Comprehensive microbial panel for molecular diagnosis of eye infections

Abstract

Methods for sensitive and reliable qualitative detection and identification of the common pathogens associated with ocular infections, including uveitis, endophthalmitis and keratitis.

Description

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 29, 2020, is named SequenceListing.txt and is 23,255 bytes in size.

TECHNICAL FIELD

Described herein are kits, devices, and methods for diagnosing eye infections.

BACKGROUND

Eye infections are among the greatest causes of blindness worldwide. The sooner effective therapy can be started, the more vision that can be saved. In an era of increasing resistance, broad-spectrum antibiotics can no longer be relied upon.

SUMMARY

New technologies have the potential to significantly increase diagnostic sensitivity and quickly detect the microbe causing an infection, as well as its antibiotic resistance, providing the physician with critical information in hours, rather than days, and for a greater number of patients. The newly developed comprehensive eye panel described here is expected to support rapid treatment with the correct drug, which will limit the often-irreversible destruction that results in loss of vision.

Provided herein are methods comprising: providing a sample comprising, or suspected of comprising, a pathogen; optionally purifying genomic DNA from the pathogen; performing PCR by contacting the sample with a plurality of sets of primers to amplify each of at least 10, 20, 30, 40, or all of the target sequences listed in Table 1 (or its complement) in genomic DNA from the pathogen; and contacting the amplified DNA with a plurality of detectable probes, wherein the plurality comprises 1, 2, or more detectable probes that bind to each of the target sequences listed in Table 1 (or its complement).

Also provided are methods for determining identity of a pathogen in a sample, the method comprising: providing a sample comprising, or suspected of comprising, a pathogen; optionally purifying genomic DNA from the pathogen; performing PCR by contacting the sample with a plurality of sets of primers to amplify each of at least 10, 20, 30, 40, or all of the target sequences listed in Table 1 (or its complement) in genomic DNA from the pathogen; and contacting the amplified DNA with a plurality of detectable probes, wherein the plurality comprises 1, 2, or more detectable probes that bind to each of the target sequences listed in Table 1 (or its complement); detecting binding of the 1, 2, or more probes to the amplified DNA; determining the identity of the probes bound to the amplified DNA; and correlating the identity of the bound probes to a pathogen.

In addition, provided herein are methods for diagnosing an infection in a subject who has uveitis or detecting the presence of a pathogen in a subject who has uveitis. The methods include providing a sample from an eye of the subject comprising, or suspected of comprising, a pathogen; optionally purifying genomic DNA from the pathogen; performing PCR by contacting the sample with a plurality of sets of primers to amplify each of at least 10, 20, 30, 40, or all of the target sequences listed in Table 1 (or its complement) in genomic DNA from the pathogen; and contacting the amplified DNA with a plurality of detectable probes, wherein the plurality comprises 1, 2, or more detectable probes that bind to each of the target sequences listed in Table 1 (or its complement); detecting binding of the 1, 2, or more probes to the amplified DNA; determining the identity of the probes bound to the amplified DNA; and correlating the identity of the bound probes to a pathogen, thereby diagnosing the infection in the subject or identifying the pathogen.

Further, provided herein are methods for selecting a treatment and optionally treating a subject who has uveitis, the methods comprising: providing a sample from an eye of the subject comprising, or suspected of comprising, a pathogen; optionally purifying genomic DNA from the pathogen; performing PCR by contacting the sample with a plurality of sets of primers to amplify each of at least 10, 20, 30, 40, or all of the target sequences listed in Table 1 (or its complement) in genomic DNA from the pathogen; and contacting the amplified DNA with a plurality of detectable probes, wherein the plurality comprises 1, 2, or more detectable probes that bind to each of the target sequences listed in Table 1 (or its complement); detecting binding of the 1, 2, or more probes to the amplified DNA; determining the identity of the probes bound to the amplified DNA; and correlating the identity of the bound probes to a pathogen; and selecting and optionally administering to the subject a treatment for the pathogen, preferably wherein the treatment is selected according to Table A.

In some embodiments of the methods described herein, determining the sequence identity comprises using high resolution melting analysis.

In some embodiments of the methods described herein, the sequence identity of the amplified portion is correlated to a pathogen according to table 1.

In some embodiments of the methods described herein, the sample comprises aqueous humor, vitreous humor, or vitreous wash.

Also provided herein is a kit for use in a method described herein comprising: a plurality of sets of primers that amplify each of at least 10, 20, 30, 40, or all of the target sequences listed in Table 1 (or its complement); a plurality of probes that bind to each of the at least 10, 20, 30, 40, or all of the target sequences listed in Table 1 (or its complement), preferably wherein the plurality comprises at least 2 probes that bind to each of the target sequences; and optionally one or more reagents for performing a multiplexed gene analysis method, e.g., a hybridization based digital barcode quantification assays.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 provides an exemplary workflow for a method described herein.

FIG. 2 is a bar graph showing detection of VZV in a sample spiked with control VZV DNA.

FIG. 3 is a trio of bar graphs showing the analytical sensitivity of detection tested with common organisms causing eye infections. Serial 10-fold dilutions ranging from 2.5 pg/reaction to 2.5 fg/reaction for a Gram-positive (MRSA USA300), a Gram-negative (P. aeruginosa) organism, and cytomegalovirus (CMV) were tested. The limit of detection for the 3 organisms was 25 femtograms of DNA per reaction.

DETAILED DESCRIPTION

Presently, infections of the eye are typically treated empirically with an antibiotic or antiviral depending on the initial clinical evaluations until a definitive diagnosis is returned from the clinical microbiology laboratory. Because many ocular pathogens cannot be readily cultured, and because of the small sample size, after several days of effort, a culture report is often returned as negative despite clear evidence of an infection in the patient. Time delays and low sensitivity of pathogen detection result in patients being treated empirically or with the wrong drug for long periods. In the interim, the infection continues its destructive path—much of it preventable if the pathogen and its susceptibilities had been more quickly identified. For these reasons, there is considerable interest in molecular detection of eye pathogens directly from ocular tissues, which has shown to significantly improve diagnosis sensitivity^1-7. Previously, detection was mainly done by regular monoplex PCR assays for detection of one target at a time, or by multiplex PCR tests that are constricted by the number of fluorescent agents that current real time PCR systems can detect at the same time.

Described herein are unique multiplex panels for sensitive, accurate, and rapid diagnosis of sight-threatening ocular infections, including uveitis, endophthalmitis and keratitis, which can be implemented using commercially-available technology such as the molecular barcoding platform NanoString. The present methods provide an unparalleled possibility for comprehensive detection of the most clinically relevant organisms that cause eye infections in just one assay. By consolidating the detection of bacteria, viruses, fungi and parasites in only one platform, the use of this panel will not only save time on detection, but also spare precious small-volume ocular specimens. The detection of a variety of organisms that otherwise would require the use of multiple specific diagnostic tests, and a relatively high volume of sample, is possible with only a tiny ocular specimen in a single reaction. In addition to being uniquely designed to detect the most important ocular pathogens in a multiplex fashion, identification of pathogen DNA takes place in a highly sensitive platform for nucleic acid detection, which is expected to considerably improve the diagnostic sensitivities. Combined, the advantages of this panel is expected to greatly impact the clinical course of eye infections by supporting quick treatment with the correct drug, which will limit the often-irreversible destruction that results in loss of vision.

Methods of Diagnosis

Included herein are methods for sensitive and reliable qualitative detection and identification of the common pathogens associated with ocular infections, including uveitis, endophthalmitis and keratitis, including those shown in Table 1, directly from intraocular fluids in a single closed-tube reaction and methods of treatment based thereon. The methods include obtaining a sample comprising intraocular fluid from a subject, e.g., a sample comprising aqueous humor, vitreous humor, or vitreous wash, and evaluating the presence and/or level of a biomarker in the sample. Vitreous wash is the mix of vitreous and balanced salt solution that collects in the cassette of the vitrectomy machine during vitrectomy surgery, i.e., diluted vitreous humor. The sample can be obtained, e.g., by anterior paracentesis (aqueous samples), posterior chamber paracentesis (undiluted vitreous samples), or during pars plana vitrectomy (both undiluted vitreous and diluted vitreous washing samples).

The methods can optionally include comparing the presence and/or level of one or more of the biomarkers that can be used to determine the presence of a pathogen as described herein with one or more references, e.g., a control reference that represents a normal level of the biomarker e.g., a level in an unaffected subject, and/or a disease reference that represents a level of the biomarker associated with a specific infection, e.g., a level in a subject having an infection with a pathogen listed in Table 1. In some embodiments, the detection of any level of pathogen DNA indicates the presence of an infection with that pathogen.

The methods include first amplifying any sequences listed in Table 1 that are present in the sample using methods known in the art, e.g., using polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative or semi-quantitative real-time RT-PCR, or digital PCR i.e. BEAMing ((Beads, Emulsion, Amplification, Magnetics) Diehl (2006) Nat Methods 3:551-559), e.g., using primers that amplify each of the followed by sequence identity analysis.

The presence of a sequence listed in Table 1 can be evaluated using methods known in the art, e.g., using polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative or semi-quantitative real-time RT-PCR, digital PCR i.e. BEAMing ((Beads, Emulsion, Amplification, Magnetics), Diehl (2006) Nat Methods 3:551-559); various types of nucleic acid sequencing (Sanger, pyrosequencing, NextGeneration Sequencing); multiplexed gene analysis methods, e.g., oligo hybridization assays including DNA microarrays; hybridization based digital barcode quantification assays such as the nCounter® System (NanoString Technologies, Inc., Seattle, WA; Kulkarni, Curr Protoc Mol Biol. 2011 April; Chapter 25:Unit25B.10) and lysate based hybridization assays utilizing branched DNA signal amplification such as the QuantiGene 2.0 Single Plex and Multiplex Assays (Affymetrix, Inc., Santa Clara, CA; see, e.g., Linton et al., J Mol Diagn. 2012 May-June; 14(3):223-32); SAGE, high-throughput sequencing, multiplex PCR, MLPA, luminex/XMAP, or branched DNA analysis methods. See, e.g., WO2012/048113, which is incorporated herein by reference in its entirety.

In some embodiments, the level of one or more of the biomarkers evaluated is comparable to or above the level of the biomarker in a disease reference, then the subject can be diagnosed with an infection with the pathogen associated with the biomarker. In some embodiments, once it has been determined that a person has an infection with an identified pathogen, then a treatment, e.g., as known in the art or as described herein, can be administered.

Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful level of the biomarker, e.g., a control reference level that represents a normal level of the biomarker, e.g., a disease reference that represents a level of the biomarker associated with a specific infection.

The predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk or presence of disease in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n-quantiles being subjects with the highest risk.

In some embodiments, the predetermined level is based on the level of detection of the assay used, and a level of the biomarker above the level of detection indicates the presence of the pathogen associated with that biomarker.

In preferred embodiments, one, two, or more detectable probes (e.g., probes that are detectable using a multiplex gene analysis method as known in the art and/or described above) that bind to each of the sequences listed in Table 1 are used in the present methods. In embodiments where two or more probes for each sequence are used, the number of false positives can be limited by requiring detection of binding of both probes in order to determine that a sequence is present in the sample.

Targeted Treatment

Once the presence of a specific pathogen has been identified using a method described herein, the method can include selecting and optionally administering a treatment specific for that pathogen as known in the art. For example, a treatment according the following Table A can be selected and/or administered:

TABLE A
Pathogen            Treatment
Herpes simplex      Systemic antiviral therapy (e.g., acyclovir, famciclovir,
virus 1 or 2        or valacyclovir) for uveitis and keratitis
(HSV1/2)            Intravitreal foscarnet or ganciclovir for uveitis
                    Topical ganciclovir gel, trifluridine and acyclovir oitments
                    for keratitis
Cytomegalovirus     Oral or IV antiviral therapy (e.g., valganciclovir, ganciclovir,
(CMV)               foscamet, or cidofovir)
                    Intravitreal foscarnet or ganciclovir for uveitis
                    Topical ganciclovir gel for keratitis
Varicella zoster    Oral or IV antiviral therapy (e.g., acyclovir, famciclovir,
virus (VZV)         valacyclovir) for uveitis and keratitis
                    Intravitreal foscarnet or ganciclovir for uveitis
Epstein-barr virus  Acyclovir, ganciclovir, valacyclovir and foscarnet
(EBV)
Toxoplasma gondii   Monotherapy or combination therapy with systemic antimicrobials.
(T. gondii)         The most commonly used are sulfadiazine, Bactrim, spiramycin,
                    clindamycin, pyrimethamine, atovaquone, andr azithromycin. The
                    traditional gold standard regimen has been triple drug therapy with
                    pyrimethamine (loading dose of 75-100 mg during the first day,
                    followed by 25-50 mg on subsequent days), sulfadiazine (loading
                    dose of 2-4 g during the first 24 h followed by 1 g qid), and
                    prednisone. Intravitreal clindamycin is also used in some cases.
Gram-positive       Intravitreal vancomycin (1 mg) for endophthalmitis. Systemic
cocci               antibiotics (e.g. linezolid and moxifloxacin) can be used.
                    Topical cefazolin (50 mg/ml), tobramycin or gentamicin (9-14
                    mg/ml) vancomycin (15-50 mg/ml), bacitracin (10,000 IU) and
                    fluoroquinolones (0.3% to 0.5%) for keratitis
Methicillin-        Intravitreal vancomycin (1 mg) for endophthalmitis.
resistant S. aureus Topical cefazolin (50 mg/ml), tobramycin or gentamicin (9-14
                    mg/ml) vancomycin (15-50 mg/ml) or bacitracin (10,000 IU) for
                    keratitis. Linezolid and daptomycin could also be used for treatment
                    of both infections.
Gram-negative       Intravitreal ceftazidime (2.25 mg) or amikacin (0.1 mg) for
bacilli             endophthalmitis. Systemic antibiotics (e.g. moxifloxacin) can be
                    used. Topical tobramycin or gentamicin (9-14 mg/ml), ceftazidime
                    (50 mg/ml) and fluoroquinolones (0.3% to 0.5%) for keratitis
Candida spp.        Intravitreal amphotericin B (5 to 10 μg) or voriconazole (100 μg)
                    plus systemic therapy with amphotericin, fluconazole or
                    voriconazole and flucytosine for endophthalmitis. Oral fluconazole.
                    Topical amphotericin B solution or flucytosine, fluconazole and
                    itraconazole for keratitis.
Aspergillus spp.    Intracameral amphotericin (5 μg) or voriconazole (50 μg) and
                    intravitreal amphotericin (5 to 10 μg) or voriconazole (100 μg), plus
                    systemic voriconazole for endophthalmitis. Oral itraconazole
                    or voriconazole. Topical natamycin, amphotericin B, or
                    flucytosine, fluconazole and itraconazole for keratitis. Topical
                    voriconazole can be used for recalcitrant cases.
Fusarium spp.       Intracameral amphotericin (5 μg) or voriconazole (50 μg) and
                    intravitreal amphotericin (5 to 10 μg) or voriconazole (100 μg), plus
                    systemic voriconazole for endophthalmitis. Oral itraconazole
                    or voriconazole. Topical natamycin or flucytosine, fluconazole
                    and itraconazole for keratitis. Topical voriconazole can be used
                    for recalcitrant cases.
Mycobacterium       Isoniazid 5 mg/kg/day, rifampicin 450 mg/day if body weight is <50
tuberculosis        kg and 600 mg if the weight is >50 kg, ethambutol 15 mg/kg/day,
                    and pyrazinamide 25-30 mg/kg/day) for 8 weeks, followed by two
                    drugs (rifampicin and isoniazid) for at least another 18 weeks
Treponema           Intravenous penicillin (4 million units every 4 h for 10 to 14 days)
pallidum
Borrelia            Intravenous ceftriaxone (2 g daily from 10 to 28 days) is the
burgdorferi         preferred therapy. Alternative includes intravenous penicillin G (15
                    to 20 million units daily for 10 to 14 days) and oral doxycycline (2
                    weeks)
Tropheryma          Intravenous therapy ceftriaxone (2 g daily for 2-4 weeks) or
whipplei            meropenem. Oral trimethoprim-sulfamethoxazole and doxycycline

