Patent Application
20120009575
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 Jun. 22, 2011, is named LT0267US.txt and is 404,503 bytes in size.
The present teachings relate to compositions, methods and kits for rapid detection and identification of various microorganisms using inducible RNA. More particularly, the specification describes compositions and kits comprising primer nucleic acid sequences having hybridization specificity for priming amplification of genes of microorganisms that are responsive to one or more RNA-inducing agents and in some embodiments further comprising probe nucleic acid sequences having hybridization specificity for genes responsive to RNA-inducing agents. Methods for rapidly detecting microorganisms (such as, but not limited to, Salmonella spp, Listeria spp. and Vibrio spp) are also described.
Detection of bacteria, particularly pathogenic bacteria, is an important parameter used to monitor for quality control and consumer safety in the food and pharmaceutical industries as well as in environmental monitoring. Earlier detection is of great benefit for quality and safety testing in industries such as the food industry where a faster time-to-result can greatly reduce total testing time as well as storage time for food prior to release of safe product to the grocery stores.
Advances in technology make detection and identification faster, more convenient, more sensitive, and more specific than traditional culture assays, at least in theory. Such assays include biochemical kits, and antibody-based and DNA-based tests. Use of probes, PCR and bacteriophage has been developed commercially for detecting pathogens. Probe assays generally target ribosomal RNA (rRNA), since rRNA provides a naturally amplified target and greater assay sensitivity. With few exceptions, almost all assays used to detect specific pathogens require some growth of the test sample in an enrichment medium before analysis. Early detection of the presence of pathogens is extremely important both from a public health perspective and from an economic perspective. Therefore, improved methods that result in saving any of time-to-result, labor, and materials are desired.
Embodiments herein demonstrate that detection of inducible RNA targets in addition to or in lieu of detection of corresponding DNA targets can shorten time-to-result when testing samples for presence of microorganisms such as bacteria and fungi.
Some embodiments herein identify inducible RNA target genes and induction conditions for detection of pathogenic microbes such as Listeria, a gram positive bacteria, and Salmonella and Vibrio, both gram negative bacteria.
Some embodiments provide compositions comprising one or more primer pairs operable to amplify one or more inducible RNA target genes. Some embodiments further provide compositions comprising one or more probes that are operable to hybridize to and identify an inducible RNA target gene.
Workflow methods using inducible RNA targets in assays for various bacteria are also described. Fast workflow methods are provided based on inducible transcription response in bacteria. An example workflow of the disclosure includes a shortened enrichment step, a rapid induction step, an automated sample preparation step, and specific detection of induced target genes using a reverse transcriptase-polymerase chain reaction (RT-PCR). Sample prep and RT-PCR in some embodiments are completed in less than 2 hours. Some embodiments comprise using real-time PCR and/or real-time RT-PCR.
A method of detecting presence of a microbe in a sample comprises exposing the sample to an RNA-inducing agent for a time to regulate a gene responsive thereto, detecting presence of RNA corresponding to the gene, and determining presence of the microbe as compared to a control sample based on the detection of RNA presence. In a further embodiment, the sample is cultured in a microbe enrichment medium to form enriched sample, and the enriched sample is exposed to the RNA-inducing agent.
Further embodiments herein include a method of detecting presence of a microbe in a sample, the method comprising culturing the sample in a microbe enrichment medium to form enriched sample, exposing the enriched sample to an inducing agent for a time to regulate a gene responsive thereto, measuring RNA expression levels of the responsive gene, and analyzing said expression levels of RNA to indicate the presence of the microbe as compared to at least one control sample, wherein a time for detecting presence of the microbe is shorter than a control method in which enriched samples are not exposed to the inducing agent.
Such methods of detecting presence of a pathogen are useful for food samples, for pharmaceutical quality control, for environmental samples, as well as in clinical samples and specimens.
In some embodiments, a microbe that is detected may be a gram positive or gram negative bacteria. In some embodiments, a microbe may be a pathogen.
In some embodiments, induced genes are stress responsive genes. In some embodiments, the pathogen is an organism of Salmonella spp. and the RNA-inducing agent-responsive gene is cspH, hilA, hsp60, dnaK, ibpAB, uspA, or agsA. In other embodiments, the pathogen is a Listeria spp. and the RNA-inducing agent-responsive gene is a inlA, a inlB, a inlC, a inlG, a inlJ, a lmo0539, a lmo2158, a lmo0596, a lmo0189, a lmo0880, a lmo1290, a lmo0514, a lmo0670, a bsh, a plcA, a clpE, a cspL, a lmo0699, a lmo0782, a lmo2230, a lmo2522, a opuCA, a cpn60, or a hlyA gene. In further embodiments, the pathogen is a Vibrio spp. and the RNA-inducing agent-responsive gene is a hsp60 gene. An RNA-inducing agent-responsive gene may be either up-regulated or down-regulated in response to exposure to the agent.
Measuring transcription may comprise measuring RNA expression levels using reverse transcriptase RT-PCR. In some embodiments, both RNA and DNA levels are measured using reverse transcriptase RT-PCR. In some embodiments, DNA expression levels are measured by RT-PCR. In further embodiments, the contribution of RNA to a signal can be determined by subtracting a CT value obtained from detecting both RNA and DNA from a CT value obtained from detecting just the DNA for a particular target. In some embodiments of the present methods, inclusion of the reverse transcriptase step improved the real time PCR signal by 4-7Ct's, enabling a shorter time-to-result in a workflow for detecting a microbe in a sample.
Several kits embodiments are also described. An example kit includes at least one primer pair having hybridization specificity for priming amplification of a RNA-inducing agent-responsive gene (or a fragment thereof) of a microbe. A kit may further comprise at least one probe specific to hybridize to and detect a RNA-inducing agent responsive gene or a fragment thereof. A kit of the disclosure may further comprise reagents for PCR, such as a PCR master mix which may include: any one of a reverse transcriptase, a DNA polymerase, dNTP's; ingredients for sample preparation including one or more of: a filtration medium, a surfactant, magnetic beads, spin columns; and various buffers.
One example kit may comprise at least one primer pair having hybridization specificity for priming amplification of a RNA-inducing agent-responsive gene (or a fragment thereof) of a Salmonella where the RNA-inducing agent-responsive gene is a cspH, a hilA, a hsp60, a dnaK, a ibpAB, a uspA, and/or a agsA gene.
Another example kit may comprise at least one primer pair having hybridization specificity for priming amplification of a RNA-inducing agent-responsive gene (or a fragment thereof) of a Listeria wherein the RNA-inducing agent-responsive gene is a inlA, a inlB, a inlC, a inlG, a inlJ, a lmo0539, a lmo2158, a lmo0596, a lmo0189, a lmo0880, a lmo1290, a lmo0514, a lmo0670, a bsh, a plcA, a clpE, a cspL, a lmo0699, a lmo0782, a lmo2230, a lmo2522, a opuCA, a cpn60, or a hlyA gene.
Yet another example kit includes at least one primer pair having hybridization specificity for priming amplification of a RNA-inducing agent-responsive gene (or a fragment thereof) of a Vibrio wherein the RNA-inducing agent-responsive gene is a hsp60 gene.
Exemplary kits described above may further comprise a probe (e.g., a TAQMAN® probe) having hybridization specificity for said RNA-inducing agent-responsive gene.
Use of inducible RNA targets provided by embodiments herein for detection of pathogens has advantages over traditional DNA targets typically used in real-time PCR based detection. For example, for RNA inducible targets, an increased copy number of the target is present upon induction. When DNA is transcribed into RNA, many copies of the target gene are generated and a greater copy number translates into more robust and potentially earlier detection of a pathogen.
Traditional culture methods as well as rapid PCR-based methods rely on an initial pre-enrichment in suitable media to revive stressed organisms in various matrices. The length of the pre-enrichment time depends on the sensitivity and limit of detection of the end assay method. Signal amplification using RNA targets, such as inducible RNA targets, allows for earlier detection of target. Workflow methods provided herein allowed for detection of 1-5 cfu of Salmonella in less then 8 hr and Listeria in less than 12 hr in food samples.
A further advantage of using inducible RNA targets for detection of pathogens is in an assessment of viability. Dead bacteria will not respond to induction. Inducible RNA targets are synthesized by living cells upon sensing a particular stimulus, such as heat, cold, acidic pH or chemical reagents. Use of inducible RNA targets therefore overcomes one disadvantage of using DNA targets, i.e., detection of dead bacteria that are incapable of causing disease. An additional advantage over traditional culture confirmation method is the ability to detect viable but non-culturable bacteria (VBNC) organisms that respond to induction.
These and other features of the present teachings will become more apparent from the description herein.
The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “contain”, and “include”, or modifications of those root words, for example but not limited to, “comprises”, “contained”, and “including”, are not intended to be limiting. Use of “or” means “and/or” unless stated otherwise. The term “and/or” means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, “X and/or Y” can mean “X” or “Y” or “X and Y”.
Whenever a range of values is provided herein, the range is meant to include the starting value and the ending value and any value or value range there between unless otherwise specifically stated. For example, “from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3-0.4, 0.2-0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26-0.39; and the like.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application including, but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines or uses a term in such a way that it contradicts that term's definition in this application, this application controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. The term “surrogate” as used herein means a product that is indicative of presence of another product. For example, an amplification product is a surrogate for a nucleic acid that has been amplified.
Various embodiments herein provide compositions, methods and kits for detection of microorganisms comprising detecting inducible RNA target genes in microbes that may be induced (transcribed) in response to an inducer (e.g., heat, pH and/or a chemical or biological agent) and thereby be detectable much faster than detecting traditional DNA targets comprised of unique signature sequences. Some embodiments relate to detection of pathogenic microbes. Some exemplary candidate bacterial pathogens that may be detected by the methods and compositions of this disclosure include gram positive and gram negative bacteria including, but not limited to, species of E. coli, Salmonella, Shigella, Campylobacter, Yersinia, Vibrio, Listeria, Staphylococcus, Bacillus, Clostridium, Pseudomonas or Cronobacter.
The term “target gene” or “inducible target gene” or “inducible RNA target gene” refers to a gene of a microorganism that has been identified as a gene that can be induced (e.g., gene expressed, RNA expressed, transcription is induced) in the microorganism in response to an inducer (an inducing agent or inducing condition). Some exemplary inducible target genes identified in the present specification include: Salmonella target genes that are responsive to one or more RNA-inducing agents such as a cspH, a hilA, a hsp60, a dnaK, a ibpAB, a uspA, and/or a agsA gene; Listeria target genes that are responsive to one or more RNA-inducing agents such as a inlA, a inlB, a inlC, a inlG, a inlJ, a lmo0539, a lmo2158, a lmo0596, a lmo0189, a lmo0880, a lmo1290, a lmo0514, a lmo0670, a bsh, a plcA, a clpE, a cspL, a lmo0699, a lmo0782, a lmo2230, a lmo2522, a opuCA, a cpn60, or a hlyA gene; and Vibrio target genes that are responsive to one or more RNA-inducing agents such as hsp60. Accordingly, one or more compositions, methods and kits of the disclosure are described in relation to these inducible genes and to the detection of these organisms. However, as will be recognized by one of skill in the art, the teachings of specification are not limited to these exemplary embodiments, and any microorganism that has an inducible gene that is inducible by an inducer may be detected by the methods described in this application. In some embodiments, induced genes are stress responsive genes of a microbe, that are induced quickly to allow the microbe to adapt and/or survive the stress (extreme heat/cold; lack of nutrients etc.).
“Induction,” or “induce a gene responsive to an RNA-inducing agent” as used herein refers to exposing and/or contacting and/or adding and/or “subjecting to” an inducing agent and/or condition (collectively referred to as an “inducer”) to a sample, containing or suspected of containing a microbe, and/or to an enriched culture, containing or suspected of containing the microbe, in response to which an inducible target gene is stimulated and/or induced to express RNA (e.g. transcription is induced) corresponding to that target gene. Inducible target genes are normally not induced without presence of the inducing agent or condition. Exemplary “inducers” may include, but are not limited to, thermal conditions including heat or cold (i.e., temperatures that an microorganism normally is not exposed to during its normal growth), activated charcoal, protein or peptide inducers, inducing chemicals (such as but not limited to homoserine lactones), metal chelators, high salt concentrations, different salts, pH values (acidic or basic conditions), nutrient concentrations, or to a physical effect such as pressure, presence of chemotactants, or the presence of a suboptimal environment, or a stress agent.
When temperature is used as an inducer, induction by heat depends on the organism whose presence is being tested for; for example, Vibrio cholerae grows normally at 30° C., therefore, heat-induction may occur at any temperature greater than 30° C., such as 37° C., or 42° C. In another example. Salmonella normally grows at 37° C., therefore, heat induction may occur at any temperature greater than 37° C., such as 42° C., 45° C., 48° C., 50° C., or 55° C. To expose a microbe to a heat inducer cultures can be transferred to incubators at higher temperatures, or cultures can be grown at the higher temperatures when the induction is desired. In some embodiments, a culture may be exposed to a cold temperature to induce genes that may be induced by colder temperatures than an organism usually grow in.
Some embodiments describe designing probes and primers of the disclosure following the identification of target inducible genes. Primer and probe sequences of the disclosure were designed using a rigorous bioinformatics assay design pipeline and are described in the nucleic acid comprised in SEQ ID NO: 1-SEQ ID NO: 1769.
Some embodiments of the present disclosure describes various nucleic acid compositions such as primers, generally comprising a primer pair, each primer pair comprising a forward primer and a reverse primer that can be used to hybridize to an inducible target gene (for example, RNA expressed by an inducible gene may be reverse transcribed and the cDNA formed therefrom can be subject to hybridization by a primer pair of the disclosure and then subject to conditions to carry out a DNA amplification reaction to obtain an amplified inducible target gene product which may then be detected). In some embodiments, the present disclosure also describes compositions of various probe sequences that are operable to bind to an inducible target gene or a fragment thereof to enable its detection.
The present disclosure, in some embodiments, relates to isolated nucleic acid molecules having: the sequences of SEQ ID NO: 1-SEQ ID NO:1769; complementary sequences thereof; and/or nucleic acid sequences having at least 90% homology with the sequences of SEQ ID NO: 1-SEQ ID NO:1769; and/or or a labeled derivative of any of these sequences.
Some compositions of the disclosure may comprise primer pairs operable to amplify one target inducible gene and may further comprise in some embodiments a corresponding probe sequence that is operable to bind to and detect the amplified product produced by the primer pairs. Many such compositions are described. In one example, Tables 1-3 describe several primer pairs, comprising at least a first primer referred to as a forward primer and a second primer referred to as a reverse primer. In each row of Tables 1-3, each primer pair also has at least one corresponding probe sequence. Compositions comprising a primer pairs (and in some embodiments, a corresponding probe) may be used in assays for specific and efficient detection of genes identified in the Tables as well. Thus, for example, an exemplary composition of the disclosure operable for detecting Salmonella by detecting an inducible gene agsA may comprise: a forward primer having SEQ ID NO: 1, a reverse primer having SEQ ID NO: 134 and optionally a probe having SEQ ID NO: 267; complementary sequences thereof; and/or nucleic acid sequences having at least 90% homology with the sequences; and/or or a labeled derivative of any of these sequences. These and other compositions are described in Tables 1-3.
Some exemplary non-limiting compositions having primer sets of the disclosure comprise the following:
A compositions for detecting the presence of Salmonella in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Salmonella wherein the RNA-inducing agent-responsive gene is agsA and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:1 and SEQ ID NO:134, or SEQ ID NO:2 and SEQ ID NO:135, or SEQ ID NO:3 and SEQ ID NO:136, or SEQ ID NO: 4 and SEQ ID NO:137, or SEQ ID NO:5 and SEQ ID NO:138, or SEQ ID NO:6 and SEQ ID NO:139, or SEQ ID NO:7 and SEQ ID NO:140, or SEQ ID NO:8 and SEQ ID NO:141, or SEQ ID NO:9 and SEQ ID NO:142, or SEQ ID NO:10 and SEQ ID NO:143, or SEQ ID NO: 11 and SEQ ID NO:144, or SEQ ID NO:12 and SEQ ID NO:145, or SEQ ID NO:13 and SEQ ID NO:146, or SEQ ID NO:14 and SEQ ID NO:147, or SEQ ID NO:15 and SEQ ID NO:148, or SEQ ID NO:16 and SEQ ID NO:149, or SEQ ID NO:17 and SEQ ID NO:150, or SEQ ID NO: 18 and SEQ ID NO:151, or SEQ ID NO:19 and SEQ ID NO:152, or SEQ ID NO:20 and SEQ ID NO:153, or SEQ ID NO:21 and SEQ ID NO:154, or SEQ ID NO:22 and SEQ ID NO:155, or SEQ ID NO:23 and SEQ ID NO:156 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 267-SEQ ID NO: 289.
A compositions for detecting the presence of Salmonella in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Salmonella wherein the RNA-inducing agent-responsive gene is cspH and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:24 and SEQ ID NO:157, or SEQ ID NO:25 and SEQ ID NO:158, or SEQ ID NO:26 and SEQ ID NO:159, or SEQ ID NO: 27 and SEQ ID NO:160, or SEQ ID NO:28 and SEQ ID NO:161, or SEQ ID NO:29 and SEQ ID NO:162, or SEQ ID NO:30 and SEQ ID NO:163, or SEQ ID NO:31 and SEQ ID NO:164, or SEQ ID NO:32 and SEQ ID NO:165, or SEQ ID NO:33 and SEQ ID NO:166, or SEQ ID NO: 34 and SEQ ID NO:167, or SEQ ID NO:35 and SEQ ID NO:168, or SEQ ID NO:36 and SEQ ID NO:169, or SEQ ID NO:37 and SEQ ID NO:170, or SEQ ID NO:38 and SEQ ID NO:171, or SEQ ID NO:39 and SEQ ID NO:172, or SEQ ID NO:40 and SEQ ID NO:173, or SEQ ID NO: 41 and SEQ ID NO:174, or SEQ ID NO:42 and SEQ ID NO:175, or SEQ ID NO:43 and SEQ ID NO:176, or SEQ ID NO:44 and SEQ ID NO:177 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 290-SEQ ID NO:310.
A compositions for detecting the presence of Salmonella in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Salmonella wherein the RNA-inducing agent-responsive gene is dnaK and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:45 and SEQ ID NO:178, or SEQ ID NO:46 and SEQ ID NO:179, or SEQ ID NO:47 and SEQ ID NO:180, or SEQ ID NO:48 and SEQ ID NO:181, or SEQ ID NO:49 and SEQ ID NO:182, or SEQ ID NO: 50 and SEQ ID NO:183, or SEQ ID NO:51 and SEQ ID NO:184, or SEQ ID NO:52 and SEQ ID NO:185, or SEQ ID NO:53 and SEQ ID NO:186, or SEQ ID NO:54 and SEQ ID NO:187, or SEQ ID NO:55 and SEQ ID NO:188, or SEQ ID NO:56 and SEQ ID NO:189, or SEQ ID NO: 57 and SEQ ID NO:190, or SEQ ID NO:58 and SEQ ID NO:191, or SEQ ID NO:59 and SEQ ID NO:192, or SEQ ID NO:60 and SEQ ID NO:193, or SEQ ID NO:61 and SEQ ID NO:194, or SEQ ID NO:62 and SEQ ID NO:195, or SEQ ID NO:63 and SEQ ID NO:196, or SEQ ID NO: 64 and SEQ ID NO:197, or SEQ ID NO:65 and SEQ ID NO:198 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 311-SEQ ID NO:331.
A compositions for detecting the presence of Salmonella in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Salmonella wherein the RNA-inducing agent-responsive gene is Hsp60 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of or SEQ ID NO:66 and SEQ ID NO:199, or SEQ ID NO:67 and SEQ ID NO:200, or SEQ ID NO:68 and SEQ ID NO:201, or SEQ ID NO:69 and SEQ ID NO:202, and SEQ ID NO:70 and SEQ ID NO:203, or SEQ ID NO:71 and SEQ ID NO:204, or SEQ ID NO:72 and SEQ ID NO:205, or SEQ ID NO:73 and SEQ ID NO:206, or SEQ ID NO:74 and SEQ ID NO:207, or SEQ ID NO:75 and SEQ ID NO:208, or SEQ ID NO:76 and SEQ ID NO:209, or SEQ ID NO:77 and SEQ ID NO:210, or SEQ ID NO:78 and SEQ ID NO:211, or SEQ ID NO:79 and SEQ ID NO:212, or SEQ ID NO:80 and SEQ ID NO:213, or SEQ ID NO:81 and SEQ ID NO:214, or SEQ ID NO:82 and SEQ ID NO:215, or SEQ ID NO:83 and SEQ ID NO:216, or SEQ ID NO:84 and SEQ ID NO:217, or SEQ ID NO:85 and SEQ ID NO:218, or SEQ ID NO:86 and SEQ ID NO:219 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 332-SEQ ID NO:352.
A compositions for detecting the presence of Salmonella in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Salmonella wherein the RNA-inducing agent-responsive gene is ibpAB and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:87 and SEQ ID NO:220, and SEQ ID NO:88 and SEQ ID NO:221, or SEQ ID NO:89 and SEQ ID NO:222, or SEQ ID NO:90 and SEQ ID NO:223, or SEQ ID NO:91 and SEQ ID NO:224, or SEQ ID NO:92 and SEQ ID NO:225, or SEQ ID NO:93 and SEQ ID NO:226, or SEQ ID NO:94 and SEQ ID NO:227, or SEQ ID NO:95 and SEQ ID NO:228, or SEQ ID NO:96 and SEQ ID NO:229, or SEQ ID NO:97 and SEQ ID NO:230, or SEQ ID NO:98 and SEQ ID NO:231, or SEQ ID NO:99 and SEQ ID NO:232, or SEQ ID NO:100 and SEQ ID NO:233, or SEQ ID NO:101 and SEQ ID NO:234, or SEQ ID NO:102 and SEQ ID NO:235, or SEQ ID NO:103 and SEQ ID NO:236, or SEQ ID NO:104 and SEQ ID NO:237, or SEQ ID NO:105 and SEQ ID NO:238, or SEQ ID NO:106 and SEQ ID NO:239, or SEQ ID NO:107 and SEQ ID NO:240, or SEQ ID NO:108 and SEQ ID NO:241, or SEQ ID NO:109 and SEQ ID NO:242 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 353-SEQ ID NO:375.
A compositions for detecting the presence of Salmonella in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Salmonella wherein the RNA-inducing agent-responsive gene is uspA and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of or SEQ ID NO:110 and SEQ ID NO:243, or SEQ ID NO:111 and SEQ ID NO:244, or SEQ ID NO:112 and SEQ ID NO:245, or SEQ ID NO:113 and SEQ ID NO:246, or SEQ ID NO:114 and SEQ ID NO:247, or SEQ ID NO:115 and SEQ ID NO:248, or SEQ ID NO:116 and SEQ ID NO:249, or SEQ ID NO:117 and SEQ ID NO:250, or SEQ ID NO:118 and SEQ ID NO:251, or SEQ ID NO:119 and SEQ ID NO:252, or SEQ ID NO:120 and SEQ ID NO:253, or SEQ ID NO:121 and SEQ ID NO:254, or SEQ ID NO:122 and SEQ ID NO:255, or SEQ ID NO:123 and SEQ ID NO:256, or SEQ ID NO:124 and SEQ ID NO:257, or SEQ ID NO:125 and SEQ ID NO:258, or SEQ ID NO:126 and SEQ ID NO:259, or SEQ ID NO:127 and SEQ ID NO:260, or SEQ ID NO:128 and SEQ ID NO:261, or SEQ ID NO:129 and SEQ ID NO:262, or SEQ ID NO:130 and SEQ ID NO:263, or SEQ ID NO:131 and SEQ ID NO:264, or SEQ ID NO:132 and SEQ ID NO:265 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 376-SEQ ID NO:398.
