Detection of Staphylococcus spp.

Abstract

A novel nucleic acid containing an oligo-nucleotide selected from a member of the group consisting of SEQ ID NOs: 1-25 and sequences complementary to SEQ ID NOs: 1-25. The nucleic acid is 10-1000 nucleotides in length. Also disclosed is a method of detecting Staphylococcus spp.

Description

BACKGROUND

[0001] Traditionally, detection of a microorganism requires time-consuming growth of the microorganism in a culture medium, followed by its isolation and identification. The entire process usually takes 24-48 hours. Many methods for rapid detection of microorganisms have recently been developed, including miniaturized biochemical analyses, antibody- and DNA-based tests, and modified conventional assays.

[0002] Staphylococci bacteria are the causative agents of many opportunistic human and animal infections. Accurate and rapid identification of Staphylococcus spp. is conducive to diagnosing and treating such infections.

SUMMARY

[0003] The present invention relates to specific nucleic acid sequences selected from the Staphylococcus spp. gap gene region for detecting Staphylococcus spp.

[0004] In one aspect, this invention features a novel nucleic acid containing an oligo-nucleotide selected from a member of the group consisting of SEQ ID NOs: 1-25 and sequences complementary to SEQ ID NOs:1-25, wherein the nucleic acid is 10-1000 (e.g., 10-500, 10-200, 10-50, and 10-20) nucleotides in length. The nucleic acid can simply be a member of the group consisting of SEQ ID NOs: 1-25 and sequences complementary to SEQ ID NOs: 1-25. These nucleic acids can be used as sequencing primers, PCR primers and hybridization probes.

[0005] In another aspect, this invention features a pair of amplification primers: one primer contains an oligo-nucleotide selected from a member of the group consisting of SEQ ID NOs:28-52, and the other primer contains an oligo-nucleotide selected from a sequence complementary to the member. Each primer is 14-40 (e.g., 14-30 and 14-20) nucleotides in length. These primers can be used to amplify a DNA template prepared from Staphylococcus spp.

[0006] Also within the scope of this invention is a method of detecting a target Staphylococcus species. The method involves (1) providing a sample having a nucleic acid from an unknown microorganism; (2) amplifying the nucleic acid with a pair of primers: one primer contains an oligo-nucleotide selected from a member of the group consisting of SEQ ID NOs:28-52, and the other primer contains an oligo-nucleotide selected from a sequence complementary to the member. Each primer is 14-40 nucleotides in length; and (3) detecting an amplification product. Detection of the amplification product indicates the presence of the target Staphylococcus species. In one example, the detecting step includes hybridizing the amplification product to a nucleic acid probe that is 10-1000 nucleotides in length and contains a sequence selected from a member of the group consisting of SEQ ID NOs: 1-27 and sequences complementary to SEQ ID NOs: 1-27. The nucleic acid probe can simply be a member of the group consisting of SEQ ID NOs: 1-27 and sequences complementary to SEQ ID NOs: 1-27.

[0007] Further within the scope of this invention is a kit for detecting Staphylococcus spp. The kit contains one or more of the nucleic acids described above. It can also include other components such as a DNA polymerase, a PCR buffer, or a solid support on which one or more of the above-described probes are immobilized.

[0008] The present invention provides a fast, accurate, and sensitive method for Staphylococcus spp. detection and typing. The details of one or more embodiments of the invention are set forth in the accompanying description below. Other advantages, features, and objects of the invention will be apparent from the detailed description, and from the claims.

DETAILED DESCRIPTION

[0009] The present invention is based on the discovery that the Staphylococcus spp. gap gene region can be used for identification of specific Staphylococcus species. The gap gene regions of 27 Staphylococcus spp. are amplified using a primer pair (GF-1: 5′-atggttttggtagaattggtcgttta-3′ and GR-2: 5′-gacatttcgttatcataccaagctg-3′; Yugueros et al., 2000, J. Clinical Microbiology 38: 4351-4355) located in the conserved region of the gap gene. Sequences of the amplified DNA fragments are shown below:

[0010] (1) Staphylococcus arlettae

1	atggttttggtagaattggtcgtttagcatttagaagaattcaaaacgttgatggaatcg	60	(SEQ ID NO:1)
61	acgttgtagcagtaaacgacttaacagatgatgaaatgttagcacacttattaaaatatg	120
121	acactatgcaaggacgtttcacaggagaagttgaagtagaaaatgacggtttccgtgtaa	180
181	atggtcaagaagttaaatcattctcagaaccagatccaaataaattaccatggggcgact	240
241	tagatatcgatgttgttttagaacgtactggtttattcgcagacaaagataaagcatcag	300
301	ctcatatcgacgcaggtgcgaaaaaagtattaatctctgctccagcatcaggtgatttaa	360
361	aaacaatcgtattcaacactaaccacaatgaattagatggttctgaaacagttgtatcag	420
421	gtgcttcatgtactactaactcattagctccagttgctaaagtattaaacgatgacttcg	480
481	gtttagtagaaggtttaatgactactatccatgcttacactggtgaccaaagcacacaag	540
541	acgctcctcacagaaaaggtgacaaacgtcgtgcgcgtgcagctgctgaaaacattattc	600
601	ctaactcaacaggtgctgctaaagcaatcggtttagttattcctgaaatcgatggtaaat	660
661	tagatggtggcgctcaacgtgtaccagtagctactggttcattaactgaattaacagttg	720
721	tattagacaaagaagtaactgttgaagacgttaatagtgcaatgaaaaatgcttcaaacg	780
781	aatcatttggttacactgaagacgaaatcgtttcttctgacgtaatcggtatgacttacg	840
841	gttcattattcgatgctacacaaactcgtgtaatgactgttggtaaccaacaaatggtta	900
901	aagtagcagcttggtatgataacgaaatgtc