Kits

Included herein are kits that can be used in the present methods. The kits can include containers holding one or more of the primers, e.g., pairs of primers that can be used to amplify each of the sequences shown in Table 1, one or more detectable probes that bind to each of the sequences shown in Table 1, as well as one or more reagents for performing the method, e.g., reagents for use in multiplexed gene analysis methods as described herein or known in the art.

Examples

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Methods

Selection of target sequences and panel design. Using sources including epidemiological information on the etiologies of ocular infections seen at our hospital, we identified 46 pathogens and 2 resistant/virulence markers that are commonly associated with ocular infections. This panel of organisms (Table 1) accounts for >90% of pathogens isolated from eye specimens. Genomic targets commonly used for PCR-based detection of these pathogens were scrutinized for stretches that are predicted to be specific for a particular species while being conserved across different strains from the same species. Regions of 150 to 300 bp in length were selected and a set of primers for pre-enrichment, and two 50 mer probes per target were designed to these sequences in collaboration with NanoString to incorporate their proprietary barcode sequences and bioinformatics algorithms to design probes that minimize cross-reactivity, and select probes with optimal thermodynamic profiles for optimized hybridization.

TABLE 1
Target	Gene	Target Sequence	#
Gram positive rods and cocci
Bacillus	RNA	GAATGCAAAGAGCGTGATGTGACGTATGCAGCACCACTTC	1
cereus group	polymerase	GTGTAAAAGTGCGTCTAATCAACAAGGAAACTGGTGAAGT
	subunit B	AAAAGAACAAGATGTGTTCATGGGAGATTTCCCACTCATG
	(rpoB)	ACAGAGACTGGAACATTCGTAATTAACGGTGCAGAACGTG
		TTATCGTTTCCCAGTTAGTTCGCTCTCCAAGCGTATACTATA
		GTGGCAAAGTGGATAAAAACGGAAAACGTGGTTTTACTGC
		TACTGTAATTCCAAACCGCGGAGCTTGGTTAGAGTATGA
Bacillus	23S	CCCAGGCGACTGTTTAGCAAAAACACAGGTCTCTGCGAAG	2
subtilis	ribosomal	CCGTAAGGCGAAGTATAGGGGCTGACGCCTGCCCGGTGCT
group	subunit	GGAAGGTTAAGAGGAGCGCTTAGCGTAAGCGAAGGTGCGA
		ATTGAAGCCCCAGTAAACGGCGGCCGTAACTATAACGGTC
		CTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCCGC
		ACGAAAGGCGCAACGATCTGGGCACTGTCTCAACGAGAGA
		CTCGGTGAAATTAT
Enterococcus	D-Ala:D-	TAGAAAGCGACATCTTTCACCACTTCACCAGGTAAAGTCGT	3
faecalis	Ala ligase	ACGGACATCTTCATTTCCTAAAATGGCTACTTCAATTTCAC
	(ddl)	GTGCTTCGATCCCTTGTTCAACAATTGCTCGGGCATCATAA
		CGGAAAGCTTCTTCCAATGCTTCTTGCAATTCTTCACGATTT
		TCCACTTTGCTAATTCCGACACTAGAACCCATATTGGCAGG
		TTTAACAAAGACCGGATAAATTAAAGAACCTTCACATTTTT
		CAAAGACTTCTTTTGGATTTCCTTTCCAGTCACTTCTTAAAA
		CTGGCACGAAT
Enterococcus	D-Ala:D-	TTATTCATTTTTTTCAAAAAAAGATTGACGCTGATGGTATC	4
faecium	Ala ligase	GATTCATTCCTAACTGGATCAGTTCTTCAATCAAATCACCG
	(ddl)	TATTTCAAGCCCATATTTTCCCATAAGAGTGGGTACATACT
		GAACTCCGTAAATCCTGGCATAGAGTTTAATTCATTCAGGA
		ATAATTCATTTTTATTTGTCAAAAAGAAATCGCACCGGCTC
		AATCCGCTTCCACCTAACATCGTGTAAGCTAACTTCGCGTA
		CTCTTGCGCTTTTTGATAAACTTCTTCTGGCACTTCGGCTGG
		AATCTGCATTTC
Mycobacterium	Immuno-	CCGTGGTGCTCAAGGTCTACCAGAACGCCGGCGGCACGCA	5
tuberculosis	genic	CCCAACGACCACGTACAAGGCCTTCGATTGGGACCAGGCC
	protein	TATCGCAAGCCAATCACCTATGACACGCTGTGGCAGGCTGA
	MPT64	CACCGATCCGCTGCCAGTCGTCTTCCCCATTGTGCAAGGTG
	(mpt64)	AACTGAGCAAGCAGACCGGACAACAGGTATCGATAGCGCC
		GAATGCCGGCTTGGACCCGGTGAATTATCAGAACTTCGCAG
		TCACGAACGACGGGGTGATTTTCTTCTTCAACCCGGGGGAG
		TTGCTGCCCGAAGCAG
Propionibacterium	Triacyl-	ATGAAGATCAACGCACGATTCGCCGTCATGGCCGCGAGTG	6
acnes	glycerol	TGGCCGTCCTGATGGCTGCCGCGCCGATTGCGCAGGCTGCT
	lipase	ACTTCGCCGGGGGATATCCATCCCCTGGTCCAGGCAGCCCA
	precursor	CAGCCCCGACGGTATTCCCGGTAACGGCGTCGGGCCGGAA
		TTCCATACGTCGTCGATGGCGCGTTCCTACAGCGAGAAGCA
		CCTGGGCGTGGCGCCGCGGGGTGTGAACGACTTCTCCTGCA
		AGGTCAAGCCCGGCGACCGACCGGTCATCCTGATTCCCGGT
		ACTGGCGGCAATGCG
Staphylococcus	23S	AGTATTTGGTCGTAGACCCGAAACCAGGTGATCTACCCWT	7
spp.	ribosomal	GGTCAGGTTGAAGTTCAGGTAACACTGAATGGAGGACCGA
	subunit	ACCGACTTACGTTGAAAAGTGAGCGGATGAACTGWGGGTA
		GCGGAGAAATTCCAATCGAACTTGGAGATAGCTGGTTCTCT
		CCGAAATAGCTTTAGGGCTAGCCTCAAGTGATGATTATTGG
		AGGTAGAGCACTGTTTGGACGAGGGGCCYCTCTCGGGTTA
		CCGAATTCAGACAAACTCCGAATGCCAAT
Staphylococcus	Immuno-	GCTGATAACAATTTCAACAAAGAACAACAAAATGCTTTCTA	8
aureus	globulin G	TGAAATCTTGAATATGCCTAACTTAAACGAAGAACAACGC
	binding	AATGGTTTCATCCAAAGCTTAAAAGATGACCCAAGCCAAA
	protein A	GTGCTAACCTATTGTCAGAAGCTAAAAAGTTAAATGAATCT
	(spa)	CAAGCACCGAAAGCGGATAACAAATTCAACAAAGAACAAC
		AAAATGCTTTCTATGAAATCTTACATTTACCTAACTTAAAC
		GAAGAACAACGCAATGGTTTCATCCAAAGCCTAAAAGATG
		ACCCAAGCCAAAGCGCT
Staphylococcus	Thermo-	TCAATTTATTAACCACGAAGGTCCATTTGGCGGTAAACAAT	9
capitis	nuclease	CAAATGAAAAAAATCTAAGCGCAGATTTAAAAGGAAAAGA
		TAAAGTTTATGTTGAACGTGTAGTAGATGGGGATACTTTTC
		TTGCTAAGAAAGATGGCGAGCGTATTAAAGTTAGAATGAT
		TGGTATGGATACACCAGAAACGGTTAAACCAAATACGCCT
		GTTCAACCCTATGGTAAAGAAGCATCAAACTATAGTAAGA
		AAGAGTTAACACATAAGTATGTTTATTTAGAATACGATAAA
		GAAAAAAATGATAGATA
Staphylococcus	Thermo-	CGATGATAGGAATACTTGTAATTATTTTCCAGTTTGTAAAC	10
epidermidis	nuclease	CATTCTGGACCGTTTAGTGATTCAGAATCTCAACATCAATC
		AGATAATTCCAATTTAAATGGTAAAGACAAAGTATATGTG
		AAACGAGTTGTAGATGGTGATACATTTGTTGCTCAAAAAAA
		TGGAGAGGAAATTAAAGTCAGATTAATTGGTGTAGATACG
		CCAGAGACTGTTAAGCCTAATACGCCAGTTCAACCATATGG
		TAAACAAGCATCTAATTATACGAAGAAGTATCTCACGCATC
		AAAATGTTTATTTAG
Staphylococcus	Thermo-	ACGCCTGAAACAGTGAAACCTAATACACCTGTACAGCCAT	11
lugdunensis	nuclease	ACGGTAAAGAAGCATCGCATTTTAGTAAAAAGAACTTAAC
		CAATAAAGATGTTTATCTGGAATATGATAAAGAAAAAAAT
		GATCGCTATGGACGTGTTTTAGCATATGTTTGGCTGGATAA
		AGATACATTATTTAATGAGCTATTAGTAAAAGAAGGGTTAG
		CTAAAGAAAAATACTTTGCACCTAATGGAAAATATAGAGA
		CGTCTTTATCAAAGCACAAAACGAGGCGCAAAAGAAAAAA
		ATCAATCTTTGGAGTTAG
Streptococcus	cAMP	ATGAACGTTAAACATATGATGTATCTATCTGGAACTCTAGT	12
agalactiae	factor (cfb)	GGCTGGTGCATTGTTATTTTCACCAGCTGTATTAGAAGTAC
		ATGCTGATCAAGTGACAACTCCACAAGTGGTAAATCATGTA
		AACAGTAATAATCAAGCCCAGCAAATGGCTCAAAAGCTTG
		ATCAAGATAGCATTCAGTTGAGAAATATCAAAGATAATGTT
		CAGGGAACAGATTATGAAAAAACGGTTAATGAGGCTATTA
		CTAGTGTTGAAAAATTAAAGACTTCATTGCGTGCCAACCCT
		GAGACAGTTTATGAT
Streptococcus	16S	AAAGGCAGTGGCTCAACCATTGTAGGCTTTGGAAACTGTTT	13
anginosus	ribosomal	AACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAG
group	subunit	CGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGA
		AAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAG
		CGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACG
		CCGTAAACGATGAGTGCTAGGTGTTAGGTCCTTTCCGGGAC
		TTAGTGCCG
Streptococcus	16S	CTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGA	14
mitts	ribosomal	TCGTAAAGCTCTGTTGTAAGAGAAGAACGRGTGTGAGAGT
group	subunit	GGAAAGTTCACACTGTGACGGTAWCTTACCAGAAAGGGAC
		GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCC
		CGAGCGTTRTCCGGATTTATTGGGCGTAAAGCGAGCGCAG
		GCGGTTAGATAAGTCTGAAGTTAAAGGCTGTGGCTTAACCA
		TAGTABGCTTTGGAAACTGTTTAACTTGAGTGCA
Streptococcus	Autolysin	ACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGT	15
pneumoniae	(lytA)	TGAACTGATTGAAAGCCATTCAACCAAAGAAGAGTTCATG
		ACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGC
		AGATGAAGCAGGTTTGCCGAAAACGCTTGATACAGGGAGT
		TTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACC
		AACCAAACAACCACTCAGACCACGTTGACCCTTATCCATAT
		CTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGA
		TATTGAGAACGGCTTGA
Streptococcus	Sodium	TCAGTGTCAAAGAAAAAGAGTTATTGACTAAAGAGCAATT	16
pyogenes	ATPase	TGATAAGCTATTGCAGGCTCCCAATACAACAACCTTAGCTC
	subunit C	GACTGTTGCACCAGTCAGTCTATCACCTAACTGTTGACGAT
	(ntpC)	CTCAACGATTTGGATCGGCTAGAATCTATCTTAATGGCTGA
		GCTAACCAAGACCTATCGCTGGGCTTTTGCAGAAACCCCTC
		AACCTGACATAGTACAATTATTTACCTTGCGTTACACCTAT
		CATAATGTCAAAGTGTTGTTAAAAGCTAAAGCTAGTCAAGC
		AGATCTGAGTCACT
Tropheryma	Repeat	AGAGAGATGGGGTGCAGGACAGGGTTTGTTTTGTACTGCTT	17
whipplei	region	GTAACAGGATCTATTAGGAGAGATACATTTGTGTTAGTTGT
	(TW625)	TACACATACTTCTTGTGTATTTGTATTACTTACTCTTGTGTA
		TTTGGTAYTAGATGAAACAGATGTAGATGAAACAGATGAT
		AGGAGGGATAGRGCAGGAGGTGTCTGTCTGGCAAAGGCTG
		TTTCATATTGTAGGGATGATAGGAGGAGAAG
Acinetobacter	16S-23S	GGGTCACAAGTTCAAGTCTTGTCAGACCCACCATGACTTTG	18
calcoaceficus-	intergenic	ACTGGTTGAAGTTATAGATAAAAGATACATGATTGATGATG
baumanii	space	TAAGCTGGGGACTTAGCTTAGTTGGTAGAGCGCCTGCTTTG
		CACGCAGGAGGTCAGGAGTTCGACTCTCCTAGTCTCCACCA
		GAACTTAAGATAAGTTCGGATTACAGAAATTAGTAAATAA
		AGATTAAGATCTTGGTTTATTAACTTCTGTGATTTCATTATC
		ACGGTAATTAGTGTGATCTGACGAAGACACATTAACTCATT
		AACAGATTGGCAA
Acinetobacter	blaOXA-	GCCTGACAGCATGTAGCCTACCCGTTTCATCTCTCCCATCTC	19
lwoffii	134	AAAGCATTTCGACTCAAGCGATTGCCAGCTTATTTGATCAG
		GCGCAAAGCTCTGGTGTTTTAGTGATTCAGCGTGATCAACA
		AGTACAGGTCTATGGCAATGATTTAAATCGTGCAAATACCG
		AATATGTTCCCGCCTCTACTTTTAAAATGCTCAATGCTCTGA
		TTGGCCTGCAACATGGCAAAGCCACAACCAATGAAATTTTT
		AAATGGGATGGCAAGAAACGCA
Enterobacter	DNA gyrase	GCTGGCGGTAACCGGTGAAACCGAAAGCACCGGTACCATG	20
aerogenes	subunit B	GTGCGTTTCTGGCCAAGCCTGGAAACCTTTACCAACGTCAC
	(gyrB)	TGAATTCGAATACGAAATCCTGGCGAAACGTCTGCGCGAG
		CTGTCGTTCCTCAACTCCGGGGTCTCTATCCGCCTGCGCGA
		TAAGCGCGACGGCAAAGAAGACCATTTCCACTACGAAGGC
		GGCATCAAGGCGTTTGTTGAGTATCTCAACAAGAACAAAA
		CGCCGATCCACCCGAATATCTTCTATTTCTCCACCGAAAAA
		GACGGTA
Escherichia	UDP-N-	CGTCATGGCATCGCCATTGCCGGAACGCACGGCAAAACGA	21
coli	acetylmura	CAACCACCGCGATGGTTTCCAGCATCTACGCAGAAGCGGG
	mate-L-	GCTCGACCCAACCTTCGTTAACGGCGGGCTGGTAAAAGCG
	alanine	GCGGGGGTTCATGCGCGTTTGGGGCATGGTCGGTACCTGAT
	ligase	TGCCGAAGCAGATGAGAGTGATGCATCGTTCCTGCATCTGC
	(murC)	AACCGATGGTGGCGATTGTCACCAATATCGAAGCCGACCA
		CATGGATACCTACCAGGGCGACTTTGAGAATTTAAAACAG
		ACTTTTATTAATTTTCTG
Haemophilus	Phosphate	TCAGGGTGGGCTGGAGCATTCGCATTAGCTTTATTGCTTAT	22
influenzae	ABC	TCCTATCGTGGTACGCACTACGGACAATATGTTATTACTTG
	transporter	TACCAAATAATTTACGTGAAGCAGCGGCAGCTCTTGGTTGC
	permease	TCTCAATGGCAAGTTATTATGATGATTTGTTATCGAGCAGC
	(pstA)	TAAATCAGGGATTTTAACTGGCGTGCTATTAGCAGTTGCCC
		GAATTTCAGGAGAAACTGCACCGCTATTATTTACCGCTCTG
		TCTAATCAATTTCTATCTTGGAATATGAATGAACCTATCGC
		AAATTTACCTGTA
Klebsiella	DNA gyrase	AGTTTCTCGCCCAGGGCACAAATACGACGATATTCCGGCCC	23
pneumoniae	subunit B	CATAATGAACTCGTTATCCAGCGGATAGTCGGTATCCACGC
	(gyrB)	CGTGGGTACGTACGCGAATAACCGGCTCAAAATGCTGCAG
		TTCTTTGTTCTCGTGGAGATCGAATTTCCACTGGCTGCCGTG
		CTGTTCTTTCTCGTTCAGCTC
Morganella	DNA gyrase	CATCCTGTACGGAGACTGAATTRTCATTGTGAATGGTCACA	24
morgani	subunit B	AYGATGTCTTTACAGTAACCGGCGAGGGCTTCGTCGATAGC
	(gyrB)	GTTGTCAACAACCTCGAAGACCATGTGGTGTAAACCGGTTC
		CGTCATCGGTATCACCAATGTACATTCCCGGGCGTTTACGC
		ACCGCGTCCAGCCCTTTTAATACTTTGATACTTGAGGAGTC
		ATAGGTATTCGACAT
Proteus	Acetate	TGTTAATACTATTCTGGCTGAGAAACCAGAACTTTCACAAC	25
mirabilis	kenase	AAATCGCAGCAATTGGTCATCGTATTGTTCACGGTGGCGAG
	(ackA)	AAATTTACTAAATCTGTCGTCATTACTGACGAAGTCATCAA
		AGGTATTGAGGCGGCTATTCCATTTGCCCCATTACATAACC
		CAGCTCACCTTATTGGTATTGAAGAAGCGCGTAAAGCCTTC
		CCTCATTTAATTAATAAAATGGTGGCAGTATTTGACACTGC
		ATTCCACCAAACAATGCCAGAAGAAGCTTATCTGTATGCTC
		TGCCATACAGCTT
Pseudomonas	Gamma-	ATGACCGAGTCCGTCCTCGACTATATGAGCCGCCTTGGCCG	26
aeruginosa	glutamyl	CGATGCCCGCGCCGCCTCGCGGTTGCTCGCGCGCGCCGCCA
	phosphate	CCGCGCAGAAGAACCGCGCCCTGCTGGCCGCGGCCGATGC
	reductase	GCTGGACGCCGCCCGCGCGGAGTTGTCCCACGCCAACGAG
	(pro A)	CAGGACCTCGCCGCCGGCCGCGCCAATGGCCTGGAGCCGG
		CGATGCTGGACCGCCTGGCGCTGACCCCGGCGCGCATCGA
		CGACATGATCGAGGGCCTGCGCCAGGTCGCCACGCTGCCC
		GACCCGATCGGCGAGATC
Serratia	DNA gvrase	CCTCAAGTATCAAGGTATTAAAAGGGCTGGATGCGGTGCG	27
marcescens	subunit B	CAAGCGCCCGGGCATGTATATCGGCGATACCGATGACGGC
	(gyrB)	ACCGGTCTGCACCACATGGTATTCGAGGTTGTGGACAACGC
		TATCGACGAAGCGCTCGCGGGCCACTGTAGYGACATTCAG
		GTCACCATCCATGCYGACAACTCGGTATCGGTRCAGGATGA
		CGGCCGCGGCATTCCGACCGGCATTCACCCGGAAGAAGGG
		GTTTCAGCCGCAGAGGTCATCATGACCGTGCTGCACGCCGG
		CGGTAAATTCGACGACAACTCCTATAA
Spirochetes
Borrelia	Flagellin	ACAGACGAAATTAATAGAATTGCTGATCAAGCTCAATATA	28
burgdorferi	(flaB)	ACCAAATGCACATGTTATCAAACAAATCTGCTTCTCAAAAT
		GTAAGAACAGCTGAAGAGCTTGGAATGCAGCCTGCAAAAA
		TTAACACACCAGCATCACTTTCAGGGTCTCAAGCGTCTTGG
		ACTTTAAGAGTTCATGTTGGAGCAAACCAAGATGAAGCTAT
		TGCTGTAAATATTTATGCAGCTAATGTTGCAAATCTTTTCTC
		TGGTGAGGGAGCTCAAACTGCTCAGGCTGCACCGGTTCAA
		GAGGGTGTTCAACAG
Treponema	Membrane	CCCAGTTGCGGTTCCTCATGAATTAAAAGGGATTGCAAAGG	29
pallidum	antigen	AGAAGTTTCACTTCGTGGAAGACTCCCGCGTTACGGAGAAT
	(tpp47)	ACCAACGGCCTTAAGACAATGCTCACTGAGGATAGTTTTTC
		TGCACGTAAGGTAAGCAGCATGGAGAGCCCGCACGACCTT
		GTGGTAGACACGGTGGGTACCGGTTACCACAGCCGTTTTGG
		TTCGGACGCAGAGGCTTCTGTGATGCTGAAAAGGGCTGAT
		GGCTCTGAGCTGTCGCACCGTGAGTTCATCGACTATGTGAT
		GAACTTCAACACGGT
Fungi
Aspergillus	Beta tubulin	TCTGACGGCAAGGATAGTTACAATGGCTCCTCCGATCTCCA	30
flavus		GCTGGAGCGTATGAACGTCTACTTCAACGAGGTGCGTACCT
		CAAAATTTCAGCATCTATGAAAACGCTTTGCAACTCCTGAC
		CGCTTCTCCAGGCCAGCGGAAACAAGTATGTCCCTCGTGCC
		GTCCTCGTTGATCTTGAGCCTGGTACCATGGACGCCGTCCG
		TGCCGGTCCCTTCGGTCAGCTCTTCCGTCCCGACAACTTCGT
		TTTCGGCCAGTCCGGTGCTGGTAACAACTGGGCCAAGGGTC
		ACTACACTGAGG
Aspergillus	Calmodulin	ATATTGAGGGTGTGCGCTGACACGAGATTTGACGTATAGG	31
fumigatus		ACAAGGATGGTGATGGTTAGTGACCCTTTTTCCACTCCTCG
		AACTTCGGCTTCCATGCGATCATGTTCAAACGCCGACTCAC
		AATATCCGGAAATGACCCKTCAGTACTGATAATATCTATGT
		TTGACTATCAGGCCAGATCACCACCAAGGAATTGGGCACT
		GTAATGCGCTCTCTGGGCCAGAACCCTTCCGAGTCAGAGCT
		GCAAGATATGATCAACGAGGTGGATGCTGACAACAACGGC
		ACCATCGATTTCCCCG
Aspergillus	Calmodulin	CGGTGAATCAGGCCAGATCACCACCAAGGAGCTCGGCACT	32
niger		GTGATGCGCTCCCTTGGCCAGAACCCCTCCGAGTCTGAGCT
		TCAGGACATGATCAACGAGGTTGACGCTGACAACAACGGA
		ACGATCGACTTCCCCGGTATGTGTTAGATTTACGCCTGTAA
		GGCGGAAATGCGGGCTGGATTGTGATTGACTTTTGCCGCCA
		GAATTCCTTACCATGATGGCTCGTAAGATGAAGGACACCG
		ACTCCGAGGAGGAAATCCGCGAGGCTTTCAAG
Candida	28S	CTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGG	33
albicans	ribosomal	CGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATG
	subunit	AAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATT
		GTTGAAAGGGAAGGGCTTGAGATCAGACTTGGTATTTTGCA
		TGYTGCTCTCTCGGGGGCGGCCGCTGCGGTTTACCGGGCCA
		GCATCGGTTTGGAGCGGCAGGATAATGGCGGAGGAATGTG
		GCACGGCTTCTGCTGTGTGTTATAGCCTCTGACGATRCTGC
		CAGCCTAGACCGAGGACTGCGGTTTT
Candida	ITS1-5.8S-	AACTTACAACCAAATTTTTTATAAACTTGTCACGAGATTAT	34
dubliniensis	ITS2	TTTTAATAGTCAAAACTTTCAACAACGGATCTCTTGGTTCTC
	intergenic	GCATCGATGAAGAACGCAGCGAAATGCGATACGTAATATG
	space	AATTGCAGATATTCGTGAATCATCGAATCTTTGAACGCACA
		TTGCGCCCTCTGGTATTCCGGAGGGCATGCCTGTTTGAGCG
		TCGTTTCTCCCTCAAACCCCTAGGGTTTGGTGTTGAGCAAT
		ACGACTTGGGTTTGCTTGAAAGATGATAGTG
Candida	28S	GTGTCAGTTCTTTGTAAAGGGTGCTCGAAGAGTCGAGTTGT	35
glabrata	ribosomal	TTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCYAAA
	subunit	GCTAAATACAGGCGAGAGACCGATAGCGAACAAGTACAGT
		GATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAA
		AGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGA
		CATGGTGTTTTGCGC
Candida	ITS1	GTTATAACTAAACCAAACTTTTTATTTACAGTCAAACTTGA	36
tropicalis	intergenic	TTTATTATTACAATAGTCAAAACTTTCAACAACGGATCTCT
	space	TGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAC
		GTAATATGAATTGCAGATATTCGTGAATCATCGAATCTTTG
		AACGCACATTGCGCCCTTTGGTATTCCAAAGGGCA
Candida	28S	CCCAGACCTATGTAAAGTTCCTTCGAAGAGTCGAGTTGTTT	37
parapsiolosis	ribosomal	GGGAATGCAGCTYTAAGTGGGTGGTAAATTCCATCTAAAG
(sensulato)	subunit	CTAAATATTGGCGAGAGACCGATAGCGAACAAGTMCAGTG
		ATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAA
		GTACGTGAAATTGTTGAAAGGGAAGGGCTTGAGATCAGAC
		TTGGTATTTTGTATGTTACTCTYTCGGGGGTGGCCTCTACAG
		TTTACCGGGCCAGCATCAGTTTGRGCGGTAGGABAAKTGCA
		AAGAAATGTGGCACTGC
Fusarium	28S	CCGAGTTGTAATTTGTAGAGGATRCTTTTGRYRMGGTGCCT	38
spp.	ribosomal	TCCGAGTWCCCTGGAACGGGACGCCATAGAGGGTGAGAGC
	subunit	CCCGTCTGGTTGGAYRCCRADYCTCTGTAAAGYTCCTTCRA
		CGAGTCGAGTAGTTTGGGAATGCTGCTCTAAATGGGAGGT
		ATATGTCTTCTAAAGCTAAATACYGGCCAGAGACCGATAG
		CGCACAAGTAGAGTGATCGAAAGATGAAAAGMACTTTGAA
		AAGAGRGTTAAAMAGYACGTGAAATTGTTGAAA
Fusarium	Calmodulin	GCCGTTGTTGTCGGCGTCGACCTCGTTGATCATGTCCTGAA	39
solani	(cmdA)	GCTCAGACTCGGAGGGGTTCTGGCCGAGGGAGCGCATGAC
		GGTGCCCAGCTCCTTGGTGGTGATCTGGCCTAGAGCGAAAC
		GTTTAGTTTACCGTCTCGAAACGATTCGCATCGACTGAAGA
		GCAGTGTGCGCATAGAGGTTCGATGCATCATGTAATTCGGA
		AAAGGAATCGGGCTGAGGCCGACGAGGGGCTTTGCGAGCA
		GGAGGGAGAATCACTCACCATCGCCATCCTTGTCCTATGCA
		AGAGGTTAGCGACAG
Viruses
Cytomegalovirus	Immediate-	TCTCAGACACTGGCTCAGACTTGACAGACACAGTGTCCTCC	40
	early gene	CGCTCCTCCTGAGCACCCTCCTCCTGTTCCTCATCACTCTGT
	(UL123)	TCACTTTCTTCCTGATCACTGTTCTCAGCCACAATTACTGAG
		GACAGAGGGATAGTCGCGGGTACAGGGGACTCTGGAGGTG
		ACACCAGAGAATCAGAGGAGCTGGCACCAGCGGTGGCCAA
		AGTGTAGGCTACAATAGCCTCTTCCTCATCTGACTCCTCGG
		CGATGGCCCGTAGGTCATCCACACTAGGAGAGCAGACTCT
		CAAAGGATCGGCCC
Epstein-barr	DNA	GACATAGAGGGGGTTGAGGTCTACGAGTTCCCATCGGAGC	41
virus	polymerase	TGGACATGCTCTACGCCTTCTTCCAGCTCATCAGAGACCTC
	(BALF5)	AGCGTGGAGATTGTGACCGGCTACAACGTGGCCAACTTTG
		ACTGGCCCTACATTCTGGACAGAGCCAGGCACATCTACAGC
		ATCAACCCAGCCTCTCTGGGCAAAATTAGGGCTGGGGGCG
		TCTGCGAGGTCAGGCGACCCCATGATGCGGGCAAGGGCTT
		CTTGCGGGCCAACACCAAGGTCCGCATCACCGGCCTCATCC
		CCATCGACATGTACGCC
Herpes	DNA	AGCTCTCGAGCTACAAGCTCAACGCCGTGGCCGAAGCCGT	42
simplex 1	polymerase	CCTGAAGGACAAGAAGAAGGACCTGAGCTATCGCGACATC
	catalytic	CCCGCCTACTACGCCACCGGGCCCGCGCAACGCGGGGTGA
	subunit	TCGGCGAGTACTGCATACAGGATTCCCTGCTGGTGGGCCAG
	(UL30)	CTGTTTTTTAAGTTTTTGCCCCATCTGGAGCTCTCGGCCGTC
		GCGCGCTTGGCGGGTATTAACATCACCCGCACCATCTACGA
		CGGCCAGCAGATCCGCGTCTTTACGTGCCTGCTGCGCCTGG
		CCGACCAGAAGGGCT
Herpes	DNA	GGCGGCGTCTGGGTTTTTTGCCCCCCACAACCCCCGGGGAG	43
simples 2	polymerase	CCACCCAGACGGCACCGCCGCCTTGCCGCCGGCAGAACTTC
	catalytic	TACAACCCCCACCTCGCTCAGACCGGAACGCAGCCAAAGG
	subunit	CCCTCGGGCCGGCTCAGCGCCATACGTACTACAGCGAGTGC
	(UL30)	GACGAATTTCGATTTATCGCCCCGCGTTCGCTGGACGAGGA
		CGCCCCCGCGGAGCAGCGCACCGGGGTCCACGACGGCCGC
		CTCCGGCGCGCCCCTAAGGTGTACTGCGGGGGGGACGAGC
		GCGACGTCCTCCGCGT
Human	Glycoprotein	AAGTGGAACTGCTTGGTGGCGGATGGCTAGTGTGCCTATTA	44
herpes virus	(U22)	ATGCTTATTTCGAAAGAGATATAGCCTTTTTATTTAACCCTC
6		GATGCGTTATCGAGACTGCCCTGGGGTCTAAGATATTATGT
		CGATACAATAAAAATATTGGTGTTGTGTTTGTGGACAATGA
		TACTACGTGTAATGTTTCCTTTCCGAGCGGTGTGCAGTTAC
		AATTACTAAATCAATCGGTGATGGAGTCAATTAGAACTAA
		AACGTATGTAGTGGATTACGCTAGGAAAACTACAGAGAGA
		GGTGATTGCTTCAT
Varicella	Regulatory	GGATACGTCGCGGGGCCCCGGCGCGTTTTGTACTCCGGGTT	45
zoster	protein	GGGAGATCCACCCGGCCAGGCTCGTTGAGGACATCAACCG
	(ORF63)	TGTTTTTTTATGTATTGCACAGTCGTCGGGACGCGTCACGC
		GAGATTCACGAAGATTGCGGCGCATATGCCTCGACTTTTAT
		CTAATGGGTCGCACCAGACAGCGTCCCACGTTAGCGTGCTG
		GGAGGAATTGTTACAGCTTCAACCCACCCAGACGCAGTGCT
		TACGCGCTACTTTAATGGAAGTGTCCCATCGACCCCCTCGG
		GGGGAAGACGGGTT
Parasite
Toxoplasma	Glycerol-3-	TTTGCATAGGTTGCAGTCACTGACGAGCTCCCCTCTGCTGG	46
gondii	phosphate	CGAAAAGTGAAATTCATGAGTATCTGTGCAACTTTGGTGTA
	dehydrogenase	TTCGCAGATTGGTCGCCTGCAATCGATAGTTGACCACGAAC
		GCTTTAAAGAACAGGAGAAGAAGATCGTGAAAGAATACGA
		GAAGAGGTACACAGAGATAGAAGTCGCTGCGGAGACAGCG
		AAGACTGCGGATGACTTCACTCCCGTCGCACCAGCAGCAG
		AGGAGTGCCGGGCAAGAAAATGAGATGCCTAGAGGAGAC
		ACAGCGTGTTATGAACAAATCTATTGAGGTTTCGCGAAGAG
		GAGGGAACATATTATATACAGAAGAAGAACAAGAGACGTG
		CCGCATGTCGCTAAGCCATCGGAAGGGATGCTCAGAAAAT
		GGCACAGTATCACATTACAGTTCCGTTGATTCGTCTGATGG
		TGACGAAAGGGGAAGAATAGTTGTCGCACCAAAACTSGCT
		AGTTGTTAYTTTGAAGAAGACGAGAGATGGAGTGAACCAC
		CAAAAATCGGAGAAAATCGATGGTGTCACGTTTTTTGTCAG
		ACTTCACTTTGTGCAGAAGCATTGCCCGTCCAAACTGCAAC
		AACTGCTCTAGCGTGTTCGTCTCCATTCCGTACAGTCTTCAA
		AAATACAAAAGAGAACATTCCAGCAACTTCTRCCTTTGTTC
		TTTTAGCCTCAATAGCAGGATGACGCCTCCCTCCTATCTTTC
		AGCCAACCCAGCAAACACCGACGAACTCTCTGTAGAGTAA
		CAA
Staphylococcus	icaA-D	TACAGTTGCCTTATTTATTGACAGTCGCTACGAAAAGAAAA	47
epidermidisica		ATATAGTTGGCCTGATATTTTTAAGTTGGTATCCAACGTTAT
locus		ACTGGGTTATCAATGCCGCAGTTGTCATTATGGCATTTCCT
		AAAGCATTAAAAAGAAAGAAAGGTGGCTATGCTACATGGT
		CAAGCCCAGACAGAGGCAATATCCAACGGTAACCTCTTATT
		TAAATATAGTTAGGGAGAGCTTATTTATTACTATATCCGGA
		GTATTTTGGATGTATTGTATCGTTGTGATGATTGTTTATATA
		GGAACTCTTAT
Staphylococci	mecA	TGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAA	48
mec A		ACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGA
		CGTCTATCCATTTATGTATGGCATGAGTAACGAAGAATATA
		ATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAA
		GTTCCAGATTACAACTTCACCAGGTTCAACTCAAAAAATAT
		TAACAGCAATGATTGGGTTAAATAACAAAACATTAGACGA
		TAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAA
		GATAAATCTTGGGGTG

#, SEQ ID NO:Multiplex-Targeted Enrichment

Because of the low pathogen biomass in ocular specimens, analytical sensitivity of an assay is critical for its clinical applications. To deal with that issue, we performed a multiplex-targeted enrichment step before detecting species-specific DNA segments with barcoded probes. Primers flanking the targeted genomic regions that bind to the specific probes were designed (described above). A 10-μl polymerase chain reaction is set up using 5 μl of 2× TaqMan PCR master mix, 1 μl of the primer mixture at 0.5 nM per oligonucleotide, 2.5 μl of purified DNA and sterile water to complete to the final volume. PCR was performed under the following conditions: 94° C. denaturation for 5 minutes followed by 20 cycles of 15 seconds at 94° C. and 4 minutes at 60° C. This number of cycles was found to improve sensitivity while maintain a low level of background noise.

DNA Detection

DNA from control organisms used for analytical validation and from clinical specimens were extracted and purified using the DNeasy blood and tissue kit (Qiagen), following the tissue protocol. Purified DNA was eluted from the silica membrane in 50 μL EB buffer and used as input for the multiplex-targeted enrichment step described above. Enriched samples were than mixed with a cocktail of 96 probes (2 50 mer probes per each of the 48 targets), containing one capture probe that secures the target of interest to the analysis matrix, and a second 50 mer that attaches a unique color-coded barcode for that pathogen or resistance/virulence gene. The hybridization was performed at 67° C. for 16 h and the resulting products analyzed in the Nanostring nCounter Digital Analyzer.

Analytical Specificity.

The analytical specificity of the panel has been tested for a subset of organisms. Purified genomic DNA from herpes virus 1 strain MacIntyre (VR-529DQ), herpes virus 2 strain MS (VR-540DQ), varicella zoster virus (VZV) strain Ellen (VR-1367DQ), cytomegalovirus (CMV) strain AD169 (VR-538DQ), Staphylococcus aureus (Strain MRSA USA300), Staphylococcus epidermidis (ATCC35984), Staphylococcus lugdunensis (clinical isolate), Staphylococcus capitis (clinical isolate), Streptococcus pyogenes (ATCC19615), Streptococcus agalactiae (ATCC13813), Enterococcus faecalis (ATCC700802) and Pseudomonas aeruginosa (ATCC27853) were used for probe specificity validation.

Analytical Sensitivity.

Analytical sensitivity was evaluated for a constricted number of targets present in the panel. Preliminary testing were performed using purified genomic DNA from one Gram-positive (S. aureus USA300 strain) and one Gram-negative pathogen (P. aeruginosa ATCC27583) and Cytomegalovirus (strain AD169; VR-538DQ), serially diluted (10-fold) from 1 pg/μl to 1 fg/μl.

Validation with Ocular Specimens.

Protocols for collection of discarded intraocular specimens were approved by the Massachusetts Eye and Ear Institutional Review Board. Samples were obtained either by anterior paracentesis (aqueous samples), posterior chamber paracentesis (undiluted vitreous samples), or during pars plana vitrectomy (both undiluted vitreous and diluted vitreous washing samples). Following collection, all specimens were immediately transported to the laboratory and stored at −20° C.

Example 1

The probes and primers have been shown to be highly specific. We tested the analytical specificity of this panel using a collection of control DNA from bacteria (8 different species plus 1 antimicrobial resistant gene) and herpesviruses (n=4) (Table 2). All the probes and oligonucleotides tested demonstrated excellent target specificity, with no cross-reactivity detected. Exemplary results are shown in FIG. 2.

TABLE 2
List of targets tested for analytical specificity
Organism                   Control Isolate
Bacteria
Staphylococcus aureus      MRSA USA300 strain
Staphylococcus epidermidis S. epidermidis ATCC35984
Staphylococcus lugdunensis Clinical isolate (identified by MicroScan)
Staphylococcus capitis     Clinical isolate (identified by MicroScan)
Streptococcus pyogenes     ATCC19615
Streptococcus agalactiae   ATCC13813
Enterococcus faecalis      ATCC 700802, V583 strain
Pseudomonas aeruginosa     P. aeruginosa ATCC27583
Virus
Cytomegalovirus            CMV-AD169
Varicella zoster           VZV-Rod
Herpes simplex 1           HSV-1
Herpes simples 2           HSV-2
Resistance Markers
mecA                       MRSA USA300 strain

The panel we tested could detect minute amounts of DNA. Serial 10-fold dilutions of DNA ranging from 2.5 pg/reaction to 2.5 fg/reaction were tested in order to determine the limits of detection (LoD). We used purified control DNA from P. aeruginosa (ATCC27853), methicillin-resistant S. aureus (MRSA strain USA300) and Cytomegalovirus (CMV), organisms that are commonly associated with eye infections. As low as 25 femtograms of DNA per reaction was detected for the 3 pathogens tested (FIG. 3).

Detection and identification were shown to be possible directly from the primary eye sample. To challenge this diagnostic panel with clinical eye specimens, we performed a small pilot study testing 3 different intraocular fluids (aqueous, vitreous and vitreous wash) collected from patients with CMV and VZV retinitis that were previously confirmed by real-time PCR. The NanoString-based multiplex panel was capable to correctly identify the causative agent from all the intraocular fluid matrices tested (Table 3). The detection was very robust, with counts for the correct pathogen 3 to 4 logs higher than the reaction noise.

TABLE 3
Specific identification and robust detection
of herpesviruses from 3 intraocular specimens
	Nanostring Detection
Clinical		Probe	Probe A	Probe B	Real Time
Diagnosis	Specimen	Identity	count	count	PCR Result
CMV	Aqueous	CMV	5 × 105	7 × 105	CMV
Retinitis	humor
CMV	Vitreous	CMV	1 × 105	1.5 × 105	CMV
Retinitis	wash
Acute	Vitreous	VZV	7 × 105	1 × 106	VZV
Retinal	tap
Necrosis

REFERENCES

• 1. Bispo P J, de Melo G B, Hofling-Lima A L, Pignatari A C. Detection and gram discrimination of bacterial pathogens from aqueous and vitreous humor using real-time PCR assays. Invest Ophthalmol Vis Sci 2011; 52:873-881.
• 2. Dabil H, Boley M L, Schmitz T M, Van Gelder R N. Validation of a diagnostic multiplex polymerase chain reaction assay for infectious posterior uveitis. Arch Ophthalmol 2001; 119:1315-1322.
• 3. De Groot-Mijnes J D, Rothova A, Van Loon A M, et al. Polymerase chain reaction and Goldmann-Witmer coefficient analysis are complimentary for the diagnosis of infectious uveitis. Am J Ophthalmol 2006; 141:313-318.
• 4. Harper T W, Miller D, Schiffman J C, Davis J L. Polymerase chain reaction analysis of aqueous and vitreous specimens in the diagnosis of posterior segment infectious uveitis. Am J Ophthalmol 2009; 147:140-147 e142.
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Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method comprising: providing a sample comprising intraocular fluid;purifying genomic DNA from the sample;performing PCR by contacting the sample with a plurality of sets of primers to amplify each of at least 40 of the target sequences SEQ ID NOs: 1-48, or its complement, in the genomic DNA; andcontacting the amplified DNA with a plurality of detectable probes that bind to each of the at least 40 of the target sequences, or its complement, wherein the plurality comprises at least 2 detectable probes that bind to each of the at least 40 of the target sequences, or its complement.

2. The method of claim 1, further comprising determining identity of the probes bound to the amplified DNA and determining sequence of the amplified DNA based on the identity of the bound probes, wherein determining the identity comprises using high resolution melting analysis.

3. The method of claim 1, wherein the sample comprises aqueous humor, vitreous humor, or vitreous wash.

4. A method of selecting a treatment and treating a subject who has uveitis, the method comprising: providing a sample from an eye of the subject comprising, or suspected of comprising, a pathogen;optionally purifying genomic DNA from the pathogen;performing PCR by contacting the sample with a plurality of sets of primers to amplify each of at least 40, of the target sequences SEQ ID NOs: 1-48, or its complement, in genomic DNA from the pathogen; andcontacting the amplified DNA with a plurality of detectable probes that bind to each of the at least 40 of the target sequences, or its complement, wherein the plurality comprises at least 2 detectable probes that bind to each of the at least 40 of the target sequences, or its complement;detecting binding of the at least 2 probes to the amplified DNA;determining identity of the probes bound to the amplified DNA and determining sequence of the amplified DNA based on the identity of the bound probes;correlating the sequence of the amplified DNA to a pathogen, thereby identifying the pathogen; andselecting and administering to the subject a treatment for the identified pathogen, wherein the treatment is an antiviral for a viral pathogen, an antifungal for a fungal pathogen, an antibacterial for a bacterial pathogen, or anti-parasitic treatment for a parasitic pathogen.

5. The method of claim 4, wherein determining the identity comprises using high resolution melting analysis.

6. The method of claim 4, wherein the sample comprises aqueous humor, vitreous humor, or vitreous wash.

7. A kit comprising: a plurality of sets of primers that amplify each of at least 40 of the target sequences SEQ ID NOs:1-48, or its complement;a plurality of probes that bind to each of the at least 40 of the target sequences, or its complement, wherein each of the probes comprises a barcode sequence; andoptionally one or more reagents for performing a multiplexed gene analysis method.

8. The kit of claim 7, wherein the kit comprises one or more reagents for performing a multiplexed gene analysis method, and wherein the multiplexed gene analysis method is a hybridization based digital barcode quantification assay.

9. The kit of claim 7, wherein the plurality of probes comprises at least 2 probes that bind to each of the at least 40 of the target sequences.