A compositions for detecting the presence of Salmonella in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Salmonella wherein the RNA-inducing agent-responsive gene is hilA and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of or SEQ ID NO:133 and SEQ ID NO:266 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 399.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is inlA and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:400 and SEQ ID NO:855, or SEQ ID NO:401 and SEQ ID NO:856, or SEQ ID NO:402 and SEQ ID NO:857, or SEQ ID NO: 403 and SEQ ID NO:858, or SEQ ID NO:404 and SEQ ID NO:859, or SEQ ID NO:405 and SEQ ID NO:860, or SEQ ID NO:406 and SEQ ID NO:861, or SEQ ID NO:407 and SEQ ID NO:862, or SEQ ID NO:408 and SEQ ID NO:863, or SEQ ID NO:409 and SEQ ID NO:864, or SEQ ID NO: 410 and SEQ ID NO:865, or SEQ ID NO:411 and SEQ ID NO:866, or SEQ ID NO:412 and SEQ ID NO:867, or SEQ ID NO:413 and SEQ ID NO:868, or SEQ ID NO:414 and SEQ ID NO:869, or SEQ ID NO:415 and SEQ ID NO:870, or SEQ ID NO:416 and SEQ ID NO:871, or SEQ ID NO:417 and SEQ ID NO:872, or SEQ ID NO:418 and SEQ ID NO:873, or SEQ ID NO:419 and SEQ ID NO:874, or SEQ ID NO: 420 and SEQ ID NO:875, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1310-SEQ ID NO: 1330.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo0539 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:421 and SEQ ID NO:876, or SEQ ID NO:422 and SEQ ID NO:877, or SEQ ID NO:423 and SEQ ID NO:878, or SEQ ID NO:424 and SEQ ID NO:879, or SEQ ID NO:425 and SEQ ID NO:880, or SEQ ID NO:426 and SEQ ID NO:881, or SEQ ID NO:427 and SEQ ID NO:882, or SEQ ID NO:428 and SEQ ID NO:883, or SEQ ID NO:429 and SEQ ID NO:884, or SEQ ID NO: 430 and SEQ ID NO:885, or SEQ ID NO:431 and SEQ ID NO:886, or SEQ ID NO:432 and SEQ ID NO:887, or SEQ ID NO:433 and SEQ ID NO:888, or SEQ ID NO:434 and SEQ ID NO:889, or SEQ ID NO:435 and SEQ ID NO:890, or SEQ ID NO:436 and SEQ ID NO:891, or SEQ ID NO:437 and SEQ ID NO:892, or SEQ ID NO:438 and SEQ ID NO:893, or SEQ ID NO:439 and SEQ ID NO:894, or SEQ ID NO: 440 and SEQ ID NO:895, or SEQ ID NO: 441 and SEQ ID NO:896, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1331-SEQ ID NO:1351.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo2158 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of or SEQ ID NO:442 and SEQ ID NO:897, or SEQ ID NO:443 and SEQ ID NO:898, or SEQ ID NO:444 and SEQ ID NO:899, or SEQ ID NO:445 and SEQ ID NO:900, or SEQ ID NO:446 and SEQ ID NO:901, or SEQ ID NO:447 and SEQ ID NO:902, or SEQ ID NO:448 and SEQ ID NO:903, or SEQ ID NO:449 and SEQ ID NO:904, or SEQ ID NO: 450 and SEQ ID NO:905, or SEQ ID NO:451 and SEQ ID NO:906, or SEQ ID NO:452 and SEQ ID NO:907, or SEQ ID NO:453 and SEQ ID NO:908, or SEQ ID NO:454 and SEQ ID NO:909, or SEQ ID NO:455 and SEQ ID NO:910, or SEQ ID NO:456 and SEQ ID NO:911, or SEQ ID NO:457 and SEQ ID NO:912, or SEQ ID NO:458 and SEQ ID NO:913, or SEQ ID NO:459 and SEQ ID NO:914, or SEQ ID NO: 460 and SEQ ID NO:915, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1352-SEQ ID NO:1370.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is bsh and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:461 and SEQ ID NO:916, or SEQ ID NO:462 and SEQ ID NO:917, or SEQ ID NO:463 and SEQ ID NO:918, or SEQ ID NO:464 and SEQ ID NO:919, or SEQ ID NO:465 and SEQ ID NO:920, or SEQ ID NO:466 and SEQ ID NO:921, or SEQ ID NO:467 and SEQ ID NO:922, or SEQ ID NO:468 and SEQ ID NO:923, or SEQ ID NO:469 and SEQ ID NO:924, or SEQ ID NO: 470 and SEQ ID NO:925, SEQ ID NO:471 and SEQ ID NO:926, or SEQ ID NO:472 and SEQ ID NO:927, or SEQ ID NO:473 and SEQ ID NO:928, or SEQ ID NO:474 and SEQ ID NO:929, or SEQ ID NO:475 and SEQ ID NO:930, or SEQ ID NO:476 and SEQ ID NO:931, or SEQ ID NO:477 and SEQ ID NO:932, or SEQ ID NO:478 and SEQ ID NO:933, or SEQ ID NO:479 and SEQ ID NO:934, or SEQ ID NO: 480 and SEQ ID NO:935, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1371-SEQ ID NO:1390.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is inlB and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:481 and SEQ ID NO:936, or SEQ ID NO:482 and SEQ ID NO:937, or SEQ ID NO:483 and SEQ ID NO:938, or SEQ ID NO:484 and SEQ ID NO:939, or SEQ ID NO:485 and SEQ ID NO:940, or SEQ ID NO:486 and SEQ ID NO:941, or SEQ ID NO:487 and SEQ ID NO:942, or SEQ ID NO:488 and SEQ ID NO:943, or SEQ ID NO:489 and SEQ ID NO:944, or SEQ ID NO: 490 and SEQ ID NO:945, SEQ ID NO:491 and SEQ ID NO:946, or SEQ ID NO:492 and SEQ ID NO:947, or SEQ ID NO:493 and SEQ ID NO:948, or SEQ ID NO:494 and SEQ ID NO:949, or SEQ ID NO:495 and SEQ ID NO:950, or SEQ ID NO:496 and SEQ ID NO:951, or SEQ ID NO:497 and SEQ ID NO:952, or SEQ ID NO:498 and SEQ ID NO:953, or SEQ ID NO:499 and SEQ ID NO:954, or SEQ ID NO: 500 and SEQ ID NO:955, or SEQ ID NO: 501 and SEQ ID NO:956 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1391-SEQ ID NO:1411.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo0596 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:502 and SEQ ID NO:957, or SEQ ID NO:503 and SEQ ID NO:958, or SEQ ID NO:504 and SEQ ID NO:959, or SEQ ID NO:505 and SEQ ID NO:960, or SEQ ID NO:506 and SEQ ID NO:961, or SEQ ID NO:507 and SEQ ID NO:962, or SEQ ID NO:508 and SEQ ID NO:963, or SEQ ID NO:509 and SEQ ID NO:964, or SEQ ID NO: 510 and SEQ ID NO:965, SEQ ID NO:511 and SEQ ID NO:966, or SEQ ID NO:512 and SEQ ID NO:967, or SEQ ID NO:513 and SEQ ID NO:968, or SEQ ID NO:514 and SEQ ID NO:969, or SEQ ID NO:515 and SEQ ID NO:970, or SEQ ID NO:516 and SEQ ID NO:971, or SEQ ID NO:517 and SEQ ID NO:972, or SEQ ID NO:518 and SEQ ID NO:973, or SEQ ID NO:519 and SEQ ID NO:974, or SEQ ID NO: 520 and SEQ ID NO:975, or SEQ ID NO: 521 and SEQ ID NO:976, or SEQ ID NO: 522 and SEQ ID NO:977, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1412-SEQ ID NO:1432.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo2230 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:523 and SEQ ID NO:978, or SEQ ID NO:524 and SEQ ID NO:979, or SEQ ID NO:525 and SEQ ID NO:980, or SEQ ID NO:526 and SEQ ID NO:981, or SEQ ID NO:527 and SEQ ID NO:982, or SEQ ID NO:528 and SEQ ID NO:983, or SEQ ID NO:529 and SEQ ID NO:984, or SEQ ID NO: 530 and SEQ ID NO:985, SEQ ID NO:531 and SEQ ID NO:986, or SEQ ID NO:532 and SEQ ID NO:987, or SEQ ID NO:533 and SEQ ID NO:988, or SEQ ID NO:534 and SEQ ID NO:989, or SEQ ID NO:535 and SEQ ID NO:990, or SEQ ID NO:536 and SEQ ID NO:991, or SEQ ID NO:537 and SEQ ID NO:992, or SEQ ID NO:538 and SEQ ID NO:993, or SEQ ID NO:539 and SEQ ID NO:994, or SEQ ID NO: 540 and SEQ ID NO:995, or SEQ ID NO: 541 and SEQ ID NO:996, or SEQ ID NO: 542 and SEQ ID NO:997, or SEQ ID NO: 543 and SEQ ID NO:998, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1433-SEQ ID NO:1453.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is clpE and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:544 and SEQ ID NO:999, or SEQ ID NO:545 and SEQ ID NO:1000, or SEQ ID NO:546 and SEQ ID NO:1001, or SEQ ID NO:547 and SEQ ID NO:1002, or SEQ ID NO:548 and SEQ ID NO:1003, or SEQ ID NO:549 and SEQ ID NO:1004, or SEQ ID NO: 550 and SEQ ID NO:1005, SEQ ID NO:551 and SEQ ID NO:1006, or SEQ ID NO:552 and SEQ ID NO:1007, or SEQ ID NO:553 and SEQ ID NO:1008, or SEQ ID NO:554 and SEQ ID NO:1009, or SEQ ID NO:555 and SEQ ID NO:1010, or SEQ ID NO:556 and SEQ ID NO:1011, or SEQ ID NO:557 and SEQ ID NO:1012, or SEQ ID NO:558 and SEQ ID NO:10013, or SEQ ID NO:559 and SEQ ID NO:1014, or SEQ ID NO: 560 and SEQ ID NO:1015, SEQ ID NO:561 and SEQ ID NO:1006, or SEQ ID NO:562 and SEQ ID NO:1017, or SEQ ID NO:563 and SEQ ID NO:1018, or SEQ ID NO:564 and SEQ ID NO:1019, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1454-SEQ ID NO:1474.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is inlC and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:565 and SEQ ID NO:1020, or SEQ ID NO:566 and SEQ ID NO:1021, or SEQ ID NO:567 and SEQ ID NO:1022, or SEQ ID NO:568 and SEQ ID NO:1023, or SEQ ID NO:569 and SEQ ID NO:1024, or SEQ ID NO: 570 and SEQ ID NO:1025, SEQ ID NO:571 and SEQ ID NO:1026, or SEQ ID NO:572 and SEQ ID NO:1027, or SEQ ID NO:573 and SEQ ID NO:1028, or SEQ ID NO:574 and SEQ ID NO:1029, or SEQ ID NO:575 and SEQ ID NO:1030, or SEQ ID NO:576 and SEQ ID NO:1031, or SEQ ID NO:577 and SEQ ID NO:1032, or SEQ ID NO:578 and SEQ ID NO:1033, or SEQ ID NO:579 and SEQ ID NO:1034, or SEQ ID NO: 580 and SEQ ID NO:1035, SEQ ID NO:581 and SEQ ID NO:1036, or SEQ ID NO:582 and SEQ ID NO:1037, or SEQ ID NO:583 and SEQ ID NO:1038, or SEQ ID NO:584 and SEQ ID NO:1039, or SEQ ID NO:585 and SEQ ID NO:1040, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1475-SEQ ID NO:1495.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo0670 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of or SEQ ID NO:586 and SEQ ID NO:1041, or SEQ ID NO:587 and SEQ ID NO:1042, or SEQ ID NO:588 and SEQ ID NO:1043, or SEQ ID NO:589 and SEQ ID NO:1044, or SEQ ID NO: 590 and SEQ ID NO:1045, SEQ ID NO:591 and SEQ ID NO:1046, or SEQ ID NO:592 and SEQ ID NO:1047, or SEQ ID NO:593 and SEQ ID NO:1048, or SEQ ID NO:594 and SEQ ID NO:1049, or SEQ ID NO:595 and SEQ ID NO:1050, or SEQ ID NO:596 and SEQ ID NO:1051, or SEQ ID NO:597 and SEQ ID NO:1052, or SEQ ID NO:598 and SEQ ID NO:1053, or SEQ ID NO:599 and SEQ ID NO:1054, or SEQ ID NO: 600 and SEQ ID NO:1055, SEQ ID NO:601 and SEQ ID NO:1056, or SEQ ID NO:602 and SEQ ID NO:1057, or SEQ ID NO:603 and SEQ ID NO:1058, or SEQ ID NO:604 and SEQ ID NO:1059, or SEQ ID NO:605 and SEQ ID NO:1060, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1496-SEQ ID NO:1515.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo2522 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:606 and SEQ ID NO:1061, or SEQ ID NO:607 and SEQ ID NO:1062, or SEQ ID NO:608 and SEQ ID NO:1063, or SEQ ID NO:609 and SEQ ID NO:1064, or SEQ ID NO: 610 and SEQ ID NO:1065, SEQ ID NO:611 and SEQ ID NO:1066, or SEQ ID NO:612 and SEQ ID NO:1067, or SEQ ID NO:613 and SEQ ID NO:1068, or SEQ ID NO:614 and SEQ ID NO:1069, or SEQ ID NO:615 and SEQ ID NO:1070, or SEQ ID NO:616 and SEQ ID NO:1071, or SEQ ID NO:617 and SEQ ID NO:1072, or SEQ ID NO:618 and SEQ ID NO:1073, or SEQ ID NO:619 and SEQ ID NO:1074, or SEQ ID NO: 620 and SEQ ID NO:1075, SEQ ID NO:621 and SEQ ID NO:1076, or SEQ ID NO:622 and SEQ ID NO:1077, or SEQ ID NO:623 and SEQ ID NO:1078, or SEQ ID NO:624 and SEQ ID NO:1079, or SEQ ID NO:625 and SEQ ID NO:1080, or SEQ ID NO:626 and SEQ ID NO:1081, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1516-SEQ ID NO:1536.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is cspL and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:627 and SEQ ID NO:1082, or SEQ ID NO:628 and SEQ ID NO:1083, or SEQ ID NO:629 and SEQ ID NO:1084, or SEQ ID NO: 630 and SEQ ID NO:1085, SEQ ID NO:631 and SEQ ID NO:1086, or SEQ ID NO:632 and SEQ ID NO:1087, or SEQ ID NO:633 and SEQ ID NO:1088, or SEQ ID NO:634 and SEQ ID NO:1089, or SEQ ID NO:635 and SEQ ID NO:1090, or SEQ ID NO:636 and SEQ ID NO:1091, or SEQ ID NO:637 and SEQ ID NO:1092, or SEQ ID NO:638 and SEQ ID NO:1093, or SEQ ID NO:639 and SEQ ID NO:1094, or SEQ ID NO: 640 and SEQ ID NO:1095, SEQ ID NO:641 and SEQ ID NO:1096, or SEQ ID NO:642 and SEQ ID NO:1097, or SEQ ID NO:643 and SEQ ID NO:1098, or SEQ ID NO:644 and SEQ ID NO:1099, or SEQ ID NO:645 and SEQ ID NO:1100, or SEQ ID NO:646 and SEQ ID NO:1101, or SEQ ID NO:647 and SEQ ID NO:1102, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1537-SEQ ID NO:1557.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is inlG and the at least one primer pair comprises isolated nucleic acid molecules having the sequences SEQ ID NO:648 and SEQ ID NO:1103, or SEQ ID NO:649 and SEQ ID NO:1104, or SEQ ID NO: 650 and SEQ ID NO:1105, SEQ ID NO:651 and SEQ ID NO:1106, or SEQ ID NO:652 and SEQ ID NO:1107, or SEQ ID NO:653 and SEQ ID NO:1108, or SEQ ID NO:654 and SEQ ID NO:1109, or SEQ ID NO:655 and SEQ ID NO:1110, or SEQ ID NO:656 and SEQ ID NO:1111, or SEQ ID NO:657 and SEQ ID NO:1112, or SEQ ID NO:658 and SEQ ID NO:1113, or SEQ ID NO:659 and SEQ ID NO:1114, or SEQ ID NO: 660 and SEQ ID NO:1115, SEQ ID NO:661 and SEQ ID NO:1116, or SEQ ID NO:662 and SEQ ID NO:1117, or SEQ ID NO:663 and SEQ ID NO:1118, or SEQ ID NO:664 and SEQ ID NO:1119, or SEQ ID NO:665 and SEQ ID NO:1120, or SEQ ID NO:666 and SEQ ID NO:1121, or SEQ ID NO:667 and SEQ ID NO:1122 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1558-SEQ ID NO:1577.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is Lmo0699 and the at least one primer pair comprises isolated nucleic acid molecules having the SEQ ID NO:668 and SEQ ID NO:1123, or SEQ ID NO:669 and SEQ ID NO:1124, or SEQ ID NO: 670 and SEQ ID NO:1125, SEQ ID NO:671 and SEQ ID NO:1126, or SEQ ID NO:672 and SEQ ID NO:1127, or SEQ ID NO:673 and SEQ ID NO:1128, or SEQ ID NO:674 and SEQ ID NO:1129, or SEQ ID NO:675 and SEQ ID NO:1130, or SEQ ID NO:676 and SEQ ID NO:1131, or SEQ ID NO:677 and SEQ ID NO:1132, or SEQ ID NO:678 and SEQ ID NO:1133, or SEQ ID NO:679 and SEQ ID NO:1134, or SEQ ID NO: 680 and SEQ ID NO:1135, or SEQ ID NO:681 and SEQ ID NO:1136, or SEQ ID NO:682 and SEQ ID NO:1137, or SEQ ID NO:683 and SEQ ID NO:1138, or SEQ ID NO:684 and SEQ ID NO:1139, or SEQ ID NO:685 and SEQ ID NO:1140, or SEQ ID NO:686 and SEQ ID NO:1141, or SEQ ID NO:687 and SEQ ID NO:1142, SEQ ID NO:688 and SEQ ID NO:1143, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1578-SEQ ID NO:1598.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is opuCA and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:689 and SEQ ID NO:1144, or SEQ ID NO: 690 and SEQ ID NO:1145, SEQ ID NO:691 and SEQ ID NO:1146, or SEQ ID NO:692 and SEQ ID NO:1147, or SEQ ID NO:693 and SEQ ID NO:1148, or SEQ ID NO:694 and SEQ ID NO:1149, or SEQ ID NO:695 and SEQ ID NO:1150, or SEQ ID NO:696 and SEQ ID NO:1151, or SEQ ID NO:697 and SEQ ID NO:1152, or SEQ ID NO:698 and SEQ ID NO:1153, or SEQ ID NO:699 and SEQ ID NO:1154, or SEQ ID NO: 700 and SEQ ID NO:1155, SEQ ID NO:701 and SEQ ID NO:1156, or SEQ ID NO:702 and SEQ ID NO:1157, or SEQ ID NO:703 and SEQ ID NO:1158, or SEQ ID NO:704 and SEQ ID NO:1159, or SEQ ID NO:705 and SEQ ID NO:1160, or SEQ ID NO:706 and SEQ ID NO:1161, or SEQ ID NO:707 and SEQ ID NO:1162, SEQ ID NO:708 and SEQ ID NO:1163, SEQ ID NO:709 and SEQ ID NO:1164, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1599-SEQ ID NO:1619.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is inlJ and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO: 710 and SEQ ID NO:1165, SEQ ID NO:711 and SEQ ID NO:1166, or SEQ ID NO:712 and SEQ ID NO:1167, or SEQ ID NO:713 and SEQ ID NO:1168, or SEQ ID NO:714 and SEQ ID NO:1169, or SEQ ID NO:715 and SEQ ID NO:1170, or SEQ ID NO:716 and SEQ ID NO:1171, or SEQ ID NO:717 and SEQ ID NO:1172, or SEQ ID NO:718 and SEQ ID NO:1173, or SEQ ID NO:719 and SEQ ID NO:1174, or SEQ ID NO: 720 and SEQ ID NO:1175, SEQ ID NO:721 and SEQ ID NO:1176, or SEQ ID NO:722 and SEQ ID NO:1177, or SEQ ID NO:723 and SEQ ID NO:1178, or SEQ ID NO:724 and SEQ ID NO:1179, or SEQ ID NO:725 and SEQ ID NO:1180, or SEQ ID NO:726 and SEQ ID NO:1181, SEQ ID NO:727 and SEQ ID NO:1182, or SEQ ID NO:728 and SEQ ID NO:1183, or SEQ ID NO:729 and SEQ ID NO:1184, or SEQ ID NO:730 and SEQ ID NO:1185, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1620-SEQ ID NO:1640.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is Lmo0782 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:731 and SEQ ID NO:1186, or SEQ ID NO:732 and SEQ ID NO:1187, or SEQ ID NO:733 and SEQ ID NO:1188, or SEQ ID NO:734 and SEQ ID NO:1189, or SEQ ID NO:735 and SEQ ID NO:1190, or SEQ ID NO:736 and SEQ ID NO:1191, or SEQ ID NO:737 and SEQ ID NO:1192, or SEQ ID NO:738 and SEQ ID NO:1193, or SEQ ID NO:739 and SEQ ID NO:1194, or SEQ ID NO: 740 and SEQ ID NO:1195, SEQ ID NO:741 and SEQ ID NO:1196, or SEQ ID NO:742 and SEQ ID NO:1197, or SEQ ID NO:743 and SEQ ID NO:1198, or SEQ ID NO:744 and SEQ ID NO:1199, or SEQ ID NO:745 and SEQ ID NO:1200, or SEQ ID NO:746 and SEQ ID NO:1201, or SEQ ID NO:747 and SEQ ID NO:1202, SEQ ID NO:748 and SEQ ID NO:1203, or SEQ ID NO:749 and SEQ ID NO:1204, or SEQ ID NO: 750 and SEQ ID NO:1205, SEQ ID NO:751 and SEQ ID NO:1206, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1641-SEQ ID NO:1661.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is plcA and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:752 and SEQ ID NO:1207, or SEQ ID NO:753 and SEQ ID NO:1208, or SEQ ID NO:754 and SEQ ID NO:1209, or SEQ ID NO:755 and SEQ ID NO:1210, or SEQ ID NO:756 and SEQ ID NO:1211, or SEQ ID NO:757 and SEQ ID NO:1212, or SEQ ID NO:758 and SEQ ID NO:1213, or SEQ ID NO:759 and SEQ ID NO:1214, or SEQ ID NO: 760 and SEQ ID NO:1215, SEQ ID NO:761 and SEQ ID NO:1216, or SEQ ID NO:762 and SEQ ID NO:1217, or SEQ ID NO:763 and SEQ ID NO:1218, or SEQ ID NO:764 and SEQ ID NO:1219, or SEQ ID NO:765 and SEQ ID NO:1220, or SEQ ID NO:766 and SEQ ID NO:1221, or SEQ ID NO:767 and SEQ ID NO:1222, or SEQ ID NO:768 and SEQ ID NO:1223, or SEQ ID NO:769 and SEQ ID NO:1224, or SEQ ID NO: 770 and SEQ ID NO:1225, SEQ ID NO:771 and SEQ ID NO:1226, or SEQ ID NO:772 and SEQ ID NO:1227, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1662-SEQ ID NO:1682.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo0189 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:773 and SEQ ID NO:1228, or SEQ ID NO:774 and SEQ ID NO:1229, or SEQ ID NO:775 and SEQ ID NO:1230, or SEQ ID NO:776 and SEQ ID NO:1231, or SEQ ID NO:777 and SEQ ID NO:1232, or SEQ ID NO:778 and SEQ ID NO:1233, or SEQ ID NO:779 and SEQ ID NO:1234, or SEQ ID NO: 780 and SEQ ID NO:1235, SEQ ID NO:881 and SEQ ID NO:1236, or SEQ ID NO:782 and SEQ ID NO:1237, or SEQ ID NO:783 and SEQ ID NO:1238, or SEQ ID NO:784 and SEQ ID NO:1239, or SEQ ID NO:785 and SEQ ID NO:1240, or SEQ ID NO:786 and SEQ ID NO:1241, or SEQ ID NO:787 and SEQ ID NO:1242, SEQ ID NO:788 and SEQ ID NO:1243, or SEQ ID NO:789 and SEQ ID NO:1244, or SEQ ID NO: 790 and SEQ ID NO:1245, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1683-SEQ ID NO:1700.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo0880 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:991 and SEQ ID NO:1246, or SEQ ID NO:792 and SEQ ID NO:1247, or SEQ ID NO:793 and SEQ ID NO:1248, or SEQ ID NO:794 and SEQ ID NO:1249, or SEQ ID NO:795 and SEQ ID NO:1250, or SEQ ID NO:796 and SEQ ID NO:1251, or SEQ ID NO:797 and SEQ ID NO:1252, or SEQ ID NO:798 and SEQ ID NO:1253, or SEQ ID NO:799 and SEQ ID NO:1254, or SEQ ID NO: 800 and SEQ ID NO:1255, SEQ ID NO:801 and SEQ ID NO:1256, or SEQ ID NO:802 and SEQ ID NO:1257, or SEQ ID NO:803 and SEQ ID NO:1258, or SEQ ID NO:804 and SEQ ID NO:1259, or SEQ ID NO:805 and SEQ ID NO:1260, or SEQ ID NO:806 and SEQ ID NO:1261, or SEQ ID NO:807 and SEQ ID NO:1262, SEQ ID NO:808 and SEQ ID NO:1263, SEQ ID NO:809 and SEQ ID NO:1264, or SEQ ID NO: 810 and SEQ ID NO:1265, SEQ ID NO:811 and SEQ ID NO:1266, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1701-SEQ ID NO:1721.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo0514 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:812 and SEQ ID NO:1267, or SEQ ID NO:813 and SEQ ID NO:1268, or SEQ ID NO:814 and SEQ ID NO:1269, or SEQ ID NO:815 and SEQ ID NO:1270, or SEQ ID NO:816 and SEQ ID NO:1271, or SEQ ID NO:817 and SEQ ID NO:1272, or SEQ ID NO:818 and SEQ ID NO:1273, or SEQ ID NO:819 and SEQ ID NO:1274, or SEQ ID NO: 820 and SEQ ID NO:1275, SEQ ID NO:821 and SEQ ID NO:1276, or SEQ ID NO:822 and SEQ ID NO:1277, or SEQ ID NO:823 and SEQ ID NO:1278, or SEQ ID NO:824 and SEQ ID NO:1279, or SEQ ID NO:825 and SEQ ID NO:1280, or SEQ ID NO:826 and SEQ ID NO:1281, SEQ ID NO:827 and SEQ ID NO:1282, or SEQ ID NO:828 and SEQ ID NO:1283, or SEQ ID NO:829 and SEQ ID NO:1284, or SEQ ID NO:830 and SEQ ID NO:1285, or SEQ ID NO:831 and SEQ ID NO:1286, or SEQ ID NO:832 and SEQ ID NO:1287, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1722-SEQ ID NO:1742.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is lmo0129 and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:833 and SEQ ID NO:1288, or SEQ ID NO:834 and SEQ ID NO:1289, or SEQ ID NO:835 and SEQ ID NO:1290, or SEQ ID NO:836 and SEQ ID NO:1291, or SEQ ID NO:837 and SEQ ID NO:1292, or SEQ ID NO:838 and SEQ ID NO:1293, or SEQ ID NO:839 and SEQ ID NO:1294, or SEQ ID NO: 840 and SEQ ID NO:1295, SEQ ID NO:841 and SEQ ID NO:1296, or SEQ ID NO:842 and SEQ ID NO:1297, or SEQ ID NO:843 and SEQ ID NO:1298, or SEQ ID NO:844 and SEQ ID NO:1299, or SEQ ID NO:845 and SEQ ID NO:1300, or SEQ ID NO:846 and SEQ ID NO:1301, or SEQ ID NO:847 and SEQ ID NO:1302, SEQ ID NO:848 and SEQ ID NO:1303, or SEQ ID NO:849 and SEQ ID NO:1304, or SEQ ID NO: 850 and SEQ ID NO:1305, SEQ ID NO:7851 and SEQ ID NO:1306, or SEQ ID NO:852 and SEQ ID NO:1307, or SEQ ID NO:853 and SEQ ID NO:1308, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1743-SEQ ID NO:1763.
A composition for detecting the presence of Listeria in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is hylA and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:854 and SEQ ID NO:1309 or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID NO:1764.
A composition for detecting the presence of Vibrio in a sample may comprise: at least one primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Vibrio wherein the RNA-inducing agent-responsive gene is a hsp60 gene and the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:1765 and SEQ ID NO: 1766, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences. The composition may also comprise at least one probe wherein the at least one probe has the sequence of SEQ ID: NO: 1767, SEQ ID NO: 1768 and/or SEQ ID NO:1769.
Some compositions of the disclosure may comprise a duplexed set of primer pairs and probes for detection of one or more microorganisms in a single assay. For example, a composition may comprise, at least two sets of primer pairs, a first primer set comprising a first primer (a first forward primer) and a second primer (a first reverse primer) and a second primer set comprising a first primer (a second forward primer) and a second primer (a second reverse primer), each primer set operable to amplify a different target inducible gene or target inducible gene nucleic acid fragment. The composition may also have a corresponding probe sequence that can hybridize to amplified target nucleic acids of each primer set (a first probe and a second probe). A duplexed primer set may be operable to amplify at least two different target nucleic acid sequences and their corresponding probes are operable to identify at least two different target nucleic acid sequences. Compositions of the disclosure may comprise additional primer pairs (such as three primer pairs, four primer pairs and optionally the same number of corresponding probes as well). In some embodiments, the target genes may be from the same organism, thereby providing a higher degree of confidence in detection. In some embodiments the target genes may be from different organisms, thereby providing the ability to detect multiple organisms in one reaction. Accordingly, in some embodiments, compositions of the disclosure may be operable to detect simultaneously the presence of two or more organisms that may be contaminating a sample, provided the same induction conditions are operable to induce the individual target inducible genes of those organisms.
In some embodiments, the disclosure describes methods of detecting in a sample the presence of a microorganism, comprising: exposing the sample to an RNA-inducing agent for a time to induce a gene responsive to the RNA-inducing agent; detecting presence of an RNA corresponding to the gene responsive to the RNA-inducing agent; and determining presence of the microorganism, wherein the detection of presence of the RNA corresponding to the gene responsive to the RNA-inducing agent in comparison to a control sample is indicative of the presence of the microorganism in the sample. In some embodiments, a method may also comprise the step of culturing the sample in a microorganism enrichment medium to form an enriched sample, and then exposing the enriched sample to the RNA-inducing agent.
Detection steps may be performed by a variety of methods, such as but not limited to, a nucleic acid amplification reaction using primers to amplify target inducible genes or fragments thereof. Detection in some embodiments may be performed by hybridization using probes specific to target sequences in a target inducible gene. Combinations of amplification and hybridization may be used for detection according to some embodiments.
Methods of the disclosure may further comprise steps of sample preparation and may also comprise identification steps (to identify a species/strain/serotype of contaminating organism following the initial detection). In some embodiments, sample preparation may comprise preparing a sample for PCR amplification (prior to hybridizing with a primer pair), comprising for example, but not limited to (1) microbial enrichment, (2) separation of microbial cells from the sample, (3) cell lysis, and (4) nucleic acid extraction (e.g. RNA extraction, total DNA extraction, genomic DNA extraction).
Samples may include without limitation, clinical samples, food/beverage samples, water samples, and environmental sample. Food sample may comprise raw produce, meats as well as a selectively enriched food matrix.
Microbial pathogens that may be detected using methods provided herein may be present in food samples, in samples from the environment including processing equipment, in pharmaceutical preparations, on processing equipment for making or assembling pharmaceutical preparations, or from animal or humans who are potential carriers. A sample for detection of a pathogen may be an uncooked food sample such as uncooked meats, fish, poultry vegetables, unpasteurized milk, foods made from unpasteurized milk, or dairy products, or cooked or processed foods such as hot dogs, deli meats, cheeses, poultry, ice cream, smoked fish, or seafood, for example; an environmental sample for detection of a pathogen may be soil, stream water, sewage, plants, or swabs from food or pharmaceutical processing equipment, or any surface that is involved with food or pharmaceutical processing.
Typically a portion of food or a swabbed or sponged sample is combined with an appropriate liquid, such as water, a buffer solution, or a culture medium such as a selective medium or an enrichment medium. In some embodiments, the food is chopped, macerated, liquefied, diced, or homogenized. In some embodiments, large volumes of sample, for example but not limited to, volumes of 100 mL, 250 mL, or more are processed or a portion of the food or beverage and appropriate liquid are typically combined to form a dilute suspension, for example but not limited to, ratios of about 1:3, 1:5, or 1:10 (w/vol). In some embodiments, a detergent, an emulsifying agent, or both, is added to enhance the solubility of high lipid foods, for example but not limited to butter and certain other dairy products. In certain embodiments, 25 grams of a solid or semi-solid food is combined with 225 mL of a suitable culture media. In some embodiments, 25 mL of a beverage or a liquefied or partially liquefied food is combined with 225 mL of a suitable culture media.
Samples may also be pooled to save on testing costs, e.g., instead of testing 15×25 g samples of food, a composite of 375 g, with 25 g coming from different lots of food will be tested. If any composite is tested positive, then the individual 15 samples are further evaluated. If the composite is negative, then the food testing lab has saved the cost of 15 individual tests.
Solid samples (e.g., 25 g), liquid samples (e.g., 10 ml-100 ml) or environmental samples (i.e., swabbed or sponged samples resuspended in e.g., 10 ml-100 ml) can be pre-enriched for a pathogenic species for a time in pathogen-specific enrichment media, or an equivalent thereof. Alternative enrichment media include Tryptic Soy Broth, Brain Heart Infusion Broth or Fraser Broth, for which ingredients can be found in, for example, “Compendium of Methods for the Microbiological Examination of Foods,” 4th Edition (2001) Downes and Ito, eds. American Public Health Association, or U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM), Media Index on the FDA web site. For example, for enrichment of Listeria species, after 4 hours of incubation, Listeria selective agents are added (see e.g., Example 2 herein). Incubation for selective enrichment is continued for a total of 24 hours enrichment, or may be continued for up to 48 h.
In an example embodiment, a method of the disclosure may comprise, following sample preparation, steps for detecting presence of a microbe by “detecting presence of an RNA corresponding to a gene responsive to an RNA-inducing agent” which may comprise a) hybridizing at least a first pair of PCR primers, comprising a forward primer and a reverse primer, that are operable to bind to and amplify at least one gene responsive to the RNA-inducing agent or a fragment thereof; b) amplifying at least one gene responsive to the RNA-inducing agent or a fragment thereof to form at least one amplified target nucleic acid product; and d) detecting the at least one amplified target polynucleotide sequence product; wherein the detection of the at least one amplified target polynucleotide sequence product is indicative of the presence of the microorganism in the sample. The step of detecting the at least one amplified target polynucleotide sequence product may further comprise using a probe that is operable to hybridize to the at least one amplified target polynucleotide sequence product. Primer and probes used may be labeled. Nucleic acid amplification reactions may be a PCR amplification and may further comprise an end-point determination, and/or maybe quantitative. In some embodiments the quantification maybe a real-time PCR. In some embodiments, the real-time PCR maybe a SYBR® Green Assay and/or a TaqMan® Assay. In some embodiments, the amplification may be by a real-time PCR assay and the probe may be a TAQMAN® probe.
In some embodiments, sample preparation may comprise extracting RNA and detecting may comprise detecting the presence of an induced RNA by a reverse transcriptase RT-PCR assay. A method of the disclosure may further comprise detecting presence of DNA corresponding to the induced gene by an RT-PCR assay. In some embodiments, a method may further comprising subtracting a CT value of RNA and DNA detection from a CT value for DNA detection. A method for identifying an RNA inducing agent-responsive gene may comprise comparing the difference in CT that results from real time reverse transcriptase PCR assays of induced samples vs uninduced samples. A method for identifying the contribution of RNA detection may comprise subtracting CT results from assays with reverse transcriptase present from the CT results from assays without reverse transcriptase present.
In some embodiments, a method for detection of a Salmonella spp. in a sample (enriched sample) may comprise: exposing the sample to an RNA-inducing agent for a time to induce at least one Salmonella gene responsive to the RNA-inducing agent, wherein the Salmonella gene responsive to the RNA-inducing agent may be a cspH, a hilA, a hsp60, a dnaK, a ibpAB, a uspA, and/or a agsA gene; detecting presence of an RNA corresponding to the gene responsive to the RNA-inducing agent; and determining presence of the Salmonella spp, wherein the detection of presence of the RNA corresponding to the Salmonella gene responsive to the RNA-inducing agent in comparison to a control sample (an identical sample known not to have the microorganism; and/or an identical sample not subject to induction) is indicative of the presence of a Salmonella in the sample.
In some embodiments, the steps of detecting presence of an RNA corresponding to the Salmonella gene responsive to the RNA-inducing agent in the method described above may comprise a) hybridizing at least a first pair of PCR primers comprising a forward primer and a reverse primer (e.g., selected for example from a row in Table 1 of the disclosure) that are operable to bind to and amplify an inducible gene or fragment thereof corresponding to at least one of the following Salmonella inducible genes: cspH, hilA, hsp60, dnaK, ibpAB, uspA, and/or agsA; b) amplifying the at least one Salmonella inducible gene or a fragment thereof (corresponding to at least one of the following genes: cspH, hilA, hsp60, dnaK, ibpAB, uspA, and/or agsA) to form at least one amplified target nucleic acid product; and d) detecting the at least one amplified target polynucleotide sequence product; wherein the detection of the at least one amplified target polynucleotide sequence product is indicative of the presence of Salmonella spp. in the sample. The step of detecting the at least one amplified target polynucleotide sequence product may further comprise using a probe that is operable to hybridize to the at least one amplified target polynucleotide sequence product (e.g., probes may be selected for example from a row in Table 1 of the disclosure, a primer pair and probe from the same row describes the probe-primer combination for an assay). Primer and probes used may be labeled. In some embodiments the amplification may be by a real-time PCR assay and the probe may be a TAQMAN® probe.
In some embodiments, a method for detection of a Listeria spp. in a sample (enriched sample) may comprise: exposing the sample to an RNA-inducing agent for a time to induce at least one Listeria gene responsive to the RNA-inducing agent, wherein the Listeria gene responsive to the RNA-inducing agent may be a inlA, a inlB, a inlC, a inlG, a inlJ, a lmo0539, a lmo2158, a lmo0596, a lmo0189, a lmo0880, a lmo1290, a lmo0514, a lmo0670, a bsh, a plcA, a clpE, a cspL, a lmo0699, a lmo0782, a lmo2230, a lmo2522, a opuCA, a cpn60, or a hlyA gene; detecting presence of an RNA corresponding to the Listeria gene responsive to the RNA-inducing agent; and determining presence of the Listeria spp, wherein the detection of presence of the RNA corresponding to the gene responsive to the RNA-inducing agent in comparison to a control sample (an identical sample known not to have the microorganism; and/or an identical sample not subject to induction) is indicative of the presence of a Listeria in the sample.
In some embodiments, the steps of detecting presence of an RNA corresponding to the Listeria gene responsive to the RNA-inducing agent in the method described above may comprise a) hybridizing at least a first pair of PCR primers comprising a forward primer and a reverse primer (e.g., selected for example from a row in Table 2 of the disclosure) that are operable to bind to and amplify an inducible gene or fragment thereof corresponding to at least one of the following genes: a inlA, a inlB, a inlC, a inlG, a inlJ, a lmo0539, a lmo2158, a lmo0596, a lmo0189, a lmo0880, a lmo1290, a lmo0514, a lmo0670, a bsh, a plcA, a clpE, a cspL, a lmo0699, a lmo0782, a lmo2230, a lmo2522, a opuCA, a cpn60, and/or a hlyA; b) amplifying at least one inducible gene or fragment thereof to form at least one amplified target nucleic acid product; and d) detecting the at least one amplified target polynucleotide sequence product; wherein the detection of the at least one amplified target polynucleotide sequence product is indicative of the presence of Listeria spp. in the sample. The step of detecting the at least one amplified target polynucleotide sequence product may further comprise using a probe that is operable to hybridize to the at least one amplified target polynucleotide sequence product (e.g., a probe may be selected for example from a row in Table 2 of the disclosure, a primer pair and probe from the same row describes the probe-primer combination for an assay). Primer and probes used may be labeled. In some embodiments the amplification may be by a real-time PCR assay and the probe may be a TAQMAN® probe.
In some embodiments, a method for detection of a Vibrio spp. in a sample (enriched sample) may comprise: exposing the sample to an RNA-inducing agent for a time to induce at least one Vibrio gene responsive to the RNA-inducing agent, wherein an example Vibrio gene responsive to the RNA-inducing agent may be a Vibrio hsp60 gene; detecting presence of an RNA corresponding to the Vibrio gene responsive to the RNA-inducing agent; and determining presence of the Vibrio spp, wherein the detection of presence of the RNA corresponding to a Vibrio gene responsive to an RNA-inducing agent in comparison to a control sample (an identical sample known not to have the microorganism; and/or an identical sample not subject to induction) is indicative of the presence of a Vibrio in the sample.
In some embodiments, the steps of detecting presence of an RNA corresponding to a Vibrio gene responsive to the RNA-inducing agent in the method described above may comprise a) hybridizing at least a first pair of PCR primers comprising a forward primer and a reverse primer (e.g., selected for example from a row in Table 3 of the disclosure) that are operable to bind to and amplify an inducible gene or fragment thereof (for e.g., a Vibrio hsp60 gene); b) amplifying at least one inducible gene or fragment thereof (for e.g., a Vibrio hsp60 gene) to form at least one amplified target nucleic acid product; and d) detecting the at least one amplified target polynucleotide sequence product; wherein the detection of the at least one amplified target polynucleotide sequence product is indicative of the presence of Vibrio spp. in the sample. The step of detecting the at least one amplified target polynucleotide sequence product may further comprise using a probe that is operable to hybridize to the at least one amplified target polynucleotide sequence product. A probe may be selected, for example, from a row in Table 3 of the disclosure, a primer pair and probe from the same row describes the probe-primer combination for an assay. Table 3 describes three probe sequences that are all operable to function with the Vibrio specific primer pair. Primer and probes used may be labeled. In some embodiments the amplification may be by a real-time PCR assay and the probe may be a TAQMAN® probe.
Methods of the disclosure may include assays such as polymerase chain reactions, wherein hybridizing and amplifying of said first pair of polynucleotide primers occurs in a first vessel and said hybridizing and amplifying of said second pair of polynucleotide primers occurs in a second vessel, or hybridizing and amplifying of said first pair of polynucleotide primers and said hybridizing and amplifying of said second pair of polynucleotide primers occurs in a single vessel. The detection may be a real-time assay and the real-time assay may be a SYBR® Green dye assay or a TaqMan® assay.
Methods of the disclosure, in various embodiments, may comprise providing at least a first probe. Some embodiments may comprise providing at least two probes, a first probe and a second probe the probes, wherein the first and second probes are different from each other, the first probe operable to identify the first amplified target polynucleotide sequence and the second probe operable to identify the second amplified target nucleotide sequence, the first probe further comprises a first label and said second probe further comprises a second label, wherein both labels are selected from a dye, a radioactive isotope, a chemiluminescent label, and an enzyme, the dye comprises a fluorescein dye, a rhodamine dye, or a cyanine dye, the dye is a fluorescein dye and first probe is labeled with FAM™ dye and said second probe is labeled with VIC® dye; and hybridizing the first and second probes to the PCR amplified fragments to detect the presence of the first amplified target polynucleotide sequence and the second amplified target polynucleotide sequence from the sample.
Methods of the disclosure comprising using inducible RNA targets for detection of microbes may also be used for assessment of viability of microbes. Dead microbes will not respond to induction. Inducible RNA targets are synthesized by living cells upon sensing a particular stimulus, such as heat, cold, acidic pH or chemical reagents. Accordingly, the disclosure provides methods of detecting viable microbes in a sample. Use of inducible RNA targets overcomes a major disadvantage of using DNA targets which detect both live and dead bacteria. If only dead bacteria are present in a sample, a method using DNA target detection will show a false positive result by amplifying and picking up dead DNA targets that are incapable of causing disease. An additional advantage over traditional culture confirmation method is the ability to detect viable but non-culturable bacteria (VBNC) organisms that respond to induction.
Use of inducible RNA targets provided by embodiments herein for detection of pathogens has other advantages over traditional DNA targets typically used in real-time PCR based detection. For example, for RNA inducible targets, an increased copy number of the target is present upon induction. When DNA is transcribed into RNA, many copies of the target gene are generated and a greater copy number translates into more robust and potentially earlier detection of a pathogen.
Traditional culture methods as well as rapid PCR-based methods rely on an initial pre-enrichment in suitable media to revive stressed organisms in various matrices. The length of the pre-enrichment time depends on the sensitivity and limit of detection of the end assay method. Signal amplification using RNA targets, such as inducible RNA targets, allows for earlier detection of target.
The present disclosure also describes workflow methods using inducible RNA targets in assays for detection of various bacteria. A fast workflow is provided based on inducible transcription response in bacteria; the workflow includes a shortened enrichment step, a rapid induction step, an automated sample preparation step, and specific detection of induced target using reverse transcriptase RT-PCR. Example workflows provided herein allowed for detection of 1-5 cfu of Salmonella in less then 8 hr and Listeria in less than 12 hr in food samples, thereby greatly reducing the time to testing, which is very important for industries such as the food industry, where valuable shelf life can be increased, if the testing time for food safety testing is reduces by the workflow methods provided herein.
The entire volume of the enriched and induced culture, or a portion thereof, may be concentrated and processed for detection of the bacterial pathogen, for example, a one mL aliquot may be taken from 250 mL of enriched and induced culture. The medium may be clarified by filtration or low speed centrifugation prior to or after concentrating. Harvested samples are lysed using, for example, the RiboPure™ RNA extraction kit (Ambion, Austin Tex.), the PrepSEQ™ Nucleic Acid Extraction Kit (Applied Biosystems) or the PrepSEQ™ RapidSpin Kit (Applied Biosystems) or any other effective lysis system that preserves nucleic acid integrity. The lysate can be amplified directly or the nucleic acid can be extracted and amplified. Amplification products may be detected, directly or indirectly, and the presence or absence of a microbe in the test sample can be determined. Sample preparation methods used are efficient in removal of PCR inhibitors.
High quality DNA and RNA can be prepared by manual low throughput methods or by automated high throughput methods, depending on the number of samples being tested. An integrated workflow for automated high-throughput sample preparation may include enrichment for the test bacterial pathogen, lysis, binding of nucleic acids to magnetic particles, magnetic separation, followed by washings and elution of DNA and/or RNA in a PCR compatible solution. An integrated workflow for manual low-throughput sample preparation may include enrichment for the test bacterial pathogen, centrifugation to pellet bacteria, resuspension of bacteria in lysis buffer, followed by amplification using primers for RNA-inducible target specific PCR as provided herein. The integrated system may also include lyophilized reagents for the assay and data analyses software.
Embodiments of detecting DNA, RNA and/or a surrogate thereof, in a lysate or extracted nucleic acid sample includes detection means using emission by an emitter that is representative of the RNA or DNA in the test sample.
In some embodiments, a lysate or extracted nucleic acid sample is mixed with a composition comprising reverse transcriptase to form a reverse transcriptase reaction mixture. A reverse transcription reaction provides a surrogate of the RNA that can be detectable. Any reverse transcriptase known to those of ordinary skill in the art can be used such as, for example, MMLV-RT (murine maloney leukemia virus-reverse transcriptase), avian myelogenous virus reverse transcriptase (AMV-RT), human immunodeficiency virus (HIV)-RT and the Tth DNA polymerase which has reverse transcriptase activity if Mn++ is provided.
A positive control for detection of RNA or DNA can be a non-homologous RNA random sequence such as XENO™ RNA (Applied Biosystems, Foster City, Calif.). A control for qPCR can be a β-actin probe/primer set, also available from Applied Biosystems, for example. The positive control can be mixed with the sample.
As used herein, “amplification” or “amplify” and the like refers to the production of multiple copies of the target nucleic acid, a surrogate of a target nucleic acid, or a portion thereof. Amplification can encompass a variety of chemical and enzymatic processes such as a polymerase chain reaction (PCR), a strand displacement amplification reaction, a transcription mediated amplification reaction, or a nucleic acid sequence-based amplification reaction, for example. Following at least one amplification cycle, the amplification products can be detected or can be separated from at least one other component of the amplification mixture based on their molecular weight or length or mobility prior to detection.
PCR includes introducing a molar excess of two or more extendable oligonucleotide primers to a reaction mixture where the primers hybridize to opposite strands of a DNA, RNA or RNA surrogate. The reaction mixture is subjected to a program of thermal cycling in the presence of a DNA polymerase, resulting in the amplification of the DNA or RNA surrogate sequence flanked by the primers. Reverse transcriptase PCR is a PCR reaction that uses an RNA template and a reverse transcriptase, or a polypeptide having reverse transcriptase activity, to first generate a single stranded DNA molecule prior to the multiple cycles of DNA-dependent DNA polymerase primer elongation as cited above. Methods for a wide variety of PCR applications are widely known in the art, and described in many sources, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, Section 15, John Wiley & Sons, Inc., New York (1994).
The present disclosure describes several primers. However, in light of the present specifications, one of skill in the art may design additional primer pairs that are specific to other inducible genes in other organisms. These additional inducible genes and primers are within the scope of the present disclosure. Criteria for designing sequence-specific primers in light of this specification are well known to persons of ordinary skill in the art. Detailed descriptions of primer design that provide for sequence-specific annealing can be found, among other places, in Diffenbach and Dveksler, PCR Primer, A Laboratory Manual, Cold Spring Harbor Press, 1995, and Kwok et al. (Nucl. Acid Res. 18:999-1005, 1990). The sequence-specific portions of the primers are of sufficient length to permit specific annealing to complementary sequences, as appropriate. A primer does not need to have 100% complementarity with a primer-specific portion for primer extension to occur. Further, a primer can be detectably labeled such that the label is detected by spectroscopy. A primer pair is sometimes said to consist of a “forward primer” and a “reverse primer,” indicating that they are initiating nucleic acid polymerization in opposing directions from different strands of a duplex template.
In some embodiments, a primer as set forth herein can comprise a universal priming sequence. The term “universal primer” refers to a primer comprising a universal sequence that is able to hybridize to all, or essentially all, potential target sequences in a multiplexed reaction. The term “semi-universal primer” refers to a primer that is capable of hybridizing with more than one (e.g., a subset), but not all, of the potential target sequences in a multiplexed reaction. The terms “universal sequence,” “universal priming sequence” or “universal primer sequence” or the like refer to a sequence contained in a plurality of primers, where the universal priming sequence that is found in a target is complementary to a universal primer.
“Hybridization” refers to a process in which single-stranded nucleic acids with complementary or near-complementary base sequences interact to form hydrogen-bonded complexes called hybrids. Hybridization reactions are sensitive and selective. In vitro, the specificity of hybridization (i.e., stringency) is controlled by the concentrations of salt or formamide in prehybridization and hybridization solutions, for example, and by the hybridization temperature; such procedures are well known in the art. In particular, stringency is increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature. For example, high stringency conditions could occur at about 50% formamide at 37° C. to 42° C. Reduced stringency conditions could occur at about 35% to 25% formamide at 30° C. to 35° C. Examples of stringency conditions for hybridization are provided in Sambrook, J., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The temperature for hybridization is about 5-10° C. less than the melting temperature (Tm) of the hybrid.
As an example of primer selection, primers can be selected by the use of any of various software programs available and known in the art for developing amplification and/or multiplex systems. Exemplary programs include, PRIMER EXPRESS® software (Applied Biosystems, Foster City, Calif.) and Primer3 software (Rozen S et al. (2000), “Primer3 on the WWW for general users and for biologist programmers,” Krawetz S et al. (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386). In the example of the use of software programs, sequence information can be imported into the software. The software then uses various algorithms to select primers that best meet the user's specifications.
Primer and probe sequences having at least 90% homology to those of Tables 1-3 are embodiments herein. “Homology,” as known by one of ordinary skill in the art, is the degree of sequence relatedness between nucleotide sequences as determined by matching the order and identity of nucleotides between the sequences. In one embodiment, the primer or probe sequences provided herein have 100% homology, at least 98% homology, at least 95% homology, at least 92% homology or at least 90% homology to their intended hybridization target. Computer methods for determining homology are designed to identify the greatest degree of matching of nucleotide sequences, for example, BLASTN (Altschul, S. F., et al. (1990) J. Mol. Biol. 215:403-410).
In certain embodiments, single-stranded amplification products can be generated by methods including, without limitation, asymmetric PCR, asymmetric reamplification, nuclease digestion, and chemical denaturation. For example, single-stranded sequences can be generated by combining at least one first primer or at least one second primer from a primer set, but not both, in an amplification reaction mixture, or by transcription, for example, when a promoter-primer is used in a first amplification mixture, a second amplification mixture, or both.
The term “polymerase,” as used herein, refers to a polypeptide that is able to catalyze the addition of nucleotides or analogs thereof to a nucleic acid in a template dependent manner, for example, the addition of deoxyribonucleotides to the 3′-end of a primer that is annealed to a nucleic acid template during a primer extension reaction. Nucleic acid polymerases can be thermostable or thermally degradable. Suitable thermostable polymerases include, but are not limited to, polymerases isolated from Thermus aquaticus, Thermus thermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritima. Suitable thermodegradable polymersases include, but are not limited to, E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, and others. Examples of other polymerizing enzymes that can be used in the methods described herein include but are not limited to T7, T3, SP6 RNA polymerases; and AMV, M-MLV and HIV reverse transcriptases.
Commercially available polymerases include, but are not limited to AMBION′S SUPERTAQ®, TAQFS®, AMPLITAQ® CS (Applied Biosystems), AMPLITAQ® FS (Applied Biosystems), KENTAQ1® (AB Peptide, St. Louis, Mo.), TAQUENASE® (Scien Tech Corp., St. Louis, Mo.), THERMOSEQUENASE® (Amersham), Bst polymerase, READER™ Taq DNA polymerase, VENT® DNA polymerase,
Descriptions of DNA polymerases can be found in, among other places, Lehninger Principles of Biochemistry, 3d ed., Nelson and Cox, Worth Publishing, New York, N.Y., 2000, particularly Chapters 26 and 29; Twyman, Advanced Molecular Biology: A Concise Reference, Bios Scientific Publishers, New York, N.Y., 1999; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., including supplements through May 2005 (hereinafter “Ausubel et al.”); Lin and Jaysena, J. Mol. Biol. 271:100-11, 1997; Pavlov et al., Trends in Biotechnol. 22:253-60, 2004; and Enzymatic Resource Guide: Polymerases, 1998, Promega, Madison, Wis.
In various detection embodiments, amplification is optionally followed by additional steps, for example, but not limited to, labeling, sequencing, purification, isolation, hybridization, size resolution, expression, detecting and/or cloning. In certain embodiments, one or both PCR primers can comprise a label, such as, for example, a fluorophore. A label can facilitate detection of an amplification product comprising a labeled PCR primer. In various detection embodiments, following the PCR, biotinylated strands can be captured, separated, and detected.
The term “multiplex assays” refers to PCR reactions that use more than two primers in a single reaction and at the same time so that more than one different amplified product is produced and detected. For example, more than two pair of amplification primers are contacted at the same time and/or in the same solution. Several target RNAs or DNAs can be detected simultaneously using multiplex assays. A multiplex reaction can also include a multiplicity of singleplex PCR reactions run in parallel, e.g., the TAQMAN® Low Density Array (TLDA). Sample preparation processes described herein have been demonstrated to be compatible with multiplex assays.
As used herein, “real-time PCR” refers to the detection and quantitation of a DNA, a RNA or a surrogate thereof in a sample. In some embodiments, the amplified segment or “amplicon” can be detected using a 5′-nuclease assay, particularly the TAQMAN® assay as described by e.g., Holland et al. (Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991); and Heid et al. (Genome Research 6:986-994, 1996). For use herein, a TAQMAN® nucleotide sequence to which a TAQMAN® probe binds can be designed into the primer portion, or known to be present in a RNA or a DNA of a sample.
“Tm” refers to the melting temperature (temperature at which 50% of the oligonucleotide is a duplex) of an oligonucleotide determined experimentally or calculated using the nearest-neighbor thermodynamic values of SantaLucia J. et al. (Biochemistry 35:3555-62, 1996) for DNA or Freier et al. (Proc. Natl. Acad. Sci. USA 83:9373-9377, 1986) for RNA. In general, the Tm of the TAQMAN® probe is about 10 degrees above the Tm of amplification primer pairs. Amplification primer sequences and double dye-labeled TAQMAN® probe sequences can be designed using PRIMER EXPRESS™ (Version 1.0, Applied Biosystems, Foster City, Calif.) or mFOLD™ software (now UNIFold™) (IDT, San Jose, Calif.).
When a TAQMAN® probe is hybridized to DNA, RNA or a surrogate thereof, the 5′-exonuclease activity of a thermostable DNA-dependent DNA polymerase such as SUPERTAQ® (a Taq polymerase from Thermus aquaticus, Ambion, Austin, Tex.) digests the hybridized TAQMAN® probe during the elongation cycle, separating the fluor from the quencher. The reporter fluor dye is then free from the quenching effect of the quencher moiety resulting in a decrease in FRET and an increase in emission of fluorescence from the fluorescent reporter dye. One molecule of reporter dye is generated for each new molecule synthesized, and detection of the free reporter dye provides the basis for quantitative interpretation of the data. In real-time PCR, the amount of fluorescent signal is monitored with each cycle of PCR. Once the signal reaches a detectable level, it has reached the “threshold or cycle threshold (Ct).” A fluorogenic PCR signal of a sample can be considered to be above background if its Ct value is at least 1 cycle less than that of a no-template control sample. The term “Ct” represents the PCR cycle number when the signal is first recorded as statistically significant. Thus, the lower the Ct value, the greater the concentration of nucleic acid target. In the TAQMAN® assay, typically each cycle almost doubles the amount of PCR product and therefore, the fluorescent signal should double if there is no inhibition of the reaction and the reaction was nearly 100% efficient with purified nucleic acid. Certain systems such as the ABI 7500, 7500FAST, 7700 and 7900HT Sequence Detection Systems (Applied Biosystems, Foster City, Calif.) conduct monitoring during each thermal cycle at a pre-determined or user-defined point.
Detection method embodiments using a TAQMAN® probe sequence comprise combining the stopped mixture or the reverse transcribed mixture with PCR reagents, including a primer set having a forward primer and a reverse primer, a DNA polymerase, and a fluorescent detector oligonucleotide TAQMAN® probe, as well as dNTP's and a salt, to form an amplification reaction mixture; subjecting the amplification reaction mixture to successive cycles of amplification to generate a fluorescent signal from the detector probe; and quantitating the nucleic acid presence based on the fluorescent signal cycle threshold of the amplification reaction.
Protocols and reagents for means of carrying out further 5′-nuclease assays are well known to one of skill in the art, and are described in various sources. For example, 5′-nuclease reactions and probes are described in U.S. Pat. Nos. 6,214,979 issued Apr. 10, 2001; 5,804,375 issued Sep. 8, 1998; 5,487,972 issued Jan. 30, 1996; and 5,210,015 issued May 11, 1993, all to Gelfand et al.
In various embodiments, a detection method can utilize any probe that can detect a nucleic acid sequence. In some configurations, a detection probe can be, for example, a TAQMAN® probe described supra, a stem-loop molecular beacon, a stemless or linear beacon, a PNA MOLECULAR BEACON™, a linear PNA beacon, non-FRET probes, SUNRISE®/AMPLIFLUOR® probes, stem-loop and duplex SCORPION™ probes, bulge loop probes, pseudo knot probes, cyclicons, MGB ECLIPSE™ probe, a probe complementary to a ZIPCODE™ sequence, hairpin probes, peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes as known by one of ordinary skill in the art. A detection probe having a sequence complementary to a detection probe hybridization sequence, such as a ZIPCODE™ sequence, a fluorophore and a mobility modifier can be, for example, a ZIPCHUTE™ probe supplied commercially by Applied Biosystems (Foster City, Calif.).
A “label” or “reporter,” as used herein, refers to a moiety or property that allows the detection of that with which it is associated and, generally, has emission spectra at between and including 300 nm to 750 nm. In certain embodiments, the emission spectra is at less than about 499 nm such as for blue emitters such as certain Alexa Fluor emitters, Cascade Blue, and Pacific Blue; at 500 nm to 549 nm emitters such as for green emitters such as certain Alexa Fluor emitters, BODIPY FL, fluorescein (FITC), CYANINE™ 2 dye, Catskill Green, 5-FAM™ dye, 6-FAM™ dye, succinimidyl ester, JOE™ dye, MFP488, the Oregon Green emitters and TET™ dye; at 550 nm to 584 nm emitters such as yellow emitters such as certain Alexa Fluor emitters, CYANINE™ 3 dye, HEX™ dye, NED™ dye, R-Phycoerythrin (R-PE), 5-TAMRA™ dye, TRITC (Rhodamine), and VIC® dye; at 585 nm to 615 nm emitters such as orange emitters such as certain Alexa Fluor emitters, CYANINE™ 3.5 dye, Lissamine Rhodamine, ROX™ dye, and R-Phycoerythrin-TEXAS RED® dye; and at 616 nm to 700 nm emitters such as red emitters such as certain Alexa Fluor emitters, CYANINE™ 5 dye, Quantum Red, Rodamine Red-X, and TEXAS RED® dye.
The label can be attached covalently or non-covalently to a DNA product, to a RNA product, or to a surrogate thereof such as an amplicon thereof. Commonly used labels include dyes that are negatively charged, such as dyes of the fluorescein family including, e.g. FAM™ dye, HEX™ dye, TET™ dye, JOE™ dye, NAN and ZOE; or dyes that are neutral in charge, such as dyes of the rhodamine family including, e.g., TEXAS RED® dye, ROX™ dye, R110, R6G, and TAMRA™ dye; or dyes that are positively charged, such as dyes of the CYANINE™ family including e.g., Cy™2 dye, Cy™3 dye, Cy™5 dye, Cy™5.5 dye and Cy™7 dye. FAM™ dye, HEX™ dye, TET™ dye, JOE™ dye, NAN, ZOE, ROX™ dye, R110, R6G, and TAMRA™ dyes are available from, e.g., Applied Biosystems (Foster City, Calif.) or Perkin-Elmer, Inc. (Wellesley, Mass.); TEXAS RED® dye is available from, e.g., Molecular Probes, Inc. (Eugene, Oreg.); and Cy™2 dye, Cy™3 dye, Cy™5 dye, Cy™5.5 dye and Cy™7 dye, and are available from, e.g., Amersham Biosciences Corp. (Piscataway, N.J.). In certain amplification embodiments, the fluorescer molecule is a fluorescein dye and the quencher molecule is a rhodamine dye.
A label or reporter can comprise both a fluorophore and a fluorescence quencher. The fluorescence quencher can be a fluorescent fluorescence quencher, such as the fluorophore TAMRA™ dye, or a non-fluorescent fluorescence quencher (NFQ), for example, a combined NFQ-minor groove binder (MGB) such as an MGB ECLIPSE™ minor groove binder supplied by Epoch Biosciences (Bothell, Wash.) and used with TAQMAN™ probes (Applied Biosystems, Foster City, Calif.). The fluorophore can be any fluorophore that can be attached to a nucleic acid, such as, for example, FAM™ dye, HEX™ dye, TET™ dye, JOE™ dye, NAN, ZOE, TEXAS RED® dye, ROX™ dye, R110, R6G, TAMRA™ dye, Cy™2 dye, Cy™3 dye, Cy™5 dye, Cy™5.5 dye and Cy™7 dye as cited above as well as VIC® dye, NED™ dye, LIZ® dye, ALEXA, Cy™9 dye, and dR6G.
Further examples of labels include black hole quenchers (BHQ) (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).
Labels can also comprise sulfonate derivatives of fluorescein dyes, phosphoramidite forms of fluorescein, phosphoramidite forms of CY™5 dye (available for example from Amersham), and intercalating labels such as ethidium bromide, SYBR™ Green I dye and PICOGREEN™ dye (Molecular Probes). Generally, an intercalating label is a molecule that reversibly inserts between two other molecules (or groups) such as between the bases of DNA.
In various embodiments, qPCR reactions can include master mixes such as the TAQMAN® Gene Expression Master Mix, TAQMAN® Universal PCR Master Mix, TAQMAN® Fast Universal PCR Master Mix, Power SYBR® Green PCR Master Mix, Fast SYBR® Green Master Mix, TAQMAN® RNA-to-CT™ 1-Step Kit, and the Power SYBR® Green RNA-to-CT™ 1-Step Kit, for example, all from Applied Biosystems.
In various embodiments, detection of emission such as fluorescence can be by any method known to skilled artisans, and can include, for example, real time detection for PCR or end point detection. Detection of fluorescence, for example, can be qualitative or quantitative. Quantitative results can be obtained, for example, with the aid of a fluorimeter, for example a fluorimeter as part of an integrated nucleic acid analysis system, such as, for example, an Applied Biosystems ABI PRISM™ 7900HT Sequence Detection System. Furthermore, quantitative results can be obtained in some configurations using a real-time PCR analysis. Some non-limiting examples of protocols for conducting fluorogenic assays such as TAQMAN® assays, including analytical methods for performing quantitative assays, can be found in publications such as, for example, “SNPLEX™ Genotyping System 48-plex”, Applied Biosystems, 2004; “User Bulletin #2 ABI PRISM™ 7700 Sequence Detection System,” Applied Biosystems 2001; “User Bulletin #5 ABI PRISM™ 7700 Sequence Detection System,” Applied Biosystems, 2001; and “Essentials of Real Time PCR,” Applied Biosystems (Foster City, Calif.). Fluorogenic PCR assays used in some configurations of the present teachings can be performed using an automated system, such as, for example, an ABI 7700 Sequence Detection System (Applied Biosystems).
For real time PCR, a passive reference dye, ROX™ dye, can be included in PCR reactions to provide an internal reference to which the reporter-dye signal can be normalized during data analysis. Normalization can be accomplished using Applied Biosystems' Sequence Detection System (SDS) software.
In general for the studies herein, the TAQMAN® probes were labeled with FAM™ dye, the TAQMAN® probe for the Internal Positive Control was labeled with VIC® dye and a Baseline Control was detected using ROX® Dye. Data analyses were carried out using the RAPIDFINDER™ Express Software (Applied Biosystems) and results are provided in an easy-to-read format with present/absent calls.
In some embodiments, detection can be achieved using microarrays or bead arrays and related software, such as the Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer, and other commercially available array systems available from Affymetrix, Agilent, and Illumina, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med. 9:140-45, including supplements, 2003).
A “kit,” as used herein, refers to a combination of at least some items for performing a reverse transcriptase RT-PCR assay for detection of inducible RNA targets of microbes, such as but not limited to, bacterial pathogens. Embodiments of kits may comprise at least one or more of the following reagents: at least one set of primers specific for detecting an inducible RNA target, at least one probe (e.g., a TAQMAN® probe) specific for detection of said inducible RNA target, internal positive control DNA to monitor presence of PCR inhibitors from various food and environmental sources, a baseline control, reagents for sample collection, reagents for isolating nucleic acid such as magnetic beads, spin columns, columns, particles, filters, lysis buffers, protease, reverse transcriptase, a reverse transcriptase buffer, a DNA polymerase or an enzymatically active mutant or variant thereof, a DNA polymerase buffer, deoxyribonucleotides dATP, dCTP, dGTP, or dTTP. In some kit embodiments, an enzyme comprising reverse transcriptase activity and thermostable DNA-dependent DNA polymerase activity are the same enzyme, e.g., Thermus sp. ZO5 polymerase or Thermus thermophilus polymerase. In certain kit embodiments, amplification primers are attached to a solid support such as a microarray.
The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other packaging means, into which a component can be placed, and in some embodiments, suitably aliquoted. Where more than one component is included in the kit (they can be packaged together), the kit also will generally contain at least one second, third or other additional container into which the additional components can be separately placed. However, various combinations of components can be packaged in a container means. The kits of the present teachings also will typically include reagent containers in close confinement for commercial sale. Such containers can include injection or blow-molded plastic containers into which the desired container means are retained.
When the components of the kit are provided in one and/or more liquid solutions, the liquid solution comprises an aqueous solution that can be a sterile aqueous solution.
In certain embodiments, at least one kit component is lyophilized and provided as dried powder(s). For example, primers and TAQMAN® probes may be lyophilized. When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. In certain embodiments, the solvent is provided in another container means. Kits can also comprise an additional container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
A kit can also include instructions for employing the kit components as well as the use of any other reagent not included in the kit. Instructions can include variations that can be implemented.
An exemplary kit may comprise one or more compositions for detecting one or more Salmonella species and may comprise at least one primer pair having hybridization specificity for amplifying an inducible gene specific to Salmonella species, or for amplifying a fragment of an inducible gene specific to Salmonella species. In some embodiments, the kit may further comprise at least one probe sequence operable to bind to an inducible gene specific to Salmonella species or a fragment thereof that may be amplified by the primer pair. A probe may be labeled for easier detection. In some embodiments, probe and primers of kits may comprise sequences selected from SEQ ID NO: 1-SEQ ID NO: 1769, including the specific primer pair combinations (and corresponding probes) described in sections above for detection of different inducible genes (also see probe primer combinations in Tables 1-3).
In one example, according to some embodiments of this disclosure a kit for detecting Salmonella may comprise: a primer pair comprising at least one Salmonella-specific primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Salmonella wherein the RNA-inducing agent-responsive gene is a cspH, a hilA, a hsp60, a dnaK, a ibpAB, a uspA, and/or a agsA gene.
Another exemplary kit may comprise one or more compositions for detecting one or more Listeria species and may comprise at least one primer pair having hybridization specificity for amplifying an inducible gene specific to Listeria species, or for amplifying a fragment of an inducible gene specific to Listeria species. In some embodiments, the kit may further comprise at least one probe sequence operable to bind to an inducible gene specific to Listeria species or a fragment thereof that may be amplified by the primer pair. A probe may be labeled for easier detection.
In one example, according to some embodiments of this disclosure a kit for detecting Listeria may comprise: a primer pair comprising at least one Listeria-specific primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Listeria wherein the RNA-inducing agent-responsive gene is a inlA, a inlB, a inlC, a inlG, a inlJ, a lmo0539, a lmo2158, a lmo0596, a lmo0189, a lmo0880, a lmo1290, a lmo0514, a lmo0670, a bsh, a plcA, a clpE, a cspL, a lmo0699, a lmo0782, a lmo2230, a lmo2522, a opuCA, a cpn60, and/or a hlyA gene.
Yet another exemplary kit may comprise one or more compositions for detecting one or more Vibrio species and may comprise at least one primer pair having hybridization specificity for amplifying an inducible gene specific to Vibrio species, or for amplifying a fragment of an inducible gene specific to Vibrio species. In some embodiments, the kit may further comprise at least one probe sequence operable to bind to an inducible gene specific to Vibrio species or a fragment thereof that may be amplified by the primer pair. A probe may be labeled for easier detection.
In one example, according to some embodiments of this disclosure a kit for detecting Vibrio may comprise: a primer pair comprising at least one Vibrio-specific primer pair having hybridization specificity for priming amplification of an RNA-inducing agent-responsive gene of Vibrio wherein the RNA-inducing agent-responsive gene is a hsp60 gene.
Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
The present example describes identification and evaluation of inducible RNA targets for detection of the pathogenic microbe Salmonella enterica using real-time RT-PCR.
Materials and Methods; Bacteria and Growth Conditions: Bacteria were routinely cultured on Brain Heart Infusion (BHI) Agar plates and in BHI broth at 37° C. overnight (Teknova, Hollister, Calif.). For spiking food samples, 25 g or 25 ml of the food sample was transferred into stomacher bags. Bacteria were spiked in the 1-100 cfu range. Samples were enriched according to standard procedures as described in the Bacteriological Analytical Manual (U.S. Food and Drug Administration, Bacteriological Analytical Manual, Chapter 5, Salmonella; see www.fda.gov). Enrichment media (225 ml) was added to each sample; for enrichment of Salmonella, Buffered Peptone Water or Brain Heart Infusion Broth was used (Teknova, Hollister, Calif.). Samples were enriched at 37° C. for a maximum of 16 hours.
Inducing Conditions: For Salmonella, samples were induced by heating at 45° C. for 15 minutes. A control (uninduced) sample was maintained at 37° C.
Sample Preparation: At the end of the induction period, tubes were placed in an ice bucket and cells were harvested by centrifugation at 16,000 rpm, 4° C., 3 minutes. Supernatants were quickly removed, and bacterial pellets were quick frozen in a dry-ice ethanol bath before proceeding for RNA extraction.
RNA was extracted using Nucleic Acid Extraction kits, e.g. RiboPure™ RNA extraction kit (Ambion, Austin Tex.). Briefly, a volume of 1 ml-20 ml of sample was centrifuged to harvest the bacteria at 4° C. The pellets were processed according to the extraction kit protocol.
Real-time PCR: Real-time PCR was performed on the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems, Foster City Calif.). For real-time RT-PCR, ARRAYSCRIPT™ reverse transcriptase (Ambion, Austin Tex.) was included in the reactions. The Environmental Master Mix v2 (Applied Biosystems) was used for all reactions. PCR was performed under standard cycling conditions with a reverse transcription step (30 minutes at 42° C.; 10 minutes at 95° C.; 40 cycles of 15 seconds at 95° C., 1 minute at 60° C.).
Primers and Probes: TAQMAN® real-time PCR assays, which include primers and probes for each assay, were designed using a rigorous bioinformatics assay design pipeline against candidate inducible targets in Salmonella enterica (Table 1). Full length genes representing each of the candidate targets were obtained from the annotated, publicly available genome sequences of Salmonella dublin or Salmonella typhi. These gene sequences were used to query the GenBank database and obtain all other publicly available, homologous Salmonella sequences. Assays were designed to match all of the related Salmonella sequences, while not matching any other sequences in the microbial subset of GenBank. The probes were labeled with a FAM™ dye to enable detection by real-time PCR.
Assays designed for Salmonella are shown in Table 1, which has a column designated as “Gene” indicating the name of the inducible target gene; a column designated as “Assay” which indicates an assay number given to the combination of “Forward Primer” and “Reverse Primer” and “Probe” in the same row as the assay number. For example, one Salmonella assay, for detecting the inducible target gene agsA, may comprise assay agsA.0 and use the primer of SEQ ID NO: 1 (forward primer), the primer of SEQ ID NO: 134 (reverse primer) and the probe of SEQ ID NO: 267. In another example, one Salmonella assay, for detecting the inducible target gene agsA, may comprise assay agsA.1 and use the primer of SEQ ID NO: 2 (forward primer), the primer of SEQ ID NO: 135 (reverse primer) and the probe of SEQ ID NO: 268.
The assays were evaluated under uninduced conditions as well as induced conditions (45° C., 15 minutes) using pure cultures of Salmonella enterica. Duplicate samples were maintained at 37° C. (uninduced) or 45° C. (heat-induced) for 15 minutes before processing for nucleic acids. The extracted nucleic acids were assayed for induction by real-time reverse-transcriptase-PCR (DNA+ RNA). To measure contribution due to DNA alone, control reactions were run without addition of the reverse transcriptase (DNA). Transcriptional activity (contribution to Ct value by RNA alone) was estimated by subtracting the Ct value obtained for DNA and RNA (with reverse transcriptase reaction) from the Ct value obtained for DNA alone (without reverse transcriptase reaction) for both uninduced and induced samples.
Induction was also evaluated with respect to enrichment time.
When combined with a larger volume of starting sample (e.g 20 ml), a Ct difference of as much as 9 Ct was observed with spinach rinse samples (
Results for enriched samples were confirmed by plating on CHROMagar™ plates (CHROMager Microbiology, Paris France). That is, following enrichment, samples were withdrawn for RNA extraction and, in parallel, samples were also plated for culture confirmation (CHROMAgar) to verify presence of pathogen. The plate results were recorded following overnight growth at 37° C. A 100% correlation was obtained between RT-PCR results and plate confirmation.
The present example describes identification and evaluation of inducible RNA targets for detection of the pathogenic microbe Listeria monocytogenes using real-time RT-PCR.
Materials and Methods; Bacteria and Growth Conditions: A Listeria monocytogenes overnight culture (Brain Heart Infusion broth (BHI), Teknova, Hollister, Calif.) was diluted and grown at 37° C. to obtain exponential phase cells (5 hours). For spiking food samples, 25 g or 25 ml of the food sample was transferred into stomacher bags. Bacteria were spiked in the 1-100 cfu range. Samples were enriched according to standard procedures as described in the Bacteriological Analytical Manual (U.S. Food and Drug Administration, Bacteriological Analytical Manual, Chapter 10, See www.fda.gov). Enrichment media (225 ml) was added to each food sample and sample; for enrichment of Listeria, samples were enriched in Buffered Listeria Enrichment Broth with selective supplements (Buffered Listeria Enrichment Broth, EMD Chemicals, Gibbstown, N.J.). Samples were enriched at 37° C. for a maximum of 24 hours.
Inducing Conditions: For Listeria, several different inducing conditions were evaluated as follows: a) heat induction at 48° C., 20 minutes, b) salt stress at 0.3M NaCl final concentration for 10 minutes at 37° C., c) activated charcoal induction for 5 hours, d) acid stress for 10 minutes at 37° C., and e) cold stress. For b), a 1:1 dilution of culture with brain heart infusion broth containing 0.6M NaCl was made to provide a final concentration of 0.3M NaCl. For c), the Listeria was grown in buffered Listeria enrichment broth treated with activated charcoal. The media was prepared by adding activated charcoal to enrichment broth to 0.2% final volume before autoclaving, stirred for 1 hour, then autoclaved and filtered through a 0.22 uM membrane. For d), a 1:3 dilution of culture in acidified brain heart infusion broth was made. Acidified BHI is prepared by adding HCl to BHI, and checking pH. For example, 100 μl HCl added to 5 ml BHI resulted in a pH of 2.5. For e), for food matrix experiments, the enriched sample was held on ice for 10 minutes. Uninduced controls were maintained at 37° C.
Sample Preparation and Real-time PCR: Sample preparation and real-time PCR procedures were performed as for Example 1.
Primers and Probes: TAQMAN® real-time PCR assays, which include primers and probes for each assay, were designed using a rigorous bioinformatics assay design pipeline against candidate inducible targets in Listeria (Table 2). Full length genes representing each of the candidate targets were obtained from the annotated, publicly available genome sequences of L. monocytogenes EGD-e. These gene sequences were used to query the GenBank database and obtain all other publicly available, homologous Listeria sequences. Assays were designed to match all of the related Listeria sequences, while not matching any other sequences in the microbial subset of GenBank. The probes were labeled with a FAM™ dye to enable detection by real-time PCR.
Assays designed for Listeria are shown in Table 2, which has a column designated as “Gene” indicating the name of the inducible target gene; a column designated as “Assay” which indicates an assay number given to the combination of “Forward Primer” and “Reverse Primer” and “Probe” in the same row as the assay number. For example, one Listeria assay, for detecting the inducible target gene inlA, may comprise assay inlA.0 and use the primer of SEQ ID NO: 400 (forward primer), the primer of SEQ ID NO: 855 (reverse primer) and the probe of SEQ ID NO: 1310. In another example, one Listeria assay, for detecting the inducible target gene lmo0539, may comprise assay lmno0539.1 and use the primer of SEQ ID NO: 422 (forward primer), the primer of SEQ ID NO: 877 (reverse primer) and the probe of SEQ ID NO: 1332.
The assays were evaluated under uninduced conditions as well as under induced conditions using pure cultures of Listeria monocytogenes. The extracted nucleic acids were assayed for induction by real-time reverse-transcriptase-PCR (DNA+RNA). To measure contribution due to DNA alone, control reactions were run without addition of the reverse transcriptase (DNA). Transcriptional activity (contribution to Ct value by RNA alone) was estimated by subtracting the Ct value obtained for DNA and RNA (with reverse transcriptase reaction) from the Ct value obtained for DNA alone (without reverse transcriptase reaction) for both uninduced and induced samples.
Heat-induction: Duplicate samples were maintained at 37° C. (uninduced) or 48° C. (heat-induced) for 20 minutes before processing for nucleic acids. Targets were initially evaluated using a pure culture of Listeria (
Charcoal-induction: A parallel sample was grown in enrichment media treated with 0.2% activated charcoal for 5 hours, along with parallel uninduced samples. Growth in media treated with activated charcoal (
Salt-Induction: One aliquot of an exponentially grown culture was treated to enrichment media containing 0.3M NaCl (final concentration) for 10 minutes. Uninduced samples were processed the same way with media alone. Brief exposure to high salt (
Acid Induction: For acid conditions, a bacterial culture (2 mL) was diluted in 6 mL acidified BHI (acidified BHI prepared by adding HCl to BHI, and checking pH: such as 100 ul HCl to 5 ml BHI resulted in pH=2.5); incubation at 37° C., 10 minutes, with shaking. The moderately inducible RNA targets were: lmo0189 (encoding a sequence highly similar to B. subtilis Veg protein) and lmo2522 (encoding a sequence similar to hypothetical cell wall binding protein from B. subtilis). For both transcripts, acid conditions produced ˜3 fold induction. At the same time, the amount of an additional lmo0699 transcript (encoding a sequence similar to flagellar switch protein FliM) was ˜3 fold reduced under the acidic conditions.
Cold Induction: For cold conditions, a culture was placed on ice for 16, 20, or 24 minutes. The moderately inducible RNA targets were: lmo0189 (encoding a sequence highly similar to B. subtilis Veg protein) and lmo2522 (encoding a sequence similar to hypothetical cell wall binding protein from B. subtilis). For both transcripts, cold conditions produced ˜4 fold induction.
The Listeria targets were also evaluated in the context of a time-course study for activated charcoal induction in a milk sample. Raw milk (50 ml) was spiked with approximately 60 cfu of L. monocytogenes. The sample was split equally into 2×25 ml samples. One 25 ml sample was enriched in 225 ml of BLEB with supplements (Buffered Listeria Enrichment Broth, EMD Chemicals, Gibbstown, N.J.). The other 25 ml aliquot was enriched in BLEB treated with 0.2% activated charcoal (supplements added at the beginning of enrichment). Samples were incubated at 37° C. and withdrawn at 16 and 24 hours post-enrichment.
Considering the long generation times for Listeria growth, an improvement of 0.5-1Ct in a detection assay can translate into reducing the enrichment times by 1-1.5 hours and a difference of 4 Ct will allow for at least a 4 hour reduction in enrichment time.
Results for enriched samples were confirmed by plating on CHROMagar™ plates (CHROMager Microbiology, Paris France). That is, following enrichment, samples were withdrawn for RNA extraction and, in parallel, samples were also plated for culture confirmation (CHROMAgar) to verify presence of pathogen. The plate results were recorded following overnight growth at 37° C. A 100% correlation was obtained between RT-PCR results and plate confirmation.
The present example provides identification and evaluation of inducible RNA targets for detection of the pathogenic microbe Vibrio cholerae using real-time RT-PCR. The normal growth temperature of Vibrio species is at 30° C.; it was discovered that growing Vibrio at 37° C. induced RNA such that reverse transcription RT-PCR detection increased sensitivity of the assay.
Sample Preparation and Real-time PCR: A single Vibrio cholerae bacterial colony was inoculated into 5 ml of nutrient enrichment broth containing 3% NaCl and grown overnight at 37° C. Nucleic acids were prepared for real-time PCR and real-time RT-PCR using PrepSEQ™ nucleic acids extraction protocol. Samples were assayed with and without the inclusion of the reverse transcriptase step.
Primers and Probes: TAQMAN® real-time PCR assay primers and probes are listed in Table 3. The hsp60 gene contains several polymorphic sequences and thus, a combination of three probes is provided for detecting all Vibrio cholerae strains. The probes were labeled with a FAM™ dye to enable detection by real-time PCR.
Results from PCR and reverse transcriptase RT-PCR assays for the hsp60 target demonstrate a significant difference in detection levels as shown by the data of
The present disclosure has evaluated food testing workflow methods using inducible RNA targets in assays for detection of various bacteria. A fast workflow method is provided based on inducible transcription response in bacteria.
A typical workflow, according to this disclosure, includes a shortened enrichment step, a rapid induction step, an automated sample preparation step, and specific detection of induced target using reverse transcriptase RT-PCR.
In one embodiment, a workflow of the disclosure for Salmonella is as follows:
Enrichment (6 hours)Induction+Sample Prep (45 min)
Real-Time RT-PCR (1 hour)
An example workflow is shown in
As shown in
Additionally,
Example workflows provided herein allowed for detection of 1-5 cfu of Salmonella in less then 8 hr (in 6 hours for the workflows shown in
The present disclosure describes methods of detecting viable bacteria as induced RNA gene targets are only expressed in live organisms thus, detecting only live organisms and not dead organisms (which cannot cause diseases in samples). Accordingly, methods of the disclosure also reduce wastage of food attributed to amplification of dead microbes by traditional testing methods.
To test viability of detected bacteria, duplicate samples of Salmonella culture were prepared. One set was heat-treated at 95° C. for 15 minutes to kill the bacteria. A control set was maintained at 37° C. Both sets were induced at 45° C. for 15 minutes, followed by sample extraction. Assays were evaluated by real-time PCR in the presence and absence of ArrayScript® M-MLV reverse transcriptase. Live and viable cells responded to the heat-induction by increase in target mRNA production, while the heat-killed cells did not. These results are shown in
The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed in part by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the compositions, kits and methods steps set forth in the present description.
When a group of materials, compositions, components or compounds is disclosed herein, it is understood that all individual members of those groups and all subgroups thereof are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure.
This patent application claims priority under 35 U.S.C. §119(e) to United States Provisional Patent Application, U.S. Ser. No. 61/360,337, filed Jun. 30, 2010, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61360337 | Jun 2010 | US |