[0011] (2) Staphylococcus auricularia

1	atggttttggtagaattggtcgtttagcattcagaagaattcaaaatgttgaaggcattg	60	(SEQ ID NO:2)
61	acgtagtagcagttaatgacttaacagatgacgatatgttagcacacttattaaaatacg	120
121	acacaatgcaaggtcgtttcacaagtgaagttgaaatcgtagaaggcggtttccgcgtta	180
181	acggtcaagaagtgaaatcattcgacgaaccagatccaagcaaattaccttggaaagact	240
241	tagacatcgacgttgtacttgaatgtactggtttattcacagacaaagaaaaagcagaag	300
301	cacacatcgacgcaggtgctaaaaaagtattaatctctgcaccagctaaaggtgacgtta	360
361	aaacagttgtttataacactaaccacgatacacttgatggtacagaaacagttgtttcag	420
421	gtgcttcatgtacaacaaactcattagcaccagttgctaaagtacttaacgatgaattcg	480
481	gtttagtagaaggattcatgactacaatccacgcatacactggtgaccaaaatacacaag	540
541	atgcaccacacaaaaaaggtgacaaacgccgtgcacgtgcagctgctcaaaacatcatcc	600
601	ctaactctacaggtgctgcaaaagcaatcggtaaagttatcccagaaatcgaaggtaaat	660
661	tagacggcggtgcacaacgtgtccctgttgctactggttcattaacagaattaacagtag	720
721	tattagaaaaagatgtttcaatcgaagacgtaaacaacgcaatgaaaaatgcatcaactg	780
781	aatcattcggttacacagatgacgaaatcgtttcttctgacgtcatcggtatgacttatg	840
841	gttcattattcgacgcaactcaaactcgtgtcatgactgttggcgatcaccaattaatca	900
901	aagttgcagcttggtatgataacgaaatgtc

[0012] (3) Staphylococcus caprae

1	atggttttggtagaattggtcgtttagcattcagaagaatccaagatgtagaaggtctta	60	(SEQ ID NO:3)
61	aagtagtagcagttaacgacttaacagacgatgaaatgttagctcatttattaaaatatg	120
121	acactatgcaaggacgcttcactggagaagttgaagttatcgaaggcggattccgcgtaa	180
181	acggtaaagaaattaaatcattcgacgaaccagatgcaaacaaattaccttggggtgacc	240
241	ttgacatcgatgtagtattagaatgtactggtttctacactgataaagataaagcacaag	300
301	cacatatcgatgcaggtgctaaaaaagtattaatctcagctccagctactggtgatttaa	360
361	aaacaatcgtattcaatactaaccatgacgaattagatggttcagaaacagttgtatcag	420
421	gtgcttcttgtacaactaactcattagctcctgttgctaaagtattaaatgatgaattcg	480
481	gtttagttgaaggtttaatgacaactatccacgcttacacaggtgaccaaaatactcaag	540
541	acgcacctcacagaaaaggtgacaaacgtcgtgctcgtgcagcagcagaaaacattatcc	600
601	ctaactcaacaggtgctgctaaagcaatcggtaaagtaattcctgaaatcgacggtaaat	660
661	tagacggtggtgcacaacgtgttccagttgctacaggttcattaacagaattaactgtag	720
721	tattagaaaaacaagatgtaactgctgatcaagttaacgaagcaatgaaacaagcttctg	780
781	acgaatcattcggttacactgaagatgaaatcgtatcttctgacgttgtaggtatcactt	840
841	atggttcattattcgatgcaactcaaactcgtgtaatgactgttggtgaccgtcaattag	900
901	ttaaagtagcagcttggtatgataacgaaatgtc

[0013] (4) Staphylococcus capitis

1	atggttttggtagaattggtcgtttagcattcagaagaatccaagatgtagaaggtctag	60	(SEQ ID NO:4)
61	aagtagttgcagttaacgacttaacagatgatgaaatgttagctcacttattaaaatatg	120
121	acactatgcaaggtcgcttcactggagaagttgaagtcatcgatggtggattccgtgtta	180
181	acggtaaagaaattaaatcattcgatgaaccagatgcaagcaaattaccttggggagacc	240
241	tagacatcgacgtagtattagaatgtactggtttctacactgacaaagataaagcacaag	300
301	ctcacatcgatgcaggtgctaaaaaagtattaatctctgctccagctacaggtgatttaa	360
361	aaacaatcgtattcaacactaaccatgatgaattagatggttctgaaacagttgtatcag	420
421	gtgcttcttgtacaactaactcattagcaccagttgctaaagttttaaatgatgagtttg	480
481	gtttagttgaaggtttaatgactactatccacgcttatactggtgaccaaaatactcaag	540
541	atgctcctcacagaaaaggcgacaaacgtcgtgctcgtgcagcagcagaaaacattatcc	600
601	ctaactctacaggtgctgctaaagcaattggtaaagttattccagaaatcgatggtaaat	660
661	tagacggtggagctcaacgtgttccagttgcaacaggttcattaactgaattaacagtag	720
721	tattagataaacaagatgtaacagctgatcaagtaaatgaagcaatgaaacaagcttctg	780
781	acgaatcattcggttacactgaagatgaaatcgtaccttctgacgttgtaggtatgactt	840
841	atggttcattattcgacgctactcaaactcgtgtaatgactgttggtgaccgtcaattag	900
901	ttaaagttgcagcttggtatgataacgaaatgtc

[0014] (5) Staphylococcus carnosus

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagatgttgaaggtatcg	60	(SEQ ID NO:5)
61	atgtagtagcagtaaacgacttaacagatgatgaaatgttagcacