAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AAGTATAGTTCTCCTTGATCCGTGACA

ACT

294
SEQ ID NO: 210
SEQ ID NO: 211
SEQ ID NO: 209

CCTGAAACGATTGAAACCAAGGTCAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACA

ACT

295
SEQ ID NO: 213
SEQ ID NO: 214
SEQ ID NO: 215

GCGCCATTGCCTGAAACGAT
AAGTCGATGCGAGCAAGACCTTTAAGACCAA
AGTATAGTTCTCCTTGATCCGTGACAAA

GACT

296
SEQ ID NO: 210
SEQ ID NO: 211
SEQ ID NO: 215

CCTGAAACGATTGAAACCAAGGTCAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACAAA

ACT

297
SEQ ID NO: 210
SEQ ID NO: 211
SEQ ID NO: 216

CCTGAAACGATTGAAACCAAGGTCAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
GTATAGTTCTCCTTGATCCGTGACAAAAA

ACT

298
SEQ ID NO: 217
SEQ ID NO: 218
SEQ ID NO: 219

CGCCATTGCCTGAAACGATTGAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TTCTCCTTGATCCGTGACAAAAAAGT

ACG

299
SEQ ID NO: 220
SEQ ID NO: 218
SEQ ID NO: 219

GCGCCATTGCCTGAAACG
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TTCTCCTTGATCCGTGACAAAAAAGT

ACG

300
SEQ ID NO: 221
SEQ ID NO: 218
SEQ ID NO: 219

GCCATTGCCTGAAACGATTGAAAC
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TTCTCCTTGATCCGTGACAAAAAAGT

ACG

301
SEQ ID NO: 222
SEQ ID NO: 218
SEQ ID NO: 219

CTGCGCCATTGCCTGAAAC
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TTCTCCTTGATCCGTGACAAAAAAGT

ACG

302
SEQ ID NO: 210
SEQ ID NO: 218
SEQ ID NO: 219

CCTGAAACGATTGAAACCAAGGTCAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TTCTCCTTGATCCGTGACAAAAAAGT

ACG

303
SEQ ID NO: 206
SEQ ID NO: 218
SEQ ID NO: 219

CGCCATTGCCTGAAACGATTG
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TTCTCCTTGATCCGTGACAAAAAAGT

ACG

304
SEQ ID NO: 222
SEQ ID NO: 218
SEQ ID NO: 216

CTGCGCCATTGCCTGAAAC
CGATGCGAGCAAGACCTTTAAGACCAAGACT
GTATAGTTCTCCTTGATCCGTGACAAAAA

ACG

305
SEQ ID NO: 217
SEQ ID NO: 218
SEQ ID NO: 223

CGCCATTGCCTGAAACGATTGAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTTCTCCTTGATCCGTGACAAAAAA

ACG

306
SEQ ID NO: 221
SEQ ID NO: 218
SEQ ID NO: 209

GCCATTGCCTGAAACGATTGAAAC
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTATAGTTCTCCTTGATCCGTGACA

ACG

307
SEQ ID NO: 224
SEQ ID NO: 211
SEQ ID NO: 216

CCTGAAACGATTGAAACCAAGGTCAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
GTATAGTTCTCCTTGATCCGTGACAAAAA

AG
ACT

308
SEQ ID NO: 225
SEQ ID NO: 211
SEQ ID NO: 216

CTGAAACGATTGAAACCAAGGTCAAAG
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
GTATAGTTCTCCTTGATCCGTGACAAAAA

ACT

309
SEQ ID NO: 226
SEQ ID NO: 211
SEQ ID NO: 209

CTGAAACGATTGAAACCAAGGTCAAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACA

GAA
ACT

310
SEQ ID NO: 222
SEQ ID NO: 218
SEQ ID NO: 209

CTGCGCCATTGCCTGAAAC
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTATAGTTCTCCTTGATCCGTGACA

ACG

311
SEQ ID NO: 206
SEQ ID NO: 218
SEQ ID NO: 227

CGCCATTGCCTGAAACGATTG
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TATAGTTCTCCTTGATCCGTGACAAAAAAG

ACG

312
SEQ ID NO: 220
SEQ ID NO: 218
SEQ ID NO: 223

GCGCCATTGCCTGAAACG
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTTCTCCTTGATCCGTGACAAAAAA

ACG

313
SEQ ID NO: 228
SEQ ID NO: 211
SEQ ID NO: 215

CCTGAAACGATTGAAACCAAGGTCAAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACAAA

ACT

314
SEQ ID NO: 206
SEQ ID NO: 218
SEQ ID NO: 209

CGCCATTGCCTGAAACGATTG
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTATAGTTCTCCTTGATCCGTGACA

ACG

315
SEQ ID NO: 217
SEQ ID NO: 218
SEQ ID NO: 227

CGCCATTGCCTGAAACGATTGAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TATAGTTCTCCTTGATCCGTGACAAAAAAG

ACG

316
SEQ ID NO: 221
SEQ ID NO: 218
SEQ ID NO: 216

GCCATTGCCTGAAACGATTGAAAC
CGATGCGAGCAAGACCTTTAAGACCAAGACT
GTATAGTTCTCCTTGATCCGTGACAAAAA

ACG

317
SEQ ID NO: 210
SEQ ID NO: 218
SEQ ID NO: 216

CCTGAAACGATTGAAACCAAGGTCAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
GTATAGTTCTCCTTGATCCGTGACAAAAA

ACG

318
SEQ ID NO: 229
SEQ ID NO: 211
SEQ ID NO: 215

CTGAAACGATTGAAACCAAGGTCAAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACAAA

GA
ACT

319
SEQ ID NO: 226
SEQ ID NO: 211
SEQ ID NO: 216

CTGAAACGATTGAAACCAAGGTCAAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
GTATAGTTCTCCTTGATCCGTGACAAAAA

GAA
ACT

320
SEQ ID NO: 222
SEQ ID NO: 218
SEQ ID NO: 223

CTGCGCCATTGCCTGAAAC
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTTCTCCTTGATCCGTGACAAAAAA

ACG

321
SEQ ID NO: 230
SEQ ID NO: 218
SEQ ID NO: 219

CCGTCCCTGCGCCATT
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TTCTCCTTGATCCGTGACAAAAAAGT

ACG

322
SEQ ID NO: 210
SEQ ID NO: 218
SEQ ID NO: 223

CCTGAAACGATTGAAACCAAGGTCAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTTCTCCTTGATCCGTGACAAAAAA

ACG

323
SEQ ID NO: 224
SEQ ID NO: 211
SEQ ID NO: 215

CCTGAAACGATTGAAACCAAGGTCAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACAAA

AG
ACT

324
SEQ ID NO: 225
SEQ ID NO: 211
SEQ ID NO: 212

CTGAAACGATTGAAACCAAGGTCAAAG
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AAGTATAGTTCTCCTTGATCCGTGACA

ACT

325
SEQ ID NO: 206
SEQ ID NO: 218
SEQ ID NO: 216

CGCCATTGCCTGAAACGATTG
CGATGCGAGCAAGACCTTTAAGACCAAGACT
GTATAGTTCTCCTTGATCCGTGACAAAAA

ACG

326
SEQ ID NO: 210
SEQ ID NO: 218
SEQ ID NO: 209

CCTGAAACGATTGAAACCAAGGTCAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTATAGTTCTCCTTGATCCGTGACA

ACG

327
SEQ ID NO: 210
SEQ ID NO: 218
SEQ ID NO: 227

CCTGAAACGATTGAAACCAAGGTCAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TATAGTTCTCCTTGATCCGTGACAAAAAAG

ACG

328
SEQ ID NO: 231
SEQ ID NO: 211
SEQ ID NO: 215

TGAAACGATTGAAACCAAGGTCAAAGA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACAAA

ACT

329
SEQ ID NO: 222
SEQ ID NO: 218
SEQ ID NO: 227

CTGCGCCATTGCCTGAAAC
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TATAGTTCTCCTTGATCCGTGACAAAAAAG

ACG

330
SEQ ID NO: 217
SEQ ID NO: 218
SEQ ID NO: 216

CGCCATTGCCTGAAACGATTGAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
GTATAGTTCTCCTTGATCCGTGACAAAAA

ACG

331
SEQ ID NO: 229
SEQ ID NO: 211
SEQ ID NO: 209

CTGAAACGATTGAAACCAAGGTCAAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACA

GA
ACT

332
SEQ ID NO: 226
SEQ ID NO: 211
SEQ ID NO: 212

CTGAAACGATTGAAACCAAGGTCAAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AAGTATAGTTCTCCTTGATCCGTGACA

GAA
ACT

333
SEQ ID NO: 217
SEQ ID NO: 218
SEQ ID NO: 209

CGCCATTGCCTGAAACGATTGAA
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTATAGTTCTCCTTGATCCGTGACA

ACG

334
SEQ ID NO: 220
SEQ ID NO: 218
SEQ ID NO: 227

GCGCCATTGCCTGAAACG
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TATAGTTCTCCTTGATCCGTGACAAAAAAG

ACG

335
SEQ ID NO: 224
SEQ ID NO: 211
SEQ ID NO: 212

CCTGAAACGATTGAAACCAAGGTCAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AAGTATAGTTCTCCTTGATCCGTGACA

AG
ACT

336
SEQ ID NO: 225
SEQ ID NO: 211
SEQ ID NO: 209

CTGAAACGATTGAAACCAAGGTCAAAG
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACA

ACT

337
SEQ ID NO: 220
SEQ ID NO: 218
SEQ ID NO: 209

GCGCCATTGCCTGAAACG
CGATGCGAGCAAGACCTTTAAGACCAAGACT
AGTATAGTTCTCCTTGATCCGTGACA

ACG

338
SEQ ID NO: 221
SEQ ID NO: 218
SEQ ID NO: 227

GCCATTGCCTGAAACGATTGAAAC
CGATGCGAGCAAGACCTTTAAGACCAAGACT
TATAGTTCTCCTTGATCCGTGACAAAAAAG

ACG

339
SEQ ID NO: 231
SEQ ID NO: 211
SEQ ID NO: 212

TGAAACGATTGAAACCAAGGTCAAAGA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AAGTATAGTTCTCCTTGATCCGTGACA

ACT

340
SEQ ID NO: 232
SEQ ID NO: 211
SEQ ID NO: 216

ACGATTGAAACCAAGGTCAAAGAACA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
GTATAGTTCTCCTTGATCCGTGACAAAAA

AG
ACT

341
SEQ ID NO: 233
SEQ ID NO: 211
SEQ ID NO: 212

TGAAACGATTGAAACCAAGGTCAAAG
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AAGTATAGTTCTCCTTGATCCGTGACA

AAC
ACT

342
SEQ ID NO: 234
SEQ ID NO: 211
SEQ ID NO: 215

TGAAACGATTGAAACCAAGGTCAAAG
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACAAA

AACA
ACT

343
SEQ ID NO: 235
SEQ ID NO: 207
SEQ ID NO: 239

AACGATTGAAACCAAGGTCAAAGAACA
AGTCTTGGTCTTAAAGGTCTTGCTCGCATCGA
AAGTATAGTTCTCCTTGATCCGTGACAAA

CT

344
SEQ ID NO: 236
SEQ ID NO: 211
SEQ ID NO: 216

ACGATTGAAACCAAGGTCAAAGAACAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
GTATAGTTCTCCTTGATCCGTGACAAAAA

ACT

345
SEQ ID NO: 237
SEQ ID NO: 211
SEQ ID NO: 215

GAAACGATTGAAACCAAGGTCAAAGA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACAAA

AC
ACT

346
SEQ ID NO: 238
SEQ ID NO: 211
SEQ ID NO: 215

GAAACGATTGAAACCAAGGTCAAAGA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACAAA

ACA
ACT

347
SEQ ID NO: 240
SEQ ID NO: 211
SEQ ID NO: 216

AAACGATTGAAACCAAGGTCAAAGAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
GTATAGTTCTCCTTGATCCGTGACAAAAA

CAAG
ACT

348
SEQ ID NO: 235
SEQ ID NO: 211
SEQ ID NO: 215

AACGATTGAAACCAAGGTCAAAGAACA
AGTCGATGCGAGCAAGA
AGTATAGTTCTCCTTGATCCGTGACAAA

ACT

349
SEQ ID NO: 237
SEQ ID NO: 211
SEQ ID NO: 209

GAAACGATTGAAACCAAGGTCAAAGA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACA

AC
ACT

350
SEQ ID NO: 235
SEQ ID NO: 207
SEQ ID NO: 241

AACGATTGAAACCAAGGTCAAAGAACA
AGTCTTGGTCTTAAAGGTCTTGCTCGCATCGA
AGTATAGTTCTCCTTGATCCGTGACAAAAA

CT

351
SEQ ID NO: 242
SEQ ID NO: 211
SEQ ID NO: 216

AAACGATTGAAACCAAGGTCAAAGAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
GTATAGTTCTCCTTGATCCGTGACAAAAA

CA
ACT

352
SEQ ID NO: 243
SEQ ID NO: 211
SEQ ID NO: 215

AAACGATTGAAACCAAGGTCAAAGAA
AGTCGATGCGAGCAAGACCTTTAAGACCAAG
AGTATAGTTCTCCTTGATCCGTGACAAA

CAA
ACT

vanG Sets

353
SEQ ID NO: 244
SEQ ID NO: 245
SEQ ID NO: 246

TCTGGCGAAATTGTATTTAATGAGGTA
CAATACCAGGCTTTACCTCGCACAGTCGC
AGACCAATGCCTTTCATCATATTTGGA

AACA

354
SEQ ID NO: 244
SEQ ID NO: 245
SEQ ID NO: 247

TCTGGCGAAATTGTATTTAATGAGGTA
CAATACCAGGCTTTACCTCGCACAGTCGC
GATAGACCAATGCCTTTCATCATATTTGGATA

AACA

355
SEQ ID NO: 244
SEQ ID NO: 245
SEQ ID NO: 248

TCTGGCGAAATTGTATTTAATGAGGTA
CAATACCAGGCTTTACCTCGCACAGTCGC
GATAGACCAATGCCTTTCATCATATTTGGA

AACA

356
SEQ ID NO: 249
SEQ ID NO: 245
SEQ ID NO: 246

ACCGTCTGGCGAAATTGTATTTAATGA
CAATACCAGGCTTTACCTCGCACAGTCGC
AGACCAATGCCTTTCATCATATTTGGA

357
SEQ ID NO: 244
SEQ ID NO: 245
SEQ ID NO: 250

TCTGGCGAAATTGTATTTAATGAGGTA
CAATACCAGGCTTTACCTCGCACAGTCGC
CGATAGACCAATGCCTTTCATCATATTTGG

AACA

358
SEQ ID NO: 244
SEQ ID NO: 245
SEQ ID NO: 251

TCTGGCGAAATTGTATTTAATGAGGTA
CAATACCAGGCTTTACCTCGCACAGTCGC
CGATAGACCAATGCCTTTCATCATATTTG

AACA

GATA

359
SEQ ID NO: 252
SEQ ID NO: 245
SEQ ID NO: 246

CACCGTCTGGCGAAATTGTATTTAATG
CAATACCAGGCTTTACCTCGCACAGTCGC
AGACCAATGCCTTTCATCATATTTGGA

360
SEQ ID NO: 253
SEQ ID NO: 245
SEQ ID NO: 246

ACACCGTCTGGCGAAATTGTAT
CAATACCAGGCTTTACCTCGCACAGTCGC
AGACCAATGCCTTTCATCATATTTGGA

361
SEQ ID NO: 244
SEQ ID NO: 245
SEQ ID NO: 254

TCTGGCGAAATTGTATTTAATGAGGTA
CAATACCAGGCTTTACCTCGCACAGTCGC
AACGATAGACCAATGCCTTTCATCATATTT

AACA

362
SEQ ID NO: 255
SEQ ID NO: 245
SEQ ID NO: 246

ACACCGTCTGGCGAAATTGTATTTA
CAATACCAGGCTTTACCTCGCACAGTCGC
AGACCAATGCCTTTCATCATATTTGGA

363
SEQ ID NO: 256
SEQ ID NO: 245
SEQ ID NO: 246

ACACCGTCTGGCGAAATTGTATT
CAATACCAGGCTTTACCTCGCACAGTCGC
AGACCAATGCCTTTCATCATATTTGGA

364
SEQ ID NO: 249
SEQ ID NO: 245
SEQ ID NO: 248

ACCGTCTGGCGAAATTGTATTTAATGA
CAATACCAGGCTTTACCTCGCACAGTCGC
GATAGACCAATGCCTTTCATCATATTTGGA

365
SEQ ID NO: 244
SEQ ID NO: 257
SEQ ID NO: 258

TCTGGCGAAATTGTATTTAATGAGGTA
AGGCTTTACCTCGCACAGTCGCTATCCAA
GAACGATAGACCAATGCCTTTCATCA

AACA

366
SEQ ID NO: 244
SEQ ID NO: 257
SEQ ID NO: 259

TCTGGCGAAATTGTATTTAATGAGGTA
AGGCTTTACCTCGCACAGTCGCTATCCAA
GAACGATAGACCAATGCCTTTCATCATA

AACA

367
SEQ ID NO: 260
SEQ ID NO: 261
SEQ ID NO: 258

ACCGTCTGGCGAAATTGTATTTAATGAG
CCAGGCTTTACCTCGCACAGTCGCTATCC
GAACGATAGACCAATGCCTTTCATCA

368
SEQ ID NO: 252
SEQ ID NO: 257
SEQ ID NO: 262

CACCGTCTGGCGAAATTGTATTTAATG
AGGCTTTACCTCGCACAGTCGCTATCCAA
GAACGATAGACCAATGCCTTTCATCAT

369
SEQ ID NO: 263
SEQ ID NO: 257
SEQ ID NO: 258

TTTATACACCGTCTGGCGAAATTGT
AGGCTTTACCTCGCACAGTCGCTATCCAA
GAACGATAGACCAATGCCTTTCATCA

370
SEQ ID NO: 264
SEQ ID NO: 265
SEQ ID NO: 266

GCCGAAGCAGAAAAACGGATACA
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

371
SEQ ID NO: 267
SEQ ID NO: 268
SEQ ID NO: 269

GCCGAAGCAGAAAAACGGATACAA
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

372
SEQ ID NO: 270
SEQ ID NO: 265
SEQ ID NO: 266

TGCCGAAGCAGAAAAACGGATAC
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

373
SEQ ID NO: 270
SEQ ID NO: 268
SEQ ID NO: 269

TGCCGAAGCAGAAAAACGGATAC
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

374
SEQ ID NO: 271
SEQ ID NO: 265
SEQ ID NO: 266

ATGCCGAAGCAGAAAAACGGATAC
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

375
SEQ ID NO: 272
SEQ ID NO: 273
SEQ ID NO: 274

AAGGATTGATGCCGAAGCAGAA
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

376
SEQ ID NO: 275
SEQ ID NO: 268
SEQ ID NO: 269

ATGCCGAAGCAGAAAAACGGATA
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

377
SEQ ID NO: 276
SEQ ID NO: 268
SEQ ID NO: 269

GATGCCGAAGCAGAAAAACGGATA
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

378
SEQ ID NO: 277
SEQ ID NO: 268
SEQ ID NO: 269

AGGATTGATGCCGAAGCAGAAAA
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

379
SEQ ID NO: 278
SEQ ID NO: 265
SEQ ID NO: 266

AAGGATTGATGCCGAAGCAGAAAAAC
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

380
SEQ ID NO: 279
SEQ ID NO: 268
SEQ ID NO: 269

AAGGATTGATGCCGAAGCAGAAA
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

381
SEQ ID NO: 280
SEQ ID NO: 265
SEQ ID NO: 266

CAAGGATTGATGCCGAAGCAGAA
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

382
SEQ ID NO: 281
SEQ ID NO: 268
SEQ ID NO: 269

AAGGATTGATGCCGAAGCAGAAAA
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

383
SEQ ID NO: 280
SEQ ID NO: 268
SEQ ID NO: 269

CAAGGATTGATGCCGAAGCAGAA
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

384
SEQ ID NO: 282
SEQ ID NO: 265
SEQ ID NO: 266

GCAAGGATTGATGCCGAAGCA
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

385
SEQ ID NO: 283
SEQ ID NO: 268
SEQ ID NO: 269

CAAGGATTGATGCCGAAGCAGAAA
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

386
SEQ ID NO: 282
SEQ ID NO: 268
SEQ ID NO: 269

GCAAGGATTGATGCCGAAGCA
TATCCACTCTGGAAAAACCCGAACAGCCCA
ACAATTTCGCCAGACGGTGTATAAAA

387
SEQ ID NO: 244
SEQ ID NO: 257
SEQ ID NO: 284

TCTGGCGAAATTGTATTTAATGAGGTA
AGGCTTTACCTCGCACAGTCGCTATCCAA
ACATACAGACCTATCAGCTTATCCAACA

AACA

388
SEQ ID NO: 285
SEQ ID NO: 257
SEQ ID NO: 259

CGGGTTTTTCCAGAGTGGATATGT
AGGCTTTACCTCGCACAGTCGCTATCCAA
GAACGATAGACCAATGCCTTTCATCATA

389
SEQ ID NO: 244
SEQ ID NO: 257
SEQ ID NO: 286

TCTGGCGAAATTGTATTTAATGAGGTA
AGGCTTTACCTCGCACAGTCGCTATCCAA
ACATACAGACCTATCAGCTTATCCAACATT

AACA

390
SEQ ID NO: 244
SEQ ID NO: 257
SEQ ID NO: 287

TCTGGCGAAATTGTATTTAATGAGGTA
AGGCTTTACCTCGCACAGTCGCTATCCAA
ACATACAGACCTATCAGCTTATCCAACAT

AACA

391
SEQ ID NO: 288
SEQ ID NO: 289
SEQ ID NO: 290

TCAAGCGGCTTTTTTGATTATACAGAG
TTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AAAT

392
SEQ ID NO: 291
SEQ ID NO: 292
SEQ ID NO: 290

TCAAGCGGCTTTTTTGATTATACAGAG
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AAATA

393
SEQ ID NO: 293
SEQ ID NO: 294
SEQ ID NO: 290

TCAAGCGGCTTTTTTGATTATACAGAGA
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

394
SEQ ID NO: 295
SEQ ID NO: 296
SEQ ID NO: 258

GTTCGGGTTTTTCCAGAGTGGAT
ACCAGGCTTTACCTCGCACAGTCGC
GAACGATAGACCAATGCCTTTCATCA

395
SEQ ID NO: 293
SEQ ID NO: 289
SEQ ID NO: 290

TCAAGCGGCTTTTTTGATTATACAGAGA
TTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

396
SEQ ID NO: 288
SEQ ID NO: 294
SEQ ID NO: 290

TCAAGCGGCTTTTTTGATTATACAGAG
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AAAT

397
SEQ ID NO: 297
SEQ ID NO: 294
SEQ ID NO: 290

TCAAGCGGCTTTTTTGATTATACAGAG
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AAATATAC

398
SEQ ID NO: 291
SEQ ID NO: 289
SEQ ID NO: 290

TCAAGCGGCTTTTTTGATTATACAGAG
TTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AAATA

399
SEQ ID NO: 249
SEQ ID NO: 257
SEQ ID NO: 286

ACCGTCTGGCGAAATTGTATTTAATGA
AGGCTTTACCTCGCACAGTCGCTATCCAA
ACATACAGACCTATCAGCTTATCCAACATT

400
SEQ ID NO: 298
SEQ ID NO: 292
SEQ ID NO: 290

GTCAAGCGGCTTTTTTGATTATACAGA
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

GA

401
SEQ ID NO: 298
SEQ ID NO: 294
SEQ ID NO: 290

GTCAAGCGGCTTTTTTGATTATACAGA
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

GA

402
SEQ ID NO: 298
SEQ ID NO: 289
SEQ ID NO: 290

GTCAAGCGGCTTTTTTGATTATACAGA
TTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

GA

403
SEQ ID NO: 299
SEQ ID NO: 294
SEQ ID NO: 290

TGTCAAGCGGCTTTTTTGATTATACAGA
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

404
SEQ ID NO: 300
SEQ ID NO: 289
SEQ ID NO: 290

TGTCAAGCGGCTTTTTTGATTATACAG
TTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AGA

405
SEQ ID NO: 249
SEQ ID NO: 257
SEQ ID NO: 301

ACCGTCTGGCGAAATTGTATTTAATGA
AGGCTTTACCTCGCACAGTCGCTATCCAA
CACATACAGACCTATCAGCTTATCCAACATT

406
SEQ ID NO: 299
SEQ ID NO: 289
SEQ ID NO: 290

TGTCAAGCGGCTTTTTTGATTATACAGA
TTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

407
SEQ ID NO: 300
SEQ ID NO: 292
SEQ ID NO: 290

TGTCAAGCGGCTTTTTTGATTATACAG
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AGA

408
SEQ ID NO: 302
SEQ ID NO: 273
SEQ ID NO: 274

ATCTTCAAAGATATATATGCCTGCAAG
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

GATTG

409
SEQ ID NO: 299
SEQ ID NO: 292
SEQ ID NO: 290

TGTCAAGCGGCTTTTTTGATTATACAGA
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

410
SEQ ID NO: 300
SEQ ID NO: 294
SEQ ID NO: 290

TGTCAAGCGGCTTTTTTGATTATACAG
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AGA

411
SEQ ID NO: 303
SEQ ID NO: 292
SEQ ID NO: 290

CTGTCAAGCGGCTTTTTTGATTATACAG
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

412
SEQ ID NO: 293
SEQ ID NO: 294
SEQ ID NO: 304

TCAAGCGGCTTTTTTGATTATACAGAGA
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
GAACAGCCCAGAGCTTTATATATGGTTAC

413
SEQ ID NO: 305
SEQ ID NO: 294
SEQ ID NO: 290

CTGTCAAGCGGCTTTTTTGATTATACA
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

GA

414
SEQ ID NO: 288
SEQ ID NO: 289
SEQ ID NO: 304

TCAAGCGGCTTTTTTGATTATACAGAG
TTTCTGCTTCGGCATCAATCCTTGCAGGC
GAACAGCCCAGAGCTTTATATATGGTTAC

AAAT

415
SEQ ID NO: 305
SEQ ID NO: 289
SEQ ID NO: 290

CTGTCAAGCGGCTTTTTTGATTATACA
TTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

GA

416
SEQ ID NO: 303
SEQ ID NO: 294
SEQ ID NO: 290

CTGTCAAGCGGCTTTTTTGATTATACAG
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

417
SEQ ID NO: 249
SEQ ID NO: 257
SEQ ID NO: 306

ACCGTCTGGCGAAATTGTATTTAATGA
AGGCTTTACCTCGCACAGTCGCTATCCAA
CCACATACAGACCTATCAGCTTATCCA

418
SEQ ID NO: 298
SEQ ID NO: 289
SEQ ID NO: 304

GTCAAGCGGCTTTTTTGATTATACAGA
TTTCTGCTTCGGCATCAATCCTTGCAGGC
GAACAGCCCAGAGCTTTATATATGGTTAC

GA

419
SEQ ID NO: 307
SEQ ID NO: 292
SEQ ID NO: 290

ACTGTCAAGCGGCTTTTTTGATTATACA
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

420
SEQ ID NO: 249
SEQ ID NO: 257
SEQ ID NO: 308

ACCGTCTGGCGAAATTGTATTTAATGA
AGGCTTTACCTCGCACAGTCGCTATCCAA
TCCACATACAGACCTATCAGCTTATCC

421
SEQ ID NO: 309
SEQ ID NO: 294
SEQ ID NO: 290

ACTGTCAAGCGGCTTTTTTGATTATAC
TTTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AG

422
SEQ ID NO: 310
SEQ ID NO: 289
SEQ ID NO: 290

ACTGTCAAGCGGCTTTTTTGATTATAC
TTTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AGA

423
SEQ ID NO: 309
SEQ ID NO: 292
SEQ ID NO: 290

ACTGTCAAGCGGCTTTTTTGATTATAC
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AG

424
SEQ ID NO: 311
SEQ ID NO: 261
SEQ ID NO: 258

GGGCTGTTCGGGTTTTTCCA
CCAGGCTTTACCTCGCACAGTCGCTATCC
GAACGATAGACCAATGCCTTTCATCA

425
SEQ ID NO: 249
SEQ ID NO: 257
SEQ ID NO: 312

ACCGTCTGGCGAAATTGTATTTAATGA
AGGCTTTACCTCGCACAGTCGCTATCCAA
TTCCACATACAGACCTATCAGCTTATCC

426
SEQ ID NO: 313
SEQ ID NO: 292
SEQ ID NO: 290

AACTGTCAAGCGGCTTTTTTGATTATA
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

CA

427
SEQ ID NO: 314
SEQ ID NO: 292
SEQ ID NO: 290

AACTGTCAAGCGGCTTTTTTGATTATA
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

CAG

428
SEQ ID NO: 303
SEQ ID NO: 292
SEQ ID NO: 304

CTGTCAAGCGGCTTTTTTGATTATACAG
TTCTGCTTCGGCATCAATCCTTGCAGGC
GAACAGCCCAGAGCTTTATATATGGTTAC

429
SEQ ID NO: 315
SEQ ID NO: 292
SEQ ID NO: 290

GAACTGTCAAGCGGCTTTTTTGATTAT
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

AC

430
SEQ ID NO: 316
SEQ ID NO: 292
SEQ ID NO: 290

CGAACTGTCAAGCGGCTTTTTTGAT
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

431
SEQ ID NO: 317
SEQ ID NO: 292
SEQ ID NO: 290

TCGAACTGTCAAGCGGCTTTTTT
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

432
SEQ ID NO: 318
SEQ ID NO: 292
SEQ ID NO: 290

ATCGAACTGTCAAGCGGCTTTTT
TTCTGCTTCGGCATCAATCCTTGCAGGC
CAGCCCAGAGCTTTATATATGGTTACAG

433
SEQ ID NO: 314
SEQ ID NO: 319
SEQ ID NO: 320

AACTGTCAAGCGGCTTTTTTGATTATA
TCTGCTTCGGCATCAATCCTTGCAGGC
CCGAACAGCCCAGAGCTTTA

CAG

434
SEQ ID NO: 321
SEQ ID NO: 261
SEQ ID NO: 258

AACCATATATAAAGCTCTGGGCTGTTC
CCAGGCTTTACCTCGCACAGTCGCTATCC
GAACGATAGACCAATGCCTTTCATCA

435
SEQ ID NO: 322
SEQ ID NO: 323
SEQ ID NO: 324

CTGCAAGGATTGATGCCGAAGCA
ATCCACTCTGGAAAAACCCGAACAGCCCAGA
GGATAGCGACTGTGCGAGGTA

436
SEQ ID NO: 325
SEQ ID NO: 273
SEQ ID NO: 274

GTCAAGCGGCTTTTTTGATTATACAGA
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

GAAATA

437
SEQ ID NO: 305
SEQ ID NO: 273
SEQ ID NO: 274

CTGTCAAGCGGCTTTTTTGATTATACA
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

GA

438
SEQ ID NO: 307
SEQ ID NO: 273
SEQ ID NO: 274

ACTGTCAAGCGGCTTTTTTGATTATACA
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

439
SEQ ID NO: 326
SEQ ID NO: 273
SEQ ID NO: 274

CGAACTGTCAAGCGGCTTTTTTGATTA
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

440
SEQ ID NO: 327
SEQ ID NO: 265
SEQ ID NO: 266

GTCAAGCGGCTTTTTTGATTATACAGA
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

GAAAT

441
SEQ ID NO: 325
SEQ ID NO: 265
SEQ ID NO: 266

GTCAAGCGGCTTTTTTGATTATACAGA
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

GAAATA

442
SEQ ID NO: 328
SEQ ID NO: 273
SEQ ID NO: 274

AATCGAACTGTCAAGCGGCTTTTTT
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

443
SEQ ID NO: 329
SEQ ID NO: 273
SEQ ID NO: 274

AAATCGAACTGTCAAGCGGCTTTTT
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

444
SEQ ID NO: 330
SEQ ID NO: 273
SEQ ID NO: 274

GAAATCGAACTGTCAAGCGGCTTTT
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

445
SEQ ID NO: 331
SEQ ID NO: 273
SEQ ID NO: 274

GAAATCGAACTGTCAAGCGGCTTTTT
CTGGAAAAACCCGAACAGCCCAGAGCTT
GCCAGACGGTGTATAAAACATATCCA

446
SEQ ID NO: 332
SEQ ID NO: 265
SEQ ID NO: 266

ATCGAACTGTCAAGCGGCTTTTTT
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

447
SEQ ID NO: 333
SEQ ID NO: 265
SEQ ID NO: 266

AATCGAACTGTCAAGCGGCTTTT
CCACTCTGGAAAAACCCGAACAGCCCAGA
CAATTTCGCCAGACGGTGTATAAAACATA

vanE Sets

448
SEQ ID NO: 334
SEQ ID NO: 335
SEQ ID NO: 336

GGTATCGGAGCTGCAGCAAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

449
SEQ ID NO: 337
SEQ ID NO: 335
SEQ ID NO: 336

TGGTGTAAAAAGCACCCCTAGTATGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

450
SEQ ID NO: 338
SEQ ID NO: 335
SEQ ID NO: 336

AGACGAAGCTTCAAAATATGATAGCC
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

GTAT

451
SEQ ID NO: 339
SEQ ID NO: 335
SEQ ID NO: 336

CAGCAATCTCCATGAATAAAATAATG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

CTCCAT

452
SEQ ID NO: 340
SEQ ID NO: 335
SEQ ID NO: 336

TGGTGTAAAAAGCACCCCTAGTATGAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

453
SEQ ID NO: 341
SEQ ID NO: 335
SEQ ID NO: 336

TTGGTGTAAAAAGCACCCCTAGTATGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

454
SEQ ID NO: 342
SEQ ID NO: 335
SEQ ID NO: 336

TTGGTGTAAAAAGCACCCCTAGTATGAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

455
SEQ ID NO: 343
SEQ ID NO: 335
SEQ ID NO: 336

TGGTGTAAAAAGCACCCCTAGTATGAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

TA

456
SEQ ID NO: 344
SEQ ID NO: 335
SEQ ID NO: 336

TTGGTGTAAAAAGCACCCCTAGTATGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

TTA

457
SEQ ID NO: 345
SEQ ID NO: 335
SEQ ID NO: 336

ACCCCTAGTATGATTATAGAAAAGGG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

ACAAGA

458
SEQ ID NO: 346
SEQ ID NO: 335
SEQ ID NO: 336

AGGGACAAGACCTACAAAAAGTCGAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

459
SEQ ID NO: 347
SEQ ID NO: 335
SEQ ID NO: 336

AAAGGGACAAGACCTACAAAAAGTCG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

AT

460
SEQ ID NO: 348
SEQ ID NO: 335
SEQ ID NO: 336

TTGGTGTAAAAAGCACCCCTAGTATGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

TT

461
SEQ ID NO: 349
SEQ ID NO: 335
SEQ ID NO: 336

TGCAGCAATCTCCATGAATAAAATAAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

GCT

462
SEQ ID NO: 350
SEQ ID NO: 335
SEQ ID NO: 336

TAGACGAAGCTTCAAAATATGATAGC
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

CGTAT

463
SEQ ID NO: 351
SEQ ID NO: 335
SEQ ID NO: 336

GAGCTGCAGCAATCTCCATGAATAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

AT

464
SEQ ID NO: 352
SEQ ID NO: 335
SEQ ID NO: 336

GAGCTGCAGCAATCTCCATGAATAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

ATAA

465
SEQ ID NO: 353
SEQ ID NO: 335
SEQ ID NO: 336

TGAGGCAGGCTCATCAAAAGG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

466
SEQ ID NO: 354
SEQ ID NO: 335
SEQ ID NO: 336

TGGTGTAAAAAGCACCCCTAGTATGATT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

467
SEQ ID NO: 355
SEQ ID NO: 335
SEQ ID NO: 336

GCAATCTCCATGAATAAAATAATGCTC
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

CATCA

468
SEQ ID NO: 356
SEQ ID NO: 335
SEQ ID NO: 336

AGCTGCAGCAATCTCCATGAATAAAAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

469
SEQ ID NO: 357
SEQ ID NO: 335
SEQ ID NO: 336

AAAAGGGACAAGACCTACAAAAAGTC
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

GAT

470
SEQ ID NO: 358
SEQ ID NO: 335
SEQ ID NO: 336

CACCCCTAGTATGATTATAGAAAAGG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

GACAA

471
SEQ ID NO: 359
SEQ ID NO: 335
SEQ ID NO: 336

CCCCTAGTATGATTATAGAAAAGGGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

CAAGAC

472
SEQ ID NO: 360
SEQ ID NO: 335
SEQ ID NO: 336

GAGCTGCAGCAATCTCCATGAATAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

ATA

473
SEQ ID NO: 361
SEQ ID NO: 335
SEQ ID NO: 336

CCCCTAGTATGATTATAGAAAAGGGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

CAAGA

474
SEQ ID NO: 362
SEQ ID NO: 335
SEQ ID NO: 336

GGCAGGCTCATCAAAAGGAATTAGC
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

475
SEQ ID NO: 363
SEQ ID NO: 335
SEQ ID NO: 336

AAGGAATTAGCAAGGTAGAACAAAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

AGTGATT

476
SEQ ID NO: 364
SEQ ID NO: 335
SEQ ID NO: 336

AGCTGCAGCAATCTCCATGAATAAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

TAAT

477
SEQ ID NO: 365
SEQ ID NO: 335
SEQ ID NO: 336

CCATCAATTTGCTGAAACAATTGGTGT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

AAAA

478
SEQ ID NO: 366
SEQ ID NO: 335
SEQ ID NO: 336

AGCTGCAGCAATCTCCATGAATAAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

TAA

479
SEQ ID NO: 367
SEQ ID NO: 335
SEQ ID NO: 336

CTGCAGCAATCTCCATGAATAAAATA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

ATGC

480
SEQ ID NO: 368
SEQ ID NO: 335
SEQ ID NO: 336

GACGAAGCTTCAAAATATGATAGCCG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

TAT

481
SEQ ID NO: 369
SEQ ID NO: 335
SEQ ID NO: 336

CCATCAATTTGCTGAAACAATTGGTGT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

AAA

482
SEQ ID NO: 370
SEQ ID NO: 335
SEQ ID NO: 336

AGCTGCAGCAATCTCCATGAATAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

483
SEQ ID NO: 371
SEQ ID NO: 335
SEQ ID NO: 336

TTGGTGTAAAAAGCACCCCTAGTATG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

484
SEQ ID NO: 372
SEQ ID NO: 335
SEQ ID NO: 336

GAAAAGGGACAAGACCTACAAAAAGT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

CGAT

485
SEQ ID NO: 373
SEQ ID NO: 335
SEQ ID NO: 336

AGCTGCAGCAATCTCCATGAATAAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

TA

486
SEQ ID NO: 374
SEQ ID NO: 335
SEQ ID NO: 336

GGTGTAAAAAGCACCCCTAGTATGATT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

487
SEQ ID NO: 375
SEQ ID NO: 335
SEQ ID NO: 336

GCTGCAGCAATCTCCATGAATAAAAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

AATGC

488
SEQ ID NO: 376
SEQ ID NO: 335
SEQ ID NO: 336

ACCCCTAGTATGATTATAGAAAAGGG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

ACAA

489
SEQ ID NO: 377
SEQ ID NO: 335
SEQ ID NO: 336

AGTATGATTATAGAAAAGGGACAAGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

CCTACA

490
SEQ ID NO: 378
SEQ ID NO: 335
SEQ ID NO: 336

TGGTGTAAAAAGCACCCCTAGTATG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

491
SEQ ID NO: 379
SEQ ID NO: 335
SEQ ID NO: 336

AGCTGCAGCAATCTCCATGAATAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

492
SEQ ID NO: 380
SEQ ID NO: 335
SEQ ID NO: 336

ATTTGCTGAAACAATTGGTGTAAAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CTGATTTGGTCACATTCTCCAACGA

GCA

493
SEQ ID NO: 347
SEQ ID NO: 335
SEQ ID NO: 381

AAAGGGACAAGACCTACAAAAAGTCG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

AT

494
SEQ ID NO: 348
SEQ ID NO: 335
SEQ ID NO: 381

TTGGTGTAAAAAGCACCCCTAGTATGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

TT

495
SEQ ID NO: 349
SEQ ID NO: 335
SEQ ID NO: 381

TGCAGCAATCTCCATGAATAAAATAAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

GCT

496
SEQ ID NO: 350
SEQ ID NO: 335
SEQ ID NO: 381

TAGACGAAGCTTCAAAATATGATAGC
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

CGTAT

497
SEQ ID NO: 382
SEQ ID NO: 335
SEQ ID NO: 381

TCCATCAATTTGCTGAAACAATTGGTG
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

TAAAA

498
SEQ ID NO: 383
SEQ ID NO: 335
SEQ ID NO: 381

GAGCTGCAGCAATCTCCATGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

499
SEQ ID NO: 355
SEQ ID NO: 335
SEQ ID NO: 381

GCAATCTCCATGAATAAAATAATGCTC
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

CATCA

500
SEQ ID NO: 359
SEQ ID NO: 335
SEQ ID NO: 381

CCCCTAGTATGATTATAGAAAAGGGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

CAAGAC

501
SEQ ID NO: 360
SEQ ID NO: 335
SEQ ID NO: 381

GAGCTGCAGCAATCTCCATGAATAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

ATA

502
SEQ ID NO: 338
SEQ ID NO: 335
SEQ ID NO: 381

AGACGAAGCTTCAAAATATGATAGCC
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

GTAT

503
SEQ ID NO: 384
SEQ ID NO: 335
SEQ ID NO: 381

TCAATTTGCTGAAACAATTGGTGTAAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

AAGC

504
SEQ ID NO: 385
SEQ ID NO: 335
SEQ ID NO: 381

GACAAGACCTACAAAAAGTCGATGAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

TTTGC

505
SEQ ID NO: 386
SEQ ID NO: 335
SEQ ID NO: 381

GAGCTGCAGCAATCTCCATGAAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

506
SEQ ID NO: 341
SEQ ID NO: 335
SEQ ID NO: 381

TTGGTGTAAAAAGCACCCCTAGTATGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

507
SEQ ID NO: 342
SEQ ID NO: 335
SEQ ID NO: 381

TTGGTGTAAAAAGCACCCCTAGTATGAT
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

508
SEQ ID NO: 344
SEQ ID NO: 335
SEQ ID NO: 381

TTGGTGTAAAAAGCACCCCTAGTATGA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

TTA

509
SEQ ID NO: 387
SEQ ID NO: 335
SEQ ID NO: 381

ATCAATTTGCTGAAACAATTGGTGTAA
TCCCACAGCCAATTTCTACCCCTTTCACTTCCT
CCACAAGACTGATTTGGTCACATTCT

AAAGC

vanD Sets

510
SEQ ID NO: 388
SEQ ID NO: 389
SEQ ID NO: 390

TGCGCCATACTGGGAAACG
TGATCCACCTCGCCAGCCATGAGATCATTT
AGCCGTGTCTCAGCTCAATC

511
SEQ ID NO: 391
SEQ ID NO: 392
SEQ ID NO: 393

CTGCGCCATACTGGGAAACG
TCTGATCCACCTCGCCAGCCATGAGA
TTAAAAAAGCCGTGTCTCAGCTCAA

512
SEQ ID NO: 394
SEQ ID NO: 392
SEQ ID NO: 395

GGTAGGCTGCGCCATACTG
TCTGATCCACCTCGCCAGCCATGAGA
AAAAGCCGTGTCTCAGCTCAA

513
SEQ ID NO: 396
SEQ ID NO: 397
SEQ ID NO: 398

GCTGCGCCATACTGGGAAAC
TCTGATCCACCTCGCCAGCCATGAGATCATTT
CTTAAAAAAGCCGTGTCTCAGCTCAA

514
SEQ ID NO: 399
SEQ ID NO: 400
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
TTCCCAGTATGGCGCAGCCTACCTCAC
CTCGCCAGCCATGAGATCATTT

515
SEQ ID NO: 399
SEQ ID NO: 402
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
CCCAGTATGGCGCAGCCTACCTCACT
CTCGCCAGCCATGAGATCATTT

516
SEQ ID NO: 399
SEQ ID NO: 403
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
TGGCGCAGCCTACCTCACTCCC
CTCGCCAGCCATGAGATCATTT

517
SEQ ID NO: 399
SEQ ID NO: 404
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
TCCCAGTATGGCGCAGCCTACCTCA
CTCGCCAGCCATGAGATCATTT

518
SEQ ID NO: 399
SEQ ID NO: 405
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
TCCCAGTATGGCGCAGCCTACCTCAC
CTCGCCAGCCATGAGATCATTT

519
SEQ ID NO: 399
SEQ ID NO: 406
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
AGTATGGCGCAGCCTACCTCACTCCC
CTCGCCAGCCATGAGATCATTT

520
SEQ ID NO: 399
SEQ ID NO: 407
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
TTTCCCAGTATGGCGCAGCCTACCTCA
CTCGCCAGCCATGAGATCATTT

521
SEQ ID NO: 399
SEQ ID NO: 408
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
TTTCCCAGTATGGCGCAGCCTACCTCAC
CTCGCCAGCCATGAGATCATTT

522
SEQ ID NO: 409
SEQ ID NO: 410
SEQ ID NO: 411

AGGTAGGCTGCGCCATACTG
CTGATCCACCTCGCCAGCCATGAGATCATTT
AAAAAAGCCGTGTCTCAGCTCAAT

523
SEQ ID NO: 399
SEQ ID NO: 412
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
CCCAGTATGGCGCAGCCTACCTCAC
CTCGCCAGCCATGAGATCATTT

524
SEQ ID NO: 399
SEQ ID NO: 413
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
ATGGCGCAGCCTACCTCACTCCC
CTCGCCAGCCATGAGATCATTT

525
SEQ ID NO: 399
SEQ ID NO: 414
SEQ ID NO: 401

AGATTTTGATTGAAGAGGCCGTTACC
TTCCCAGTATGGCGCAGCCTACCTCA
CTCGCCAGCCATGAGATCATTT

526
SEQ ID NO: 415
SEQ ID NO: 406
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
AGTATGGCGCAGCCTACCTCACTCCC
CTCGCCAGCCATGAGATCATTT

527
SEQ ID NO: 415
SEQ ID NO: 407
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
TTTCCCAGTATGGCGCAGCCTACCTCA
CTCGCCAGCCATGAGATCATTT

528
SEQ ID NO: 415
SEQ ID NO: 408
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
TTTCCCAGTATGGCGCAGCCTACCTCAC
CTCGCCAGCCATGAGATCATTT

529
SEQ ID NO: 394
SEQ ID NO: 410
SEQ ID NO: 393

GGTAGGCTGCGCCATACTG
CTGATCCACCTCGCCAGCCATGAGATCATTT
TTAAAAAAGCCGTGTCTCAGCTCAA

530
SEQ ID NO: 415
SEQ ID NO: 412
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
CCCAGTATGGCGCAGCCTACCTCAC
CTCGCCAGCCATGAGATCATTT

531
SEQ ID NO: 415
SEQ ID NO: 413
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
ATGGCGCAGCCTACCTCACTCCC
CTCGCCAGCCATGAGATCATTT

532
SEQ ID NO: 415
SEQ ID NO: 414
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
TTCCCAGTATGGCGCAGCCTACCTCA
CTCGCCAGCCATGAGATCATTT

533
SEQ ID NO: 415
SEQ ID NO: 400
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
TTCCCAGTATGGCGCAGCCTACCTCAC
CTCGCCAGCCATGAGATCATTT

534
SEQ ID NO: 394
SEQ ID NO: 389
SEQ ID NO: 393

GGTAGGCTGCGCCATACTG
TGATCCACCTCGCCAGCCATGAGATCATTT
TTAAAAAAGCCGTGTCTCAGCTCAA

535
SEQ ID NO: 415
SEQ ID NO: 402
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
CCCAGTATGGCGCAGCCTACCTCACT
CTCGCCAGCCATGAGATCATTT

536
SEQ ID NO: 415
SEQ ID NO: 403
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
TGGCGCAGCCTACCTCACTCCC
CTCGCCAGCCATGAGATCATTT

537
SEQ ID NO: 415
SEQ ID NO: 404
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
TCCCAGTATGGCGCAGCCTACCTCA
CTCGCCAGCCATGAGATCATTT

538
SEQ ID NO: 394
SEQ ID NO: 397
SEQ ID NO: 393

GGTAGGCTGCGCCATACTG
TCTGATCCACCTCGCCAGCCATGAGATCATTT
TTAAAAAAGCCGTGTCTCAGCTCAA

539
SEQ ID NO: 415
SEQ ID NO: 405
SEQ ID NO: 401

AAGATTTTGATTGAAGAGGCCGTTACC
TCCCAGTATGGCGCAGCCTACCTCAC
CTCGCCAGCCATGAGATCATTT

540
SEQ ID NO: 416
SEQ ID NO: 400
SEQ ID NO: 417

CAAGATTTTGATTGAAGAGGCCGTTACC
TTCCCAGTATGGCGCAGCCTACCTCAC
CCTCGCCAGCCATGAGAT

541
SEQ ID NO: 396
SEQ ID NO: 418
SEQ ID NO: 419

GCTGCGCCATACTGGGAAAC
CAATCTGATCCACCTCGCCAGCCATGAGA
CCTGATGAATCTTAAAAAAGCCGTGTCT

542
SEQ ID NO: 396
SEQ ID NO: 397
SEQ ID NO: 419

GCTGCGCCATACTGGGAAAC
TCTGATCCACCTCGCCAGCCATGAGATCATTT
CCTGATGAATCTTAAAAAAGCCGTGTCT

543
SEQ ID NO: 391
SEQ ID NO: 420
SEQ ID NO: 421

CTGCGCCATACTGGGAAACG
CTCAATCTGATCCACCTCGCCAGCCATGAGA
TCCTGATGAATCTTAAAAAAGCCGTGTCT

544
SEQ ID NO: 396
SEQ ID NO: 420
SEQ ID NO: 419

GCTGCGCCATACTGGGAAAC
CTCAATCTGATCCACCTCGCCAGCCATGAGA
CCTGATGAATCTTAAAAAAGCCGTGTCT

545
SEQ ID NO: 396
SEQ ID NO: 422
SEQ ID NO: 419

GCTGCGCCATACTGGGAAAC
CTCAATCTGATCCACCTCGCCAGCCATGAGAT
CCTGATGAATCTTAAAAAAGCCGTGTCT

546
SEQ ID NO: 396
SEQ ID NO: 423
SEQ ID NO: 419

GCTGCGCCATACTGGGAAAC
CTCAATCTGATCCACCTCGCCAGCCATGAG
CCTGATGAATCTTAAAAAAGCCGTGTCT

547
SEQ ID NO: 396
SEQ ID NO: 424
SEQ ID NO: 419

GCTGCGCCATACTGGGAAAC
TCAATCTGATCCACCTCGCCAGCCATGAGA
CCTGATGAATCTTAAAAAAGCCGTGTCT

548
SEQ ID NO: 425
SEQ ID NO: 406
SEQ ID NO: 426

AGCAAGATTTTGATTGAAGAGGCCGTTA
AGTATGGCGCAGCCTACCTCACTCCC
CACCTCGCCAGCCATGA

549
SEQ ID NO: 396
SEQ ID NO: 427
SEQ ID NO: 419

GCTGCGCCATACTGGGAAAC
TCAATCTGATCCACCTCGCCAGCCATGAGAT
CCTGATGAATCTTAAAAAAGCCGTGTCT

550
SEQ ID NO: 428
SEQ ID NO: 429
SEQ ID NO: 430

GGCTTGCTTGAATTGTCAGGCATT
CACGGAGCTTTGAATATCGCATCCCACATAC
AACGGTATATGCAAGCGCCTTATC

GG

551
SEQ ID NO: 391
SEQ ID NO: 431
SEQ ID NO: 421

CTGCGCCATACTGGGAAACG
AGCTCAATCTGATCCACCTCGCCAGCC
TCCTGATGAATCTTAAAAAAGCCGTGTCT

552
SEQ ID NO: 396
SEQ ID NO: 432
SEQ ID NO: 419

GCTGCGCCATACTGGGAAAC
TCAATCTGATCCACCTCGCCAGCCATGAG
CCTGATGAATCTTAAAAAAGCCGTGTCT

553
SEQ ID NO: 391
SEQ ID NO: 433
SEQ ID NO: 434

CTGCGCCATACTGGGAAACG
TCTGATCCACCTCGCCAGCCATGAGAT
CGGCTGTGCTTCCTGATGA

554
SEQ ID NO: 435
SEQ ID NO: 436
SEQ ID NO: 437

CCATACAAGGCTTGCTTGAATTGTCA
ACGGAGCTTTGAATATCGCATCCCACATACG
ATACCCGCATTTTTCACAACGGTAT

GAA

555
SEQ ID NO: 438
SEQ ID NO: 420
SEQ ID NO: 439

GGCCGTTACCGGGAGTGA
CTCAATCTGATCCACCTCGCCAGCCATGAGA
TTCCTGATGAATCTTAAAAAAGCCGTGTCT

556
SEQ ID NO: 440
SEQ ID NO: 441
SEQ ID NO: 442

AGGAAGCACAGCCGGAGAA
CCTCATCCGGTAAGGCGGCTGGAACT
AGCCAAGTATCCGGTAAATCTTCATTG

557
SEQ ID NO: 443
SEQ ID NO: 392
SEQ ID NO: 434

CCGTTACCGGGAGTGAGGTA
TCTGATCCACCTCGCCAGCCATGAGA
CGGCTGTGCTTCCTGATGA

558
SEQ ID NO: 399
SEQ ID NO: 397
SEQ ID NO: 444

AGATTTTGATTGAAGAGGCCGTTACC
TCTGATCCACCTCGCCAGCCATGAGATCATTT
TGATGAATCTTAAAAAAGCCGTGTCTCA

559
SEQ ID NO: 399
SEQ ID NO: 410
SEQ ID NO: 444

AGATTTTGATTGAAGAGGCCGTTACC
CTGATCCACCTCGCCAGCCATGAGATCATTT
TGATGAATCTTAAAAAAGCCGTGTCTCA

560
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 447

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
TTCCGGCTGTGCTTCCTGAT

561
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 448

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
CCGGCTGTGCTTCCTGATG

562
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 449

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
TCCGGCTGTGCTTCCTGAT

563
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 450

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
TCCTACCTCACTCCCGGAAAC

564
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 451

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
CCTACCTCACTCCCGGAAAC

565
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 452

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
GACCGCTGCCTGCAGTTC

566
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 453

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
ATCCTACCTCACTCCCGGAAAC

567
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 454

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
CATCCTACCTCACTCCCGGAAA

568
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 455

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
CCGGCTGTGCTTCCTGAT

569
SEQ ID NO: 456
SEQ ID NO: 457
SEQ ID NO: 458

GGTTGTGAAAAATGCGGGAATTGAG
CTGCCCGTTCCGGCTCCTCTTTTGG
TCTGCCCGGCATACCTTATTCAC

570
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 459

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
CAGTATGGCACATCCTACCTCACT

571
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 460

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
CAGCCAAGTACCCGGTAAATCTTC

572
SEQ ID NO: 445
SEQ ID NO: 446
SEQ ID NO: 461

CGCTCTCGTATCCGGTCTTTG
CCGTTCCGGCTCCTCTTTTGGCGTGAATAA
GCTGTGCTTCCTGATGGATCTTAAAAA

573
SEQ ID NO: 462
SEQ ID NO: 463
SEQ ID NO: 464

CGTTCCGGCTCCTCTTTTGG
ACCGCTGCCTGCAGTTCCTCTGC
CGGAAACGGCCTCCTCAAC

574
SEQ ID NO: 465
SEQ ID NO: 466
SEQ ID NO: 467

GGCTCCTCTTTTGGCGTGAA
TGCAGTTCCTCTGCCCGGCATACCT
CTACCTCACTCCCGGAAACG

575
SEQ ID NO: 468
SEQ ID NO: 469
SEQ ID NO: 470

CCGTTCCGGCTCCTCTTTT
CTCTGCCCGGCATACCTTATTCACGCC
ACCGCTGCCTGCAGTT

576
SEQ ID NO: 471
SEQ ID NO: 472
SEQ ID NO: 473

CTGCTTGAGCTGTCCGGCATT
AGAACTCGAAACCCAGGTACCTCAATTCCCGC
TCCAGGCTGTCCCCCTTTT

577
SEQ ID NO: 474
SEQ ID NO: 475
SEQ ID NO: 476

TAAGGTATGCCGGGCAGAGGAA
TCCCGGAAACGGCCTCCTCAACCAAT
CCCAGTATGGCACATCCTACCT

578
SEQ ID NO: 477
SEQ ID NO: 478
SEQ ID NO: 479

GTCGCTCGCTTATATGGTTGTGAA
ACTCGAAACCCAGGTACCTCAATTCCCGCAT
AGGCTGTCCCCCTTTTGTAGA

579
SEQ ID NO: 480
SEQ ID NO: 481
SEQ ID NO: 482

CTCCTCTTTTGGCGTGAATAAGGT
TCTGTGACCGCTGCCTGCAGTTCC
AAACGGCCTCCTCAACCAAT

580
SEQ ID NO: 483
SEQ ID NO: 484
SEQ ID NO: 479

TGGATAAGTCGCTCGCTTATATGGT
ACTCGAAACCCAGGTACCTCAATTCCCGCA
AGGCTGTCCCCCTTTTGTAGA

581
SEQ ID NO: 483
SEQ ID NO: 485
SEQ ID NO: 486

TGGATAAGTCGCTCGCTTATATGGT
AACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

582
SEQ ID NO: 483
SEQ ID NO: 487
SEQ ID NO: 486

TGGATAAGTCGCTCGCTTATATGGT
ACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

583
SEQ ID NO: 488
SEQ ID NO: 489
SEQ ID NO: 490

CCTCTTTTGGCGTGAATAAGGTATGC
CTCTGTGACCGCTGCCTGCAGTTCC
GAAACGGCCTCCTCAACCA

584
SEQ ID NO: 491
SEQ ID NO: 492
SEQ ID NO: 493

TCGCTCGCTTATATGGTTGTGAAAA
TCGAAACCCAGGTACCTCAATTCCCGCA
CAGGCTGTCCCCCTTTTGT

585
SEQ ID NO: 494
SEQ ID NO: 485
SEQ ID NO: 486

ATGGATAAGTCGCTCGCTTATATGGT
ACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

586
SEQ ID NO: 494
SEQ ID NO: 487
SEQ ID NO: 486

ATGGATAAGTCGCTCGCTTATATGGT
AACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

587
SEQ ID NO: 495
SEQ ID NO: 492
SEQ ID NO: 493

GATAAGTCGCTCGCTTATATGGTTGT
TCGAAACCCAGGTACCTCAATTCCCGCA
CAGGCTGTCCCCCTTTTGT

588
SEQ ID NO: 496
SEQ ID NO: 485
SEQ ID NO: 486

TATGGATAAGTCGCTCGCTTATATGGT
ACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

589
SEQ ID NO: 496
SEQ ID NO: 487
SEQ ID NO: 486

TATGGATAAGTCGCTCGCTTATATGGT
AACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

590
SEQ ID NO: 497
SEQ ID NO: 485
SEQ ID NO: 486

GTATGGATAAGTCGCTCGCTTATATGGT
ACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

591
SEQ ID NO: 497
SEQ ID NO: 487
SEQ ID NO: 486

GTATGGATAAGTCGCTCGCTTATATGGT
AACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

592
SEQ ID NO: 498
SEQ ID NO: 499
SEQ ID NO: 486

TGTATGGATAAGTCGCTCGCTTATATGG
ACCCAGGTACCTCAATTCCCGCATTTTTCACAA
CAGGCTGTCCCCCTTTTGTA

593
SEQ ID NO: 498
SEQ ID NO: 485
SEQ ID NO: 486

TGTATGGATAAGTCGCTCGCTTATATGG
ACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

594
SEQ ID NO: 498
SEQ ID NO: 487
SEQ ID NO: 486

TGTATGGATAAGTCGCTCGCTTATATGG
AACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

595
SEQ ID NO: 500
SEQ ID NO: 499
SEQ ID NO: 486

GTATGGATAAGTCGCTCGCTTATATGG
ACCCAGGTACCTCAATTCCCGCATTTTTCACAA
CAGGCTGTCCCCCTTTTGTA

596
SEQ ID NO: 500
SEQ ID NO: 485
SEQ ID NO: 486

GTATGGATAAGTCGCTCGCTTATATGG
ACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

597
SEQ ID NO: 500
SEQ ID NO: 487
SEQ ID NO: 486

GTATGGATAAGTCGCTCGCTTATATGG
AACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

598
SEQ ID NO: 501
SEQ ID NO: 499
SEQ ID NO: 486

CTGTATGGATAAGTCGCTCGCTTATAT
ACCCAGGTACCTCAATTCCCGCATTTTTCACAA
CAGGCTGTCCCCCTTTTGTA

GG

599
SEQ ID NO: 501
SEQ ID NO: 485
SEQ ID NO: 486

CTGTATGGATAAGTCGCTCGCTTATAT
ACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

GG

600
SEQ ID NO: 501
SEQ ID NO: 487
SEQ ID NO: 486

CTGTATGGATAAGTCGCTCGCTTATAT
AACCCAGGTACCTCAATTCCCGCATTTTTCACA
CAGGCTGTCCCCCTTTTGTA

GG

601
SEQ ID NO: 502
SEQ ID NO: 492
SEQ ID NO: 493

TGTATGGATAAGTCGCTCGCTTATATG
TCGAAACCCAGGTACCTCAATTCCCGCA
CAGGCTGTCCCCCTTTTGT

GTT

A PCR primer set for amplifying a vanC1 gene comprises at least one of the following sets of primer sequences: (1) SEQ ID NOS: 123 and 125; (2) SEQ ID NOS: 127 and 129; (3) SEQ ID NOS: 130 and 132; (4) SEQ ID NOS: 133 and 135; (5) SEQ ID NOS: 133 and 137; (6) SEQ ID NOS: 138 and 140; (7) SEQ ID NOS: 141 and 137; (8) SEQ ID NOS: 141 and 143; (9) SEQ ID NOS: 141 and 147; (10) SEQ ID NOS: 141 and 179; (11) SEQ ID NOS: 144 and 137; (12) SEQ ID NOS: 144 and 146; (13) SEQ ID NOS: 144 and 147; (14) SEQ ID NOS: 144 and 157; (15) SEQ ID NOS: 148 and 137; (16) SEQ ID NOS: 148 and 150; (17) SEQ ID NOS: 151 and 153; (18) SEQ ID NOS: 151 and 155; (19) SEQ ID NOS: 156 and 150; (20) SEQ ID NOS: 158 and 160; (21) SEQ ID NOS: 161 and 137; (22) SEQ ID NOS: 161 and 147; (23) SEQ ID NOS: 161 and 150; (24) SEQ ID NOS: 161 and 153; (25) SEQ ID NOS: 161 and 190; (26) SEQ ID NOS: 161 and 192; (27) SEQ ID NOS: 162 and 164; (28) SEQ ID NOS: 165 and 167; (29) SEQ ID NOS: 168 and 169; (30) SEQ ID NOS: 170 and 169; (31) SEQ ID NOS: 171 and 173; (32) SEQ ID NOS: 171 and 174; (33) SEQ ID NOS: 175 and 177; (34) SEQ ID NOS: 178 and 179; (35) SEQ ID NOS: 180 and 146; (36) SEQ ID NOS: 183 and 150; (37) SEQ ID NOS: 184 and 174; (38) SEQ ID NOS: 185 and 187; (39) SEQ ID NOS: 188 and 137; (40) SEQ ID NOS: 188 and 150; (41) SEQ ID NOS: 188 and 190; (42) SEQ ID NOS: 191 and 137; (43) SEQ ID NOS: 191 and 150; (44) SEQ ID NOS: 191 and 153; (45) SEQ ID NOS: 191 and 190; (46) SEQ ID NOS: 191 and 192; (47) SEQ ID NOS: 193 and 137; (48) SEQ ID NOS: 193 and 150; (49) SEQ ID NOS: 193 and 153; (50) SEQ ID NOS: 193 and 190; (51) SEQ ID NOS: 194 and 147; (52) SEQ ID NOS: 194 and 160; (53) SEQ ID NOS: 196 and 147; (54) SEQ ID NOS: 196 and 160; (55) SEQ ID NOS: 198 and 179; (56) SEQ ID NOS: 200 and 179; (57) SEQ ID NOS: 201 and 179; (58) SEQ ID NOS: 202 and 179; (59) SEQ ID NOS: 203 and 147; (60) SEQ ID NOS: 204 and 179; and (61) SEQ ID NOS: 204 and 187.

A PCR primer set for amplifying a vanC2/3 gene comprises at least one of the following sets of primer sequences: (1) SEQ ID NOS: 206 and 208; (2) SEQ ID NOS: 206 and 209; (3) SEQ ID NOS: 206 and 216; (4) SEQ ID NOS: 206 and 219; (5) SEQ ID NOS: 206 and 227; (6) SEQ ID NOS: 210 and 209; (7) SEQ ID NOS: 210 and 212; (8) SEQ ID NOS: 210 and 215; (9) SEQ ID NOS: 210 and 216; (10) SEQ ID NOS: 210 and 219; (11) SEQ ID NOS: 210 and 223; (12) SEQ ID NOS: 210 and 227; (13) SEQ ID NOS: 213 and 215; (14) SEQ ID NOS: 217 and 209; (15) SEQ ID NOS: 217 and 216; (16) SEQ ID NOS: 217 and 219; (17) SEQ ID NOS: 217 and 223; (18) SEQ ID NOS: 217 and 227; (19) SEQ ID NOS: 220 and 209; (20) SEQ ID NOS: 220 and 219; (21) SEQ ID NOS: 220 and 223; (22) SEQ ID NOS: 220 and 227; (23) SEQ ID NOS: 221 and 209; (24) SEQ ID NOS: 221 and 216; (25) SEQ ID NOS: 221 and 219; (26) SEQ ID NOS: 221 and 227; (27) SEQ ID NOS: 222 and 209; (28) SEQ ID NOS: 222 and 216; (29) SEQ ID NOS: 222 and 219; (30) SEQ ID NOS: 222 and 223; (31) SEQ ID NOS: 222 and 227; (32) SEQ ID NOS: 224 and 212; (33) SEQ ID NOS: 224 and 215; (34) SEQ ID NOS: 224 and 216; (35) SEQ ID NOS: 225 and 209; (36) SEQ ID NOS: 225 and 212; (37) SEQ ID NOS: 225 and 216; (38) SEQ ID NOS: 226 and 209; (39) SEQ ID NOS: 226 and 212; (40) SEQ ID NOS: 226 and 216; (41) SEQ ID NOS: 228 and 215; (42) SEQ ID NOS: 229 and 209; (43) SEQ ID NOS: 229 and 215; (44) SEQ ID NOS: 230 and 219; (45) SEQ ID NOS: 231 and 212; (46) SEQ ID NOS: 231 and 215; (47) SEQ ID NOS: 232 and 216; (48) SEQ ID NOS: 233 and 212; (49) SEQ ID NOS: 234 and 215; (50) SEQ ID NOS: 235 and 215; (51) SEQ ID NOS: 235 and 239; (52) SEQ ID NOS: 235 and 241; (53) SEQ ID NOS: 236 and 216; (54) SEQ ID NOS: 237 and 209; (55) SEQ ID NOS: 237 and 215; (56) SEQ ID NOS: 238 and 215; (57) SEQ ID NOS: 240 and 216; (58) SEQ ID NOS: 242 and 216; and (59) SEQ ID NOS: 243 and 215.

A PCR primer set for amplifying a vanD gene comprises at least one of the following sets of primer sequences: (1) SEQ ID NOS: 388 and 390; (2) SEQ ID NOS: 391 and 393; (3) SEQ ID NOS: 391 and 434; (4) SEQ ID NOS: 394 and 393; (5) SEQ ID NOS: 396 and 398; (6) SEQ ID NOS: 396 and 419; (7) SEQ ID NOS: 396 and 419; (8) SEQ ID NOS: 399 and 401; (9) SEQ ID NOS: 399 and 401; (10) SEQ ID NOS: 399 and 401; (11) SEQ ID NOS: 399 and 401; (12) SEQ ID NOS: 399 and 444; (13) SEQ ID NOS: 399 and 444; (14) SEQ ID NOS: 415 and 401; (15) SEQ ID NOS: 416 and 417; (16) SEQ ID NOS: 435 and 437; (17) SEQ ID NOS: 438 and 439; (18) SEQ ID NOS: 440 and 442; (19) SEQ ID NOS: 443 and 434; (20) SEQ ID NOS: 445 and 447; (21) SEQ ID NOS: 445 and 448; (22) SEQ ID NOS: 445 and 449; (23) SEQ ID NOS: 445 and 450; (24) SEQ ID NOS: 445 and 451; (25) SEQ ID NOS: 445 and 452; (26) SEQ ID NOS: 445 and 453; (27) SEQ ID NOS: 445 and 454; (28) SEQ ID NOS: 445 and 455; (29) SEQ ID NOS: 445 and 459; (30) SEQ ID NOS: 445 and 460; (31) SEQ ID NOS: 445 and 461; (32) SEQ ID NOS: 456 and 458; (33) SEQ ID NOS: 462 and 464; (34) SEQ ID NOS: 465 and 467; (35) SEQ ID NOS: 468 and 470; (36) SEQ ID NOS: 471 and 473; (37) SEQ ID NOS: 474 and 476; (38) SEQ ID NOS: 477 and 479; (39) SEQ ID NOS: 480 and 482; (40) SEQ ID NOS: 483 and 479; (41) SEQ ID NOS: 483 and 486; (42) SEQ ID NOS: 488 and 490; (43) SEQ ID NOS: 491 and 493; (44) SEQ ID NOS: 494 and 486; (45) SEQ ID NOS: 495 and 493; (46) SEQ ID NOS: 496 and 486; (47) SEQ ID NOS: 497 and 486; (48) SEQ ID NOS: 498 and 486; (49) SEQ ID NOS: 500 and 486; (50) SEQ ID NOS: 501 and 486; and (51) SEQ ID NOS: 502 and 493.

A PCR primer set for amplifying a vanE gene comprises at least one of the following sets of primer sequences: (1) SEQ ID NOS: 334 and 336; (2) SEQ ID NOS: 337 and 336; (3) SEQ ID NOS: 338 and 336; (4) SEQ ID NOS: 338 and 381; (5) SEQ ID NOS: 339 and 336; (6) SEQ ID NOS: 340 and 336; (7) SEQ ID NOS: 341 and 336; (8) SEQ ID NOS: 341 and 381; (9) SEQ ID NOS: 342 and 336; (10) SEQ ID NOS: 342 and 381; (11) SEQ ID NOS: 343 and 336; (12) SEQ ID NOS: 344 and 336; (13) SEQ ID NOS: 344 and 381; (14) SEQ ID NOS: 345 and 336; (15) SEQ ID NOS: 346 and 336; (16) SEQ ID NOS: 347 and 336; (17) SEQ ID NOS: 347 and 381; (18) SEQ ID NOS: 348 and 336; (19) SEQ ID NOS: 348 and 381; (20) SEQ ID NOS: 349 and 336; (21) SEQ ID NOS: 349 and 381; (22) SEQ ID NOS: 350 and 336; (23) SEQ ID NOS: 350 and 381; (24) SEQ ID NOS: 351 and 336; (25) SEQ ID NOS: 352 and 336; (26) SEQ ID NOS: 353 and 336; (27) SEQ ID NOS: 354 and 336; (28) SEQ ID NOS: 355 and 336; (29) SEQ ID NOS: 355 and 381; (30) SEQ ID NOS: 356 and 336; (31) SEQ ID NOS: 357 and 336; (32) SEQ ID NOS: 358 and 336; (33) SEQ ID NOS: 359 and 336; (34) SEQ ID NOS: 359 and 381; (35) SEQ ID NOS: 360 and 336; (36) SEQ ID NOS: 360 and 381; (37) SEQ ID NOS: 361 and 336; (38) SEQ ID NOS: 362 and 336; (39) SEQ ID NOS: 363 and 336; (40) SEQ ID NOS: 364 and 336; (41) SEQ ID NOS: 365 and 336; (42) SEQ ID NOS: 366 and 336; (43) SEQ ID NOS: 367 and 336; (44) SEQ ID NOS: 368 and 336; (45) SEQ ID NOS: 369 and 336; (46) SEQ ID NOS: 370 and 336; (47) SEQ ID NOS: 371 and 336; (48) SEQ ID NOS: 372 and 336; (49) SEQ ID NOS: 373 and 336; (50) SEQ ID NOS: 374 and 336; (51) SEQ ID NOS: 375 and 336; (52) SEQ ID NOS: 376 and 336; (53) SEQ ID NOS: 377 and 336; (54) SEQ ID NOS: 378 and 336; (55) SEQ ID NOS: 379 and 336; (56) SEQ ID NOS: 380 and 336; (57) SEQ ID NOS: 382 and 381; (58) SEQ ID NOS: 383 and 381; (59) SEQ ID NOS: 384 and 381; (60) SEQ ID NOS: 385 and 381; (61) SEQ ID NOS: 386 and 381; and (62) SEQ ID NOS: 387 and 381.

A PCR primer set for amplifying a vanG gene comprises at least one of the following sets of primer sequences: (1) SEQ ID NOS: 244 and 246; (2) SEQ ID NOS: 244 and 247; (3) SEQ ID NOS: 244 and 248; (4) SEQ ID NOS: 244 and 250; (5) SEQ ID NOS: 244 and 251; (6) SEQ ID NOS: 244 and 254; (7) SEQ ID NOS: 244 and 258; (8) SEQ ID NOS: 244 and 259; (9) SEQ ID NOS: 244 and 284; (10) SEQ ID NOS: 244 and 286; (11) SEQ ID NOS: 244 and 287; (12) SEQ ID NOS: 249 and 246; (13) SEQ ID NOS: 249 and 248; (14) SEQ ID NOS: 249 and 286; (15) SEQ ID NOS: 249 and 301; (16) SEQ ID NOS: 249 and 306; (17) SEQ ID NOS: 249 and 308; (18) SEQ ID NOS: 249 and 312; (19) SEQ ID NOS: 252 and 246; (20) SEQ ID NOS: 252 and 262; (21) SEQ ID NOS: 253 and 246; (22) SEQ ID NOS: 255 and 246; (23) SEQ ID NOS: 256 and 246; (24) SEQ ID NOS: 260 and 258; (25) SEQ ID NOS: 263 and 258; (26) SEQ ID NOS: 264 and 266; (27) SEQ ID NOS: 267 and 269; (28) SEQ ID NOS: 270 and 266; (29) SEQ ID NOS: 270 and 269; (30) SEQ ID NOS: 271 and 266; (31) SEQ ID NOS: 272 and 274; (32) SEQ ID NOS: 275 and 269; (33) SEQ ID NOS: 276 and 269; (34) SEQ ID NOS: 277 and 269; (35) SEQ ID NOS: 278 and 266; (36) SEQ ID NOS: 279 and 269; (37) SEQ ID NOS: 280 and 266; (38) SEQ ID NOS: 280 and 269; (39) SEQ ID NOS: 281 and 269; (40) SEQ ID NOS: 282 and 266; (41) SEQ ID NOS: 282 and 269; (42) SEQ ID NOS: 283 and 269; (43) SEQ ID NOS: 285 and 259; (44) SEQ ID NOS: 288 and 290; (45) SEQ ID NOS: 288 and 304; (46) SEQ ID NOS: 291 and 290; (47) SEQ ID NOS: 293 and 290; (48) SEQ ID NOS: 293 and 304; (49) SEQ ID NOS: 295 and 258; (50) SEQ ID NOS: 297 and 290; (51) SEQ ID NOS: 298 and 290; (52) SEQ ID NOS: 298 and 304; (53) SEQ ID NOS: 299 and 290; (54) SEQ ID NOS: 300 and 290; (55) SEQ ID NOS: 302 and 274; (56) SEQ ID NOS: 303 and 290; (57) SEQ ID NOS: 303 and 304; (58) SEQ ID NOS: 305 and 274; (59) SEQ ID NOS: 305 and 290; (60) SEQ ID NOS: 307 and 274; (61) SEQ ID NOS: 307 and 290; (62) SEQ ID NOS: 309 and 290; (63) SEQ ID NOS: 310 and 290; (64) SEQ ID NOS: 311 and 258; (65) SEQ ID NOS: 313 and 290; (66) SEQ ID NOS: 314 and 290; (67) SEQ ID NOS: 314 and 320; (68) SEQ ID NOS: 315 and 290; (69) SEQ ID NOS: 316 and 290; (70) SEQ ID NOS: 317 and 290; (71) SEQ ID NOS: 318 and 290; (72) SEQ ID NOS: 321 and 258; (73) SEQ ID NOS: 322 and 324; (74) SEQ ID NOS: 325 and 266; (75) SEQ ID NOS: 325 and 274; (76) SEQ ID NOS: 326 and 274; (77) SEQ ID NOS: 327 and 266; (78) SEQ ID NOS: 328 and 274; (79) SEQ ID NOS: 329 and 274; (80) SEQ ID NOS: 330 and 274; (81) SEQ ID NOS: 331 and 274; (82) SEQ ID NOS: 332 and 266; and (83) SEQ ID NOS: 333 and 266.

The preceding numbering of the sets of primers does not correspond exactly to the “Group” numbering scheme in Table 6 because certain groups use the same primer set, but different internal probes. For example, Groups 213 and 214 of Table 6 each employ the forward primer of SEQ ID NO: 123 and the reverse primer of SEQ ID NO: 125, but different internal probes in each instance, e.g., SEQ ID NOS: 124 and 126. Accordingly, primer set “(1)” of the preceding passage relating to the vanC1 primers implies any one of Groups 213 or 214 of Table 6.

Any set of primers can be used simultaneously in a multiplex reaction with one or more other primer sets, so that multiple amplicons are amplified simultaneously.

A probe for binding to an amplicon(s) of a vanC, vanD, vanE and/or vanG gene, or to a vanC, vanD, vanE and/or vanG gene target, comprises at least one of the following probe sequences: SEQ ID NOS: 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205, 207, 211 (vanC probes); SEQ ID NOS: 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 478, 481, 484, 485, 487, 489, 492, 499 (vanD probes); SEQ ID NOS: 335 (vanE probe) and SEQ ID NOS: 245, 257, 261, 265, 268, 273, 292, 294, 296, 319, 323 (vanG probes).

Any set of primers can be used simultaneously in a multiplex reaction with one or more other primer sets, so that multiple amplicons are amplified simultaneously.

Primer sets for simultaneously amplifying the vanA and/or vanB and/or vanC and/or vanD and/or vanE and/or vanG comprises a nucleotide sequence selected from the primer sets consisting of: Groups 1-601 of Tables 5 and 6. Oligonucleotide probes for binding to the vanA and/or vanB and/or vanC and/or vanD and/or vanE and/or vanG genes comprises a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 7, 9, 11-18, 20, 24, 25, 27, 30, 35, 43, 46, 49, 50, 54, 56, 57, 58 (vanA probes); 63, 65, 66, 74, 77, 85-88, 90, 91, 95, 97-102 (vanB probes); 124, 126, 128, 131, 134, 136, 139, 142, 145, 149, 152, 154, 159, 163, 166, 172, 176, 181, 182, 186, 189, 195, 197, 199, 205, 207, 211 (vanC probes); SEQ ID NOS: 389, 392, 397, 400, 402-408, 410, 412-414, 418, 420, 422-424, 427, 429, 431-433, 436, 441, 446, 457, 463, 466, 469, 472, 478, 481, 484, 485, 487, 489, 492, 499 (vanD probes); SEQ ID NOS: 335 (vanE probe); and SEQ ID NOS: 245, 257, 261, 265, 268, 273, 292, 294, 296, 319, 323 (vanG probes).

The internal control is detected by a forward primer (SEQ ID NO: 504), a reverse primer (SEQ ID NO: 506) and a probe (SEQ ID NO: 505). A plasmid vector containing the internal control target sequence (SEQ ID NO: 503): GCGAAGTGAGAATACGCCGTGTCGCAGTTTCCTTGAGCAGTGTCTCTAAATGCCTCA AACCGTCGCATTTTTGGTTATAGCAGTAACTATATGGAGGTCCGTAGGCGGCGTGCG TGGGGGCACCAAACTCATCCAACGGTCGACTGCGCCTGTAGGGTCTTAAGAAGCGG CACCTCAGACCGATAGCATAGCACTTAAAGAGGAATTGAATAATCAAGATGGGTAT CCGACCGACGCGGAGTGACCGAGGAAGAGGACCCTGCATGTATCCTGAGAGTATAG TTGTCAGAGCAGCAATTGATTCACCACCAAGGGACTTAGTCT is included in the assay. The internal control plasmid is added directly to the reaction mix to monitor the integrity of the PCR reagents and the presence of PCR inhibitors.

TABLE 7

Internal Control.

Group

No.
Forward primer
Probe
Reverse primer

602
SEQ ID NO: 504
SEQ ID NO: 505
SEQ ID NO: 506

CAGACCGATAGCA
TGCTGCTCTGACA
TCCCTTGGTGGTG

TAGCACTTAAA
ACTATACTCTCAG
AATCAAT

GATACA

Example 5

Enterococcus Species-Specific Markers

The vanA and vanB markers, which are carried on a transferable element, are indicative of the presence of VRE. vanA is almost always associated with VRE, while vanB is usually associated with VRE. vanB can also occur in species other than Enterococcus (e.g. Clostridium). In either case, a direct link cannot be made between Enterococcus and the detection of vanA or vanB in a mixed flora population. In some cases, detection of vanA or vanB harboring organisms is followed by an attempt to isolate the vancomycin resistant organism and conclusively identify it as Enterococcus.

Thus, in one respect, a species-specific marker is useful for identifying vancomycin-resistant clinical isolates as Enterococcus faecium (E. faecium or Efm) and/or Enterococcus faecalis (E. faecalis or Efs), which are the two most common Type A and Type B Enterococcus species. These two species are also the most important with regard to VRE.

One embodiment is directed to species-specific markers for the detection of Efm, Efs or both Efm and Efs (“Efm/Efs dual”). Two approaches were utilized within this embodiment. One approach targeted the sodA gene, which encodes the enzyme superoxide dismutase A. The sodA gene is frequently used as a bacterial species-specific marker. A second approach targeted novel genes from Efm, Efs that were identified through in silico analyses. Below are the sodA markers for Efm and Efs, the novel marker for Efm and Efs and a dual marker (dual Efm/Efs dual). The dual marker detects both Efm and Efs. Table 8A-12 describe the nucleic acid primers and probes used for detection and screening of Efm and/or Efs based on the target. Below are the sequences of the sodA for Efm and Efs, novel genes for Efm and Efs, and dual genes for Efm and Efs.

Efm_sodA

(SEQ ID NO: 507)

TAGAAAGATTATTATCTGATATGGACGCTATTCCAACAGATATCAAGACAGCTGTAC

GTAACAATGGTGGCGGACATGCTAACCATTCATTTTTCTGGGAAATCATGGCACCAA

ATGCTGGTGGCGAACCTACAGGAGAAATAAAAGAAGCGATTAATGAAGCTTTTGGT

GATTTTTCTTCTTTTAAAGAAGAATTCAAAAAAGCAGCCGCTGGACGATTTGGTTCT

GGATGGGCTTGGCATGTAATGGAAATTGGAAAATTAGCTATTACCTCTACTGCAAAT

CAAGATTCTCCATT

Efs_sodA

(SEQ ID NO: 508)

TCTGTAGAAAACCTAATTTCAGATATGAATGCTATTCCTGAAGATATCCGCACAGCT

GTTCGTAACAATGGTGGCGGTCACGCAAACCATACATTCTTCTGGGAAATTATGGCA

CCAAATGCTGGTGGACAACCAACTGGCGCTATTAAAGAAGCAATCGATGAAACATT

TGGTAGCTTTGATGAAATGAAAGCTGCTTTCAAAACAGCTGCAACTGGCCGCTTTGG

TTCAGGTTGGGCTTGGTTAGTTGTGAATAACGGTAAATTAGAAATCACTTCAACACC

AA

Efm_novel_marker

(SEQ ID NO: 509)

ACGCGTTCGTTTTCGTTCTCTTCTAGCAAAAATACTCCCCGTATCCCGCTTCAGAGGT

TTCGCCTTCTTATGGATGTCCTTATAGCGATCCATCCGACTCATTGTGGTTCCTCCTC

GTTTCTTACTGATAAATAAAGGATACGATAACCAATCGACAAAAGCCTATCATTTTT

TGATAATTTAATAAAAAATAACGAAATAAATGCTTCGATACATAAAG

Efs_novel_marker

(SEQ ID NO: 510)

CTCGTTCCTTTTAGCAAAAGTAATTGGGACAAGCGTCGCACATCTTCAGGAGCTAAA

TCAATTTTTTTAATCGTATGATACAGCGTTTCTAATTGTTTCACTGTCTCTGGATTAG

GTACTCCGTCAAGTACTCGTACTTGAAAATCATCTAAAATATTTTCACCATTTTCAAT

ATACGCATCTAAAAAAGAAGTATCCAATGACTGTTGCAATAACTGGATTGCTTGTTC

CATTGGCTCGAAAGCCTCTTCAATTTTTTCTGGGAACAAATTAGCACCTCTATTCTAT

TCAAAATGTAACAACTTCCATACCTGTTTTATTTTAACGAAAAAAGAAAGAATAATC

AATGCCTTTACTGTCTTTCTTCTAAAATCTAATAATTTGTAGAAAAACAACGCTATTC

CAGTATTGTTTCCGCTTTTTTTTGATGAGAAACTCTCTCTTGATATTGGTATTTTCGAC

CAGATTTGTTTAATAAAACAGTTATTCTTAATTGTTCATTTTTGTATGTATAATCGAG

GGTCACTTGCGCGTATACGAGCCTTTCTTCTGTTTTTAACTTTCGTCTATTTTGTTTAA

CATCTGCAAGAAATAAATGAAAGATACTTTTCCCTGCATAGTATTCCTGGGTAGCTT

GCGTAAATTTTTGTGTTAACTGACGTTCTTCTAGTAACTGAAGGGCGAGAAAACTCA

TCAAATAAACAATGGCCATGGCCGTGAACAATAAGTTTCCTGAATATTTTTGCCTCA

TTAAAATTCTCCCTTTATCGGAAAAAAACTATCAAATGTTTTTCCAGAAGTAAAAGT

AACTTCCAAAGTAAACGATTGTCCTTCATTTTTAAACTGTCCATTTTTGATACCAATT

AACAGCGGTTGATAGCCTTTACCATTGACTCGCTTAATAATTTTATCTTCTTTGATTT

GAATTGAAATAGTCTTGTTTGTTTCTGAGTCGAGGAAAGAAATCTCTTGAGCAGAGC

CTGTTTGGAAAACGAGCTTTTGACATTCTTTTTCTAATTGAATTAAAAAAATATGCCA

GGATTTCGAATCATTGTTTTTCAAATATTGATTTCCGATAAAACTTTGTTGAATCATC

AACTGAAACAATTGACACATACAACTTAAAACGACTAGTGCTACTAAACACTCTAA

CATCGTAAAACCCGCATATTTTTTATTGACCAATCAAAATCGCTCCTCTTGAATCTGT

GACTTTTAGTATCCTTCTTCCATCGTTCTCCATCAAAGAAAATCGGTAAGTTTCTCCT

TGATATATCCGCTCTTTGAAACGTAAATCACCCGTCTGCTCTAAAAATAAAATTGCT

TCGTATCCTAGACGAGTGCGCGTTAATTCTTGCTCCCGTTGATAATTTTGCTGAATTA

ATTGTGTAATAGTCAGGGAGAATATCCCTGCTATCACACATACAATACTAAAACTAA

CGAGACTCTCTAATAAAATAAAACCGCTATAAATAGTTAATTTTCTTCGTATATTTGC

CACTCCCCATCT

Efm_Efs_dual_novel_marker (from Efm)

(SEQ ID NO: 511)

CGAGCGTTCTCGAGAATACTCGACGCTGAAATTTTTGCACCATTTATTGCTTTATCGT

GACGTGACTTTGCAATAACAAATCCTAAACCAAGACCGACAATCAAACCGACGATA

GCGAGGAGAATATTTAAAACCATATTTCCACCTCCATACTATCTTTTT

Efm_Efs_dual_novel_marker (from Efs)

(SEQ ID NO: 512)

GCTGATGATTGTGCACCAGCTATTTCTTTCTCGTGACGTGACTTTGCGACCATAAACC

CTAAACCAAGACCGACAATTAAACCGATGATAGCGAGGAGAATGTTGAATACCATA

AAATCCACCTCCATACTATCTTTTT

TABLE 8A

Efm sodA gene nucleic acid primers and probes

Primer

SEQ ID NO: 513

GGACATGCTAACCATTCATTT

SEQ ID NO: 514

CGCTGGACGATTTGGTTCTG

SEQ ID NO: 515

AGCGATTAATGAAGCTTTTGGTGA

SEQ ID NO: 516

GGAAATCATGGCACCGAATG

SEQ ID NO: 517

TGGTGGACATGCTAACCATTCA

SEQ ID NO: 518

AGCGATTAATGAAGCTTTTGGTGAT

SEQ ID NO: 519

CAGTAGAGGTAATAGCTAATTTTCC

SEQ ID NO: 520

ATTTCTCCTGTAGGTTCGC

SEQ ID NO: 521

GCCAAGCCCATCCAGA

SEQ ID NO: 522

CCATTACAAGCCAAGCC

SEQ ID NO: 523

AGCTTCATTAATCGCTTCTTTT

SEQ ID NO: 524

GCCAAGCCCATCCAGAAC

SEQ ID NO: 525

GCCAAGCCCAACCAGA

SEQ ID NO: 526

GCTTCATTAATTGCTTCTTTTATTG

SEQ ID NO: 527

GGCAGTAGAGGTAATAGCTAA

SEQ ID NO: 528

GGCAGTAGAGGTAATAGCTAAT

SEQ ID NO: 529

CCAAAAGCTTCATTAATCGCTTCTT

SEQ ID NO: 530

CATCCAGAACCAAATCGTCCAG

SEQ ID NO: 531

TCACCAAAAGCTTCATTAATCGCT

SEQ ID NO: 532

TCTTTTATTTCTCCTGTAGGTTCGC

SEQ ID NO: 533

CATTAATCGCTTCTTTTATTTCTCC

SEQ ID NO: 534

TAATCGCTTCTTTTATTTCTCCTGT

SEQ ID NO: 535

CATTTTCCATTACAAGCCAAGCC

SEQ ID NO: 536

CAAGCCCATCCAGAACCAAATC

SEQ ID NO: 537

GTCCAGCGGCTGCTTTT

SEQ ID NO: 538

CATCCAGAACCAAATCGTCCA

SEQ ID NO: 539

TGCCAAGCCCATCCAGAAC

SEQ ID NO: 540

CAACCAGAACCAAAACGTCCA

SEQ ID NO: 541

TCGCCAAAAGCTTCATTAATTGCTT

SEQ ID NO: 542

AAAATCGCCAAAAGCTTCATT

SEQ ID NO: 543

GCCAAGCCCAACCAGAAC

SEQ ID NO: 544

ATTACAAGCCAAGCCCAACCA

SEQ ID NO: 545

ATTCTCCATTACAAGCCAAGCC

SEQ ID NO: 546

CGTCCAGCTGCTGCTTTT

SEQ ID NO: 547

TAATTGCTTCTTTTATTGCCCCAGT

SEQ ID NO: 548

TTTCCCATTCTCCATTACAAGCCA

SEQ ID NO: 549

AGCTAATTTCCCATTCTCCATTACA

SEQ ID NO: 550

TGGAGAATCTTGATTGGCAGTAGAG

SEQ ID NO: 551

TTGATTGGCAGTAGAGGTAATAGC

SEQ ID NO: 552

GATTGGCAGTAGAGGTAATAGCTAA

SEQ ID NO: 553

TATTACAAGCCAAGCCCATCCAG

Probe

SEQ ID NO: 554

CATGGCACCAAATGCTGGT

SEQ ID NO: 555

CGGTGCCATGATTTCCCAGAAAA

SEQ ID NO: 556

CCAAAACGTCCAGCTGCTGC

SEQ ID NO: 557

ATGGCACCGAATGCGGG

SEQ ID NO: 558

AGAAGCAATTAATGAAGCTTTTGGCGA

SEQ ID NO: 559

ATTGCTTCTTTTATTGCCCCAGTAGG

SEQ ID NO: 560

AGCTAATTTTCCATTTTCCATTACAAGCCAAGCCC

SEQ ID NO: 561

TGGGCTTGGCTTGTAATGGAAAATGGAAAATTAGC

SEQ ID NO: 562

CCAGCATTTGGTGCCATGATTTCCCAGAAAA

SEQ ID NO: 563

TTTTCCATTTTCCATTACAAGCCAAGCCCATCCA

SEQ ID NO: 564

TTTTCCATTACAAGCCAAGCCCATCCAGAACC

SEQ ID NO: 565

ATTACAAGCCAAGCCCATCCAGAACCAAAT

SEQ ID NO: 566

ACCAAATGCTGGTGGCGAACCTACA

SEQ ID NO: 567

ATTTGGTTCTGGATGGGCTTGGCTTGTA

SEQ ID NO: 568

CAACCAGAACCAAAACGTCCAGCTGC

SEQ ID NO: 569

CGCCAAAAGCTTCATTAATTGCTTCTTTTATTGCC

SEQ ID NO: 570

CGCATTCGGTGCCATGATTTCCCAGAA

SEQ ID NO: 571

TTTTCTGGGAAATCATGGCACCGAATGC

SEQ ID NO: 572

TTCTGGGAAATCATGGCACCGAATGCG

SEQ ID NO: 573

CAATAAAAGAAGCAATTAATGAAGCTTTTGGCGAT

SEQ ID NO: 574

AGCTTCATTAATTGCTTCTTTTATTGCCCCAGTAG

TABLE 8B

Efm sodA gene solutions

Group

No.
Forward Primer
Probe
Reverse Primer

603
SEQ ID NO: 517
SEQ ID NO: 571
SEQ ID NO: 529

TGGTGGACATGC
TTTTCTGGGAAATC
CCAAAAGCTTCATT

TAACCATTCA
ATGGCACCGAATGC
AATCGCTTCTT

604
SEQ ID NO: 517
SEQ ID NO: 555
SEQ ID NO: 529

TGGTGGACATG
CGGTGCCATGATTT
CCAAAAGCTTCATT

CTAACCATTCA
CCCAGAAAA
AATCGCTTCTT

605
SEQ ID NO: 517
SEQ ID NO: 562
SEQ ID NO: 529

TGGTGGACATGC
CCAGCATTTGGTG
CCAAAAGCTTCATT

TAACCATTCA
CCATGATTTCCCA
AATCGCTTCTT

GAAAA

TABLE 9A

Efs sodA gene nucleic acid primers and probes

Primer

SEQ ID NO: 575

CTGGCCGCTTTGGTT

SEQ ID NO: 576

ACATTCTTCTGGGAAATTATGG

SEQ ID NO: 577

TGAATGCTATTCCTGAAGATATCCG

SEQ ID NO: 578

AAAGAAGCAATCGATGAAACATTTG

SEQ ID NO: 579

GGGAAATTATGGCACCAAAT

SEQ ID NO: 580

TTCTGGGAAATTATGGCACCAAATG

SEQ ID NO: 581

TGGCGCTATTAAAGAAGCAATCGA

SEQ ID NO: 582

TTCTTCTGGGAAATTATGGCACCAA

SEQ ID NO: 583

GCAATCGATGAAACATTTGGTAGC

SEQ ID NO: 584

CAACTGGCGCTATTAAAGAAGCA

SEQ ID NO: 585

ACAGCTGTTCGTAACAATGGTG

SEQ ID NO: 586

TCAGATATGAATGCTATTCCTGAAG

SEQ ID NO: 587

GGTAGCTTTGATGAAATGAAAGCTG

SEQ ID NO: 588

TGCTGGTGGACAACCAACTG

SEQ ID NO: 589

ACCAAATGCTGGTGGACAAC

SEQ ID NO: 590

GGTCACGCAAACCATACATTCTTC

SEQ ID NO: 591

TGAAAGCTGCTTTCAAAACAGCTG

SEQ ID NO: 592

TTGATGAAATGAAAGCTGCTTTCAA

SEQ ID NO: 593

GAAATGAAAGCTGCTTTCAAAACAG

SEQ ID NO: 594

CTATTCCTGAAGATATCCGTACTGC

SEQ ID NO: 595

AACATTCTTCTGGGAAATTATGGCA

SEQ ID NO: 596

GCAAACCAAACATTCTTCTGGGAAA

SEQ ID NO: 597

CAAACATTCTTCTGGGAAATTATGG

SEQ ID NO: 598

GGTCACGCAAACCAAACATTCT

SEQ ID NO: 599

GATGAAACATTTGGCAGCTTTGATG

SEQ ID NO: 600

ACATTTGGCAGCTTTGATGAAATG

SEQ ID NO: 601

TGGCGGGCACGCAA

SEQ ID NO: 602

CAACCAACTGGCGCTATTAAAGA

SEQ ID NO: 603

GGACAACCAACTGGCGCTA

SEQ ID NO: 604

TGCGACTGGCCGCTTT

SEQ ID NO: 605

TTTCAAAACAGCTGCGACTGG

SEQ ID NO: 606

GTTTCATCGATTGCTTCTTTAATAG

SEQ ID NO: 607

CCAAAGCGGCCAGT

SEQ ID NO: 608

AGAAGAATGTATGGTTTGCG

SEQ ID NO: 609

GCCATAATTTCCCAGAAGAATG

SEQ ID NO: 610

TTCTAATTTACCGTTATTCACAACT

SEQ ID NO: 611

GAAAGCAGCTTTCATTTCATC

SEQ ID NO: 612

GGTGATTTCTAATTTACCGTTATTC

SEQ ID NO: 613

AGCGCCAGTTGGTTG

SEQ ID NO: 614

TTCTTTAATAGCGCCAGTTGGTTG

SEQ ID NO: 615

CCAAATGTTTCATCGATTGCTTCTT

SEQ ID NO: 616

TTTCATCGATTGCTTCTTTAATAGC

SEQ ID NO: 617

TTTGGTGCCATAATTTCCCAGAAGA

SEQ ID NO: 618

GATTGCTTCTTTAATAGCGCCAGTT

SEQ ID NO: 619

TGAACCAAAGCGGCCAGT

SEQ ID NO: 620

CAAAGCGGCCAGTTGCA

SEQ ID NO: 621

AGCGGCCAGTTGCAG

SEQ ID NO: 622

TACCGTTATTCACAACTAACCAAGC

SEQ ID NO: 623

ATTTACCGTTATTCACAACTAACCA

SEQ ID NO: 624

AATTTACCGTTATTCACAACTAACC

SEQ ID NO: 625

CCGTTATTCACAACTAACCAAGCC

SEQ ID NO: 626

AAGCAGCTTTCATTTCATCAAAGC

SEQ ID NO: 627

CCAGTTGGTTGTCCACCAG

SEQ ID NO: 628

ACAACTAACCAAGCCCAACC

SEQ ID NO: 629

CAGTTGCAGCTGTTTTGAAAGCA

SEQ ID NO: 630

TAACCAAGCCCAACCTGAACC

SEQ ID NO: 631

GCTGTTTTGAAAGCAGCTTTCATT

SEQ ID NO: 632

GCCCAACCTGAACCAAAGC

SEQ ID NO: 633

TCAAAGCTACCAAATGTTTCATCGA

SEQ ID NO: 634

ATTTCCCAGAAGAATGTTTGGTTTG

SEQ ID NO: 635

TTTCCCAGAAGAATGTTTGGTTTGC

SEQ ID NO: 636

GCTGCCAAATGTTTCATCGATTG

SEQ ID NO: 637

TGCCAAATGTTTCATCGATTGCTT

SEQ ID NO: 638

TTTCATCAAAGCTGCCAAATGTTTC

SEQ ID NO: 639

TTCATCAAAGCTGCCAAATGTTTCA

SEQ ID NO: 640

CCAGCATTTGGTGCCATAATTTC

Probe

SEQ ID NO: 641

CTTCTTTAATAGCGCCAGTTGGTTG

SEQ ID NO: 642

CGCTATTAAAGAAGCAATCGATGAAACATTTG

SEQ ID NO: 643

CAGGTTGGGCTTGGTTAGTTGT

SEQ ID NO: 644

CTGGGAAATTATGGCACCAAATGCT

SEQ ID NO: 645

AGAAGCAATCGATGAAACATTTGGTAGCTTTGATG

SEQ ID NO: 646

CCAACTGGCGCTATTAAAGAAGCAATCGATGAAAC

SEQ ID NO: 647

AATGTTTCATCGATTGCTTCTTTAATAGCGCCAGT

SEQ ID NO: 648

ATCGATGAAACATTTGGTAGCTTTGATGAAATGAA

SEQ ID NO: 649

ATCGATTGCTTCTTTAATAGCGCCAGTTGGTTG

SEQ ID NO: 650

TGGCCGCTTTGGTTCAGGTTGGG

SEQ ID NO: 651

TGCCATAATTTCCCAGAAGAATGTATGGTTTGCG

SEQ ID NO: 652

CAGCATTTGGTGCCATAATTTCCCAGAAGAATGT

SEQ ID NO: 653

CCAACCTGAACCAAAGCGGCCAG

SEQ ID NO: 654

CAGCTGTTCGTAACAATGGTGGCGG

SEQ ID NO: 655

TTCTTTAATAGCGCCAGTTGGTTGTCCACCAG

SEQ ID NO: 656

TAACCAAGCCCAACCTGAACCAAAGCG

SEQ ID NO: 657

TGGTGGACAACCAACTGGCGCTATTAAAGAAG

SEQ ID NO: 658

AGCAGCTTTCATTTCATCAAAGCTACCAAATGTTT

SEQ ID NO: 659

CAAATGCTGGTGGACAACCAACTGGC

SEQ ID NO: 660

AAACATTTGGTAGCTTTGATGAAATGAAAGCTGCT

SEQ ID NO: 661

CGTGACCGCCACCATTGTTACGAACA

SEQ ID NO: 662

CGCCAGTTGGTTGTCCACCAGC

SEQ ID NO: 663

CTGCTTTCAAAACAGCTGCAACTGGCC

SEQ ID NO: 664

AGAAGAATGTATGGTTTGCGTGACCGCC

SEQ ID NO: 665

AAAGCGGCCAGTTGCAGCTGT

SEQ ID NO: 666

AACCAAACATTCTTCTGGGAAATTATGGCACCAAA

SEQ ID NO: 667

CGCCACCATTGTTACGAACGGCTGT

SEQ ID NO: 668

CACGCAAACCATACATTCTTCTGGGAAATTATGGC

SEQ ID NO: 669

TTCATTTCATCAAAGCTGCCAAATGTTTCATCGAT

SEQ ID NO: 670

CGCTATTAAAGAAGCAATCGATGAAACATTTGGCA

SEQ ID NO: 671

AAGCTGCCAAATGTTTCATCGATTGCTTCTTTAAT

SEQ ID NO: 672

TCATCAAAGCTGCCAAATGTTTCATCGATTGCTTC

SEQ ID NO: 673

CCAGTTGCAGCTGTTTTGAAAGCAGC

SEQ ID NO: 674

CATTCTTCTGGGAAATTATGGCACCAAATGCTGG

SEQ ID NO: 675

TGGGAAATTATGGCACCAAATGCTGGCG

SEQ ID NO: 676

CTGCGACTGGCCGCTTTGGTTCA

SEQ ID NO: 677

TGAACCAAAGCGGCCAGTCGCAG

TABLE 9B

Efs sodA gene solutions

Group

No.
Forward
Probe
Reverse

606
SEQ ID NO: 577
SEQ ID NO: 661
SEQ ID NO: 640

TGAATGCTATTCCTGAAGATATCCG
CGTGACCGCCACCATTGTTACGAACA
CCAGCATTTGGTGCCATAATTTC

607
SEQ ID NO: 577
SEQ ID NO: 654
SEQ ID NO: 640

TGAATGCTATTCCTGAAGATATCCG
CAGCTGTTCGTAACAATGGTGGCGG
CCAGCATTTGGTGCCATAATTTC

608
SEQ ID NO: 600
SEQ ID NO: 650
SEQ ID NO: 623

ACATTTGGCAGCTTTGATGAAATG
TGGCCGCTTTGGTTCAGGTTGGG
ATTTACCGTTATTCACAACTAACCA

609
SEQ ID NO: 590
SEQ ID NO: 675
SEQ ID NO: 637

GGTCACGCAAACCATACATTCTTC
TGGGAAATTATGGCACCAAATGCTGGCG
TGCCAAATGTTTCATCGATTGCTT

610
SEQ ID NO: 598
SEQ ID NO: 644
SEQ ID NO: 637

GGTCACGCAAACCAAACATTCT
CTGGGAAATTATGGCACCAAATGCT
TGCCAAATGTTTCATCGATTGCTT

611
SEQ ID NO: 577
SEQ ID NO: 664
SEQ ID NO: 640

TGAATGCTATTCCTGAAGATATCCG
AGAAGAATGTATGGTTTGCGTGACCGCC
CCAGCATTTGGTGCCATAATTTC

612
SEQ ID NO: 577
SEQ ID NO: 667
SEQ ID NO: 640

TGAATGCTATTCCTGAAGATATCCG
CGCCACCATTGTTACGAACGGCTGT
CCAGCATTTGGTGCCATAATTTC

613
SEQ ID NO: 590
SEQ ID NO: 659
SEQ ID NO: 637

GGTCACGCAAACCATACATTCTTC
CAAATGCTGGTGGACAACCAACTGGC
TGCCAAATGTTTCATCGATTGCTT

614
SEQ ID NO: 590
SEQ ID NO: 662
SEQ ID NO: 637

GGTCACGCAAACCATACATTCTTC
CGCCAGTTGGTTGTCCACCAGC
TGCCAAATGTTTCATCGATTGCTT

615
SEQ ID NO: 600
SEQ ID NO: 676
SEQ ID NO: 624

ACATTTGGCAGCTTTGATGAAATG
CTGCGACTGGCCGCTTTGGTTCA
AATTTACCGTTATTCACAACTAACC

616
SEQ ID NO: 599
SEQ ID NO: 673
SEQ ID NO: 625

GATGAAACATTTGGCAGCTTTGATG
CCAGTTGCAGCTGTTTTGAAAGCAGC
CCGTTATTCACAACTAACCAAGCC

617
SEQ ID NO: 600
SEQ ID NO: 673
SEQ ID NO: 624

ACATTTGGCAGCTTTGATGAAATG
CCAGTTGCAGCTGTTTTGAAAGCAGC
AATTTACCGTTATTCACAACTAACC

618
SEQ ID NO: 600
SEQ ID NO: 677
SEQ ID NO: 624

ACATTTGGCAGCTTTGATGAAATG
TGAACCAAAGCGGCCAGTCGCAG
AATTTACCGTTATTCACAACTAACC

619
SEQ ID NO: 599
SEQ ID NO: 676
SEQ ID NO: 625

GATGAAACATTTGGCAGCTTTGATG
CTGCGACTGGCCGCTTTGGTTCA
CCGTTATTCACAACTAACCAAGCC

620
SEQ ID NO: 599
SEQ ID NO: 677
SEQ ID NO: 625

GATGAAACATTTGGCAGCTTTGATG
TGAACCAAAGCGGCCAGTCGCAG
CCGTTATTCACAACTAACCAAGCC

621
SEQ ID NO: 586
SEQ ID NO: 667
SEQ ID NO: 617

TCAGATATGAATGCTATTCCTGAAG
CGCCACCATTGTTACGAACGGCTGT
TTTGGTGCCATAATTTCCCAGAAGA

622
SEQ ID NO: 599
SEQ ID NO: 665
SEQ ID NO: 625

GATGAAACATTTGGCAGCTTTGATG
AAAGCGGCCAGTTGCAGCTGT
CCGTTATTCACAACTAACCAAGCC

623
SEQ ID NO: 599
SEQ ID NO: 663
SEQ ID NO: 625

GATGAAACATTTGGCAGCTTTGATG
CTGCTTTCAAAACAGCTGCAACTGGCC
CCGTTATTCACAACTAACCAAGCC

624
SEQ ID NO: 600
SEQ ID NO: 665
SEQ ID NO: 624

ACATTTGGCAGCTTTGATGAAATG
AAAGCGGCCAGTTGCAGCTGT
AATTTACCGTTATTCACAACTAACC

625
SEQ ID NO: 586
SEQ ID NO: 661
SEQ ID NO: 617

TCAGATATGAATGCTATTCCTGAAG
CGTGACCGCCACCATTGTTACGAACA
TTTGGTGCCATAATTTCCCAGAAGA

626
SEQ ID NO: 586
SEQ ID NO: 654
SEQ ID NO: 617

TCAGATATGAATGCTATTCCTGAAG
CAGCTGTTCGTAACAATGGTGGCGG
TTTGGTGCCATAATTTCCCAGAAGA

627
SEQ ID NO: 600
SEQ ID NO: 663
SEQ ID NO: 624

ACATTTGGCAGCTTTGATGAAATG
CTGCTTTCAAAACAGCTGCAACTGGCC
AATTTACCGTTATTCACAACTAACC

TABLE 10A

Efm novel gene nucleic acid primers and probes

Primer

SEQ ID NO: 678

CCTCCTCGTTTCTTACTGAT

SEQ ID NO: 679

GGTTCCTCCTCGTTTCTT

SEQ ID NO: 680

GTTTTCGTTCTCTTCTAGCAAAA

SEQ ID NO: 681

CGATCCATCCGACTCATTG

SEQ ID NO: 682

CGATCCATCCGACTCATT

SEQ ID NO: 683

TTTCGCCTTCTTATGGATGTCCTTA

SEQ ID NO: 684

GATCCATCCGACTCATTGTGGT

SEQ ID NO: 685

TTATAGCGATCCATCCGACTCATTG

SEQ ID NO: 686

TTATAGCGATCCATCCGACTCATT

SEQ ID NO: 687

TCTTATGGATGTCCTTATAGCGATC

SEQ ID NO: 688

TGGATGTCCTTATAGCGATCCATC

SEQ ID NO: 689

TTGTGGTTCCTCCTCGTTTCTTAC

SEQ ID NO: 690

TCATTGTGGTTCCTCCTCGTTTC

SEQ ID NO: 691

TCCGACTCATTGTGGTTCCTC

SEQ ID NO: 692

TTCAGAGGTTTCGCCTTCTTATGG

SEQ ID NO: 693

GCTTCAGAGGTTTCGCCTTCTTA

SEQ ID NO: 694

GCTTCAGAGGTTTCGCCTTCTT

SEQ ID NO: 695

GTATCCCGCTTCAGAGGTTTC

SEQ ID NO: 696

TCCCGCTTCAGAGGTTTCG

SEQ ID NO: 697

CGTATCCCGCTTCAGAGG

SEQ ID NO: 698

GGTTTCGCCTTCTTATGGATGTC

SEQ ID NO: 699

TACTCCCCGTATCCCGCTTCA

SEQ ID NO: 700

CCCGTATCCCGCTTCAGA

SEQ ID NO: 701

AAGAAACGAGGAGGAACC

SEQ ID NO: 702

CGCTATAAGGACATCCATAAGA

SEQ ID NO: 703

CGAGGAGGAACCACAATG

SEQ ID NO: 704

CTTTTGTCGATTGGTTATCGTA

SEQ ID NO: 705

CTTTTGTCGATTGGTTATCGTAT

SEQ ID NO: 706

TATCAGTAAGAAACGAGGAGGAACC

SEQ ID NO: 707

ATGATAGGCTTTTGTCGATTGGTTA

SEQ ID NO: 708

CACAATGAGTCGGATGGATCG

SEQ ID NO: 709

ACCACAATGAGTCGGATGGA

SEQ ID NO: 710

CAATGAGTCGGATGGATCGCTAT

SEQ ID NO: 711

CAATGAGTCGGATGGATCGCTA

SEQ ID NO: 712

GTCGGATGGATCGCTATAAGG

SEQ ID NO: 713

ATTTATCAGTAAGAAACGAGGAGGA

SEQ ID NO: 714

GGATGGATCGCTATAAGGACATCC

SEQ ID NO: 715

TAAGAAACGAGGAGGAACCACAAT

SEQ ID NO: 716

GAGGAGGAACCACAATGAGTCG

SEQ ID NO: 717

ATAAGGACATCCATAAGAAGGCGAA

SEQ ID NO: 718

ATCGCTATAAGGACATCCATAAGAA

SEQ ID NO: 719

TGTATCGAAGCATTTATTTCGTTAT

SEQ ID NO: 720

GGCTTTTGTCGATTGGTTATCGTAT

SEQ ID NO: 721

GGCTTTTGTCGATTGGTTATCGTA

SEQ ID NO: 722

TTTGTCGATTGGTTATCGTATCCTT

SEQ ID NO: 723

TGTCGATTGGTTATCGTATCCTTTA

SEQ ID NO: 724

CGAGGAGGAACCACAATGAG

SEQ ID NO: 725

AAATGATAAGCTTTTGTCGATTGGT

Probe

SEQ ID NO: 726

CGGATGGATCGCTATAAGGACATCCAT

SEQ ID NO: 727

AGGATACGATAACCAATCGACAAAAGC

SEQ ID NO: 728

CGATCCATCCGACTCATTGTGGT

SEQ ID NO: 729

ACTGATAAATAAAGGATACGATAACCAATCGACAA

SEQ ID NO: 730

CCCGTATCCCGCTTCAGAGG

SEQ ID NO: 731

AGGCGAAACCTCTGAAGCG

SEQ ID NO: 732

TTCTTATGGATGTCCTTATAGCGATCCATCCGACT

SEQ ID NO: 733

TGGATGTCCTTATAGCGATCCATCCGACTCATTG

SEQ ID NO: 734

TGGTTCCTCCTCGTTTCTTACTGATAAATAAAGGA

SEQ ID NO: 735

TTCGCCTTCTTATGGATGTCCTTATAGCGATCCA

SEQ ID NO: 736

AATGATAGGCTTTTGTCGATTGGTTATCGTATCCT

SEQ ID NO: 737

CAATGAGTCGGATGGATCGCTATAAGGACATCCAT

SEQ ID NO: 738

CCTTTATTTATCAGTAAGAAACGAGGAGGAACCAC

SEQ ID NO: 739

TTATAGCGATCCATCCGACTCATTGTGGTTCCTC

SEQ ID NO: 740

TCAGTAAGAAACGAGGAGGAACCACAATGAGTCG

SEQ ID NO: 741

TCGCTATAAGGACATCCATAAGAAGGCGAAACCTC

SEQ ID NO: 742

CTCCTCGTTTCTTACTGATAAATAAAGGATACGAT

SEQ ID NO: 743

TCGTATCCTTTATTTATCAGTAAGAAACGAGGAGG

SEQ ID NO: 744

AAATAAAGGATACGATAACCAATCGACAAAAGCCT

SEQ ID NO: 745

ATAAATAAAGGATACGATAACCAATCGACAAAAGC

SEQ ID NO: 746

CTTCAGAGGTTTCGCCTTCTTATGGATGTCCTTAT

SEQ ID NO: 747

TAAGGACATCCATAAGAAGGCGAAACCTCTGAAGC

SEQ ID NO: 748

TTGTCGATTGGTTATCGTATCCTTTATTTATCAGT

SEQ ID NO: 749

CATCCATAAGAAGGCGAAACCTCTGAAGCG

SEQ ID NO: 750

CGAGGAGGAACCACAATGAGTCGGATG

SEQ ID NO: 751

CGACTCATTGTGGTTCCTCCTCGTTTCTTACTG

SEQ ID NO: 752

AGGATACGATAACCAATCGACAAAAGCTTATCATT

SEQ ID NO: 753

AAGCTTTTGTCGATTGGTTATCGTATCCTTTATTT

TABLE 10B

Efm novel gene solutions

Group

No.
Forward
Probe
Reverse

628
SEQ ID NO: 687
SEQ ID NO: 750
SEQ ID NO: 723

TCTTATGGATGTCCTTATAGCGATC
CGAGGAGGAACCACAATGAGTCGGATG
TGTCGATTGGTTATCGTATCCTTTA

629
SEQ ID NO: 687
SEQ ID NO: 750
SEQ ID NO: 720

TCTTATGGATGTCCTTATAGCGATC
CGAGGAGGAACCACAATGAGTCGGATG
GGCTTTTGTCGATTGGTTATCGTAT

630
SEQ ID NO: 683
SEQ ID NO: 728
SEQ ID NO: 723

TTTCGCCTTCTTATGGATGTCCTTA
CGATCCATCCGACTCATTGTGGT
TGTCGATTGGTTATCGTATCCTTTA

631
SEQ ID NO: 683
SEQ ID NO: 750
SEQ ID NO: 723

TTTCGCCTTCTTATGGATGTCCTTA
CGAGGAGGAACCACAATGAGTCGGATG
TGTCGATTGGTTATCGTATCCTTTA

632
SEQ ID NO: 687
SEQ ID NO: 750
SEQ ID NO: 707

TCTTATGGATGTCCTTATAGCGATC
CGAGGAGGAACCACAATGAGTCGGATG
ATGATAGGCTTTTGTCGATTGGTTA

633
SEQ ID NO: 683
SEQ ID NO: 728
SEQ ID NO: 720

TTTCGCCTTCTTATGGATGTCCTTA
CGATCCATCCGACTCATTGTGGT
GGCTTTTGTCGATTGGTTATCGTAT

634
SEQ ID NO: 683
SEQ ID NO: 750
SEQ ID NO: 720

TTTCGCCTTCTTATGGATGTCCTTA
CGAGGAGGAACCACAATGAGTCGGATG
GGCTTTTGTCGATTGGTTATCGTAT

635
SEQ ID NO: 692
SEQ ID NO: 728
SEQ ID NO: 723

TTCAGAGGTTTCGCCTTCTTATGG
CGATCCATCCGACTCATTGTGGT
TGTCGATTGGTTATCGTATCCTTTA

636
SEQ ID NO: 692
SEQ ID NO: 750
SEQ ID NO: 723

TTCAGAGGTTTCGCCTTCTTATGG
CGAGGAGGAACCACAATGAGTCGGATG
TGTCGATTGGTTATCGTATCCTTTA

TABLE 11A

Efs novel gene nucleic acid primers and probes

Primer

SEQ ID NO: 754

CATCTTCAGGAGCTAAATCAAT

SEQ ID NO: 755

AAGTACTCGTACTTGAAAATCATCT

SEQ ID NO: 756

GTCGCACATCTTCAGGAGCTAAA

SEQ ID NO: 757

CTAATTGTTTCACTGTCTCTGGATT

SEQ ID NO: 758

AAGAAGTATCCAATGACTGTTGCAA

SEQ ID NO: 759

TCCAATGACTGTTGCAATAACTGGA

SEQ ID NO: 760

TTCACTGTCTCTGGATTAGGTACTC

SEQ ID NO: 761

AATTGTTTCACTGTCTCTGGATTAG

SEQ ID NO: 762

TCACCATTTTCAATATACGCATCTA

SEQ ID NO: 763

TTCACCATTTTCAATATACGCATCT

SEQ ID NO: 764

CGTATGATACAGCGTTTCTAATTGT

SEQ ID NO: 765

AGCGTTTCTAATTGTTTCACTGTCT

SEQ ID NO: 766

CAGCGTTTCTAATTGTTTCACTGTC

SEQ ID NO: 767

TTAGGTACTCCGTCAAGTACTCGT

SEQ ID NO: 768

GACAAGCGTCGCACATCTTC

SEQ ID NO: 769

AAAAGAAGTATCCAATGACTGTTGC

SEQ ID NO: 770

TTTTAATCGTATGATACAGCGTTTC

SEQ ID NO: 771

GGAACAAATTAGCACCTCTATTCTA

SEQ ID NO: 772

GATTGCTTGTTCCATTGGCT

SEQ ID NO: 773

TGTTGCAATAACTGGATTGCTTGTT

SEQ ID NO: 774

TCTGGATTAGGTACTCCGTCAAGT

SEQ ID NO: 775

TAACTGGATTGCTTGTTCCATTGG

SEQ ID NO: 776

TAATTGGGACAAGCGTCGCA

SEQ ID NO: 777

TTTCTGGGAACAAATTAGCACCTCT

SEQ ID NO: 778

TCCGTCAAGTACTCGTACTTGAAAA

SEQ ID NO: 779

ATTGTTTCACTGTCTCTGGATTAGG

SEQ ID NO: 780

GCTTTCGAGCCAATGGAACA

SEQ ID NO: 781

CGGAGTACCTAATCCAGAG

SEQ ID NO: 782

TTCGTTAAAATAAAACAGGTATGGA

SEQ ID NO: 783

AGTAAAGGCATTGATTATTCTTTCT

SEQ ID NO: 784

ACAGTAAAGGCATTGATTATTCTTT

SEQ ID NO: 785

ATGGAAGTTGTTACATTTTGAATAG

SEQ ID NO: 786

TTGCAACAGTCATTGGATACTTCTT

SEQ ID NO: 787

TTAGATGATTTTCAAGTACGAGTAC

SEQ ID NO: 788

GAGCCAATGGAACAAGCAATCCA

SEQ ID NO: 789

GAGGTGCTAATTTGTTCCCAGAAAA

SEQ ID NO: 790

GTACGAGTACTTGACGGAGTACCTA

SEQ ID NO: 791

AAAATAAAACAGGTATGGAAGTTG

SEQ ID NO: 792

TCCAGTTATTGCAACAGTCATTGGA

SEQ ID NO: 793

AGAAGAAAGACAGTAAAGGCATTGA

SEQ ID NO: 794

TAAAACAGGTATGGAAGTTGTTACA

SEQ ID NO: 795

TTTCGAGCCAATGGAACAAGCA

SEQ ID NO: 796

TGGAACAAGCAATCCAGTTATTGCA

SEQ ID NO: 797

TGAATAGAATAGAGGTGCTAATTTG

SEQ ID NO: 798

GAGCCAATGGAACAAGCAAT

SEQ ID NO: 799

AACAGGTATGGAAGTTGTTACATTT

SEQ ID NO: 800

CAAGCAATCCAGTTATTGCAACAG

SEQ ID NO: 801

AGACAGTAAAGGCATTGATTATTCT

SEQ ID NO: 802

GCAACAGTCATTGGATACTTCTTTT

SEQ ID NO: 803

TAGAGGTGCTAATTTGTTCCCAGAA

SEQ ID NO: 804

AAAGGCATTGATTATTCTTTCTTTT

SEQ ID NO: 805

GAATAGAATAGAGGTGCTAATTTGT

SEQ ID NO: 806

ATAGAATAGAGGTGCTAATTTGTTC

SEQ ID NO: 807

GTACTTGACGGAGTACCTAATCCAG

SEQ ID NO: 808

TTTTCAAGTACGAGTACTTGACGGA

SEQ ID NO: 809

AGATGCGTATATTGAAAATGGTGAA

Probe

SEQ ID NO: 810

CCAGTTATTGCAACAGTCATTGGATACTTC

SEQ ID NO: 811

CGTTAAAATAAAACAGGTATGGAAGTTGTTACATT

SEQ ID NO: 812

CCGTCAAGTACTCGTACTTGAAAATCATCTAAAAT

SEQ ID NO: 813

ATGTAACAACTTCCATACCTGTTTTATTTTAACGA

SEQ ID NO: 814

ACATTTTGAATAGAATAGAGGTGCTAATTTGTTCC

SEQ ID NO: 815

AATAGAGGTGCTAATTTGTTCCCAGAAAA

SEQ ID NO: 816

CCAGAGACAGTGAAACAATTAGAAACGCTGTATCA

SEQ ID NO: 817

CGTATGATACAGCGTTTCTAATTGTTTCACTGTCT

SEQ ID NO: 818

AGAAGTATCCAATGACTGTTGCAATAACTGGATTG

SEQ ID NO: 819

ACAGTGAAACAATTAGAAACGCTGTATCATACGAT

SEQ ID NO: 820

CTGTCTCTGGATTAGGTACTCCGTCAAGTACTCGT

SEQ ID NO: 821

CTGGATTAGGTACTCCGTCAAGTACTCGTACTTGA

SEQ ID NO: 822

AGGTACTCCGTCAAGTACTCGTACTTGAAAATCAT

SEQ ID NO: 823

AGCGTTTCTAATTGTTTCACTGTCTCTGGATTAGG

SEQ ID NO: 824

ATCCAATGACTGTTGCAATAACTGGATTGCTTGTT

SEQ ID NO: 825

AAACAGGTATGGAAGTTGTTACATTTTGAATAGAA

SEQ ID NO: 826

AATGGAACAAGCAATCCAGTTATTGCAACAGTCAT

SEQ ID NO: 827

CTTTCGAGCCAATGGAACAAGCAATCCAGTTATT

SEQ ID NO: 828

ACCTAATCCAGAGACAGTGAAACAATTAGAAACGC

SEQ ID NO: 829

AGCACCTCTATTCTATTCAAAATGTAACAACTTCC

SEQ ID NO: 830

ATTGTTTCACTGTCTCTGGATTAGGTACTCCGTCA

SEQ ID NO: 831

TGTTGCAATAACTGGATTGCTTGTTCCATTGGCTC

SEQ ID NO: 832

TAACTGGATTGCTTGTTCCATTGGCTCGAAAGC

SEQ ID NO: 833

ATTCTATTCAAAATGTAACAACTTCCATACCTGTT

SEQ ID NO: 834

ATGATTTTCAAGTACGAGTACTTGACGGAGTACCT

SEQ ID NO: 835

CTGGGAACAAATTAGCACCTCTATTCTATTCAAAA

SEQ ID NO: 836

CAAGTACGAGTACTTGACGGAGTACCTAATCCAGA

SEQ ID NO: 837

TGACGGAGTACCTAATCCAGAGACAGTGAAACAAT

SEQ ID NO: 838

TGGAAGTTGTTACATTTTGAATAGAATAGAGGTGC

SEQ ID NO: 839

TGAATAGAATAGAGGTGCTAATTTGTTCCCAGAAA

SEQ ID NO: 840

CGAGTACTTGACGGAGTACCTAATCCAGAGACAG

SEQ ID NO: 841

ATTTTAGATGATTTTCAAGTACGAGTACTTGACGG

SEQ ID NO: 842

AGCAATCCAGTTATTGCAACAGTCATTGGATACTT

TABLE 11B

Efs novel gene solutions

Group

No.
Forward
Probe
Reverse

637
SEQ ID NO: 758
SEQ ID NO: 832
SEQ ID NO: 803

AAGAAGTATCCAATGACTGTTGCAA
TAACTGGATTGCTTGTTCCATTGGCTCGAAAGC
TAGAGGTGCTAATTTGTTCCCAGAA

638
SEQ ID NO: 775
SEQ ID NO: 815
SEQ ID NO: 785

TAACTGGATTGCTTGTTCCATTGG
AATAGAGGTGCTAATTTGTTCCCAGAAAA
ATGGAAGTTGTTACATTTTGAATAG

639
SEQ ID NO: 772
SEQ ID NO: 815
SEQ ID NO: 799

GATTGCTTGTTCCATTGGCT
AATAGAGGTGCTAATTTGTTCCCAGAAAA
AACAGGTATGGAAGTTGTTACATTT

640
SEQ ID NO: 758
SEQ ID NO: 832
SEQ ID NO: 797

AAGAAGTATCCAATGACTGTTGCAA
TAACTGGATTGCTTGTTCCATTGGCTCGAAAGC
TGAATAGAATAGAGGTGCTAATTTG

641
SEQ ID NO: 775
SEQ ID NO: 815
SEQ ID NO: 799

TAACTGGATTGCTTGTTCCATTGG
AATAGAGGTGCTAATTTGTTCCCAGAAAA
AACAGGTATGGAAGTTGTTACATTT

642
SEQ ID NO: 772
SEQ ID NO: 815
SEQ ID NO: 791

GATTGCTTGTTCCATTGGCT
AATAGAGGTGCTAATTTGTTCCCAGAAAA
AAAATAAAACAGGTATGGAAGTTG

643
SEQ ID NO: 773
SEQ ID NO: 815
SEQ ID NO: 785

TGTTGCAATAACTGGATTGCTTGTT
AATAGAGGTGCTAATTTGTTCCCAGAAAA
ATGGAAGTTGTTACATTTTGAATAG

TABLE 12

Efm/Efs dual nucleic acid primers and probes

Group

No.
Forward
Probe
Reverse

644
SEQ ID NO: 843
SEQ ID NO: 844
SEQ ID NO: 845

TTGCTTTATCGT
CATTCTCCTCGCT
GATAGTATGGAGGT

GACGTGACTTTG
ATCATCGGTTTAA
GGAAATATGGT

TTGTCGG
SEQ ID NO: 846

TATGGAGGTGGAT

TTTATGGTATTC

Example 6
Testing vanA and vanB Primer and Probe Sequences for their Ability to Amplify their Intended Target Sequences

The oligonucleotide sequences listed in Table 5 were tested for their ability to amplify their intended target sequences. About 25 μA PCRs were formulated using iQTM Supermix for qPCR (BioRad) and oligos at a final concentration of 400 nM each.

Genomic DNA isolated from E. faecium (ATCC No. 5159, strain MMC4, vanA) and E. faecalis (ATCC No. 700802, strain V583, vanB) were loaded into real-time PCRs using oligonucleotide solutions specific for vanA and vanB. The specificity of the oligonucleotide solutions was assessed by attempting to amplify E. faecalis (vanB) gDNA with vanA oligos and E. faecium (vanA) gDNA with vanB oligos. Amplification plots are illustrated in FIGS. 1 and 2, which show detection of vanA and vanB. Synthetic constructs encoding the vanA and vanB targets were amplified and compared to duplicate reactions where E. faecium (ATCC No. 5159, strain MMC4, vanA) and E. faecalis (ATCC No. 700802, strain V583, vanB) gDNA was amplified.

PCRs were run on the ABI 7500 with the following Thermal protocol:

- 95° C. 5 minute, initial denaturation
- 50 cycles of:
  - 95° C. 15 sec, denaturation, and
  - 60° C. 1 min, annealing/extension

In FIG. 1, the vanA solution (SEQ ID NO: 1, 2, and 5) included amplification of E. faecium gDNA (*) and the E. faecium vanA synthetic construct (§), while the vanB solution (SEQ ID NOS: 103, 108, and 66) in FIG. 2 included amplification of the E. faecalis vanB synthetic construct (¥) and E. faecalis gDNA (+). PCR products were not evident in any of the No template controls (NTC). Ct values corresponding to the presence of PCR product are shown in Table 13 below.

TABLE 13

Ct values showing presence of vanA and vanB.

Oligo solution

Input
vanA Ct
vanB Ct

Synthetic construct
23.36
24.04

NTC
UND
UND

E. faecalis (ATCC No. 700802, strain V583, vanB)
UND
21.85

UND
21.53

E. faecium (ATCC No. 5159, strain MMC4 vanA)
19.67
UND

19.80
UND

Example 7
Gel Electrophoresis Analysis for vanA and vanB

A gel electrophoresis analysis was performed using 20 μL aliquots of post-amplification vanA and vanB PCR products on an agarose gel plate. The gel electrophoresis of FIG. 3 illustrates that the molecular weight marker was a 25 bp ladder (Invitrogen); gel, 4% agarose e-gel (Invitrogen), EtBr stained; inputs are shown in the table of FIG. 3. The arrows on the gel point to the 100 bp and 150 bp markers. The gel illustrates that the vanA and vanB PCR products migrate according to their predicted sizes.

Example 8
Testing sodA and Efm/EFs Dual Sequences for Ability to Amplify their Intended Target Sequences

The nucleic acid primers and probes listed in Tables 8B, 9B and 12 were tested for their ability to amplify their intended target sequences. About 25 μL PCRs were formulated using iQTM Supermix for qPCR (BioRad) and oligonucleotides at a final concentration of 400 nM each.

In this example, 1×10⁴copies of genomic DNA from E. faecium (ATCC No. 51559), E. faecalis (ATCC. No 700802) and C. difficile (ATCC No. 43598) were loaded into real-time PCRs using oligonucleotide solutions directed against E. faecium sodA, E. faecalis sodA novel markers for both E. faecalis and E. faecium (Efm/Efs dual). PCRs were run on the ABI 7500 with the following Thermal protocol:

- 95° C. 5 minute, initial denaturation
- 50 cycles of:
  - 95° C. 15 sec, denaturation
  - 60° C. 1 min, annealing/extension

In FIG. 4, amplification plots show detection of E. faecium and not E. faecalis or C. difficile using the E. faecium sodA oligonucleotide solution (♦—SEQ ID NO: 517, 571, and 529); detection of E. faecalis and not E. faecium or C. difficile using the E. faecalis sodA oligonulceotide solution (X—SEQ ID NO: 599, 663, and 625); and detection of both E. faecium and E. faecalis but not C. difficile using the Efm/Efs dual oligonucleotide solution (·—SEQ ID NO: 843, 844, 845 and 846). Ct values corresponding to the presence of PCR product are shown in Table 14 below. PCR products were not evident in any of the No template controls (NTC).

TABLE 14

Ct values showing presence of sodA for E. faecium,

E. faecalis, and Efs/Efm dual.

Target Input
Oligos
Ct

E. faecium

E. faecium

31.45

E. faecalis

UND

C. difficile

UND

NTC

UND

E. faecium

E. faecalis

UND

E. faecalis

36.52

C. difficile

UND

NTC

UND

E. faecium

Dual
32.45

E. faecalis

34.93

C. difficile

UND

NTC

UND

Example 9
Gel Electrophoresis Analysis for Soda of E. faecium, E. faecalis, and Efm/Efs Dual

A gel electrophoresis analysis was performed using 20 μL aliquots of post-amplification PCR sodA and dual Efs/Efm products on an agarose gel plate. The gel electrophoresis was shown in FIG. 5 where molecular weight markers were the 100 bp ladder and 50 bp ladder (Invitrogen); gel, 4% agarose e-gel (Invitrogen), EtBr stained; inputs as shown in the table of FIG. 5. The arrow on the gel of FIG. 5 points to the 100 bp marker. The gel illustrates that the PCR products migrate according to their predicted sizes.

TABLE 15

Additional Sequences

SEQ ID NO: 113

0000

SEQ ID NO: 114

0000

SEQ ID NO: 115

0000

SEQ ID NO: 116

0000

SEQ ID NO: 117

0000

SEQ ID NO: 118

0000

SEQ ID NO: 119

0000

SEQ ID NO: 120

0000

SEQ ID NO: 121

0000

SEQ ID NO: 122

0000

Other Embodiments

Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims. The contents of all references cited herein are incorporated by reference in their entireties.

OPTIMIZED PROBES AND PRIMERS AND METHODS OF USING SAME FOR THE DETECTION, SCREENING, ISOLATION AND SEQUENCING OF VANCOMYCIN RESISTANCE GENES AND VANCOMYCIN RESISTANT ENTEROCOCCI

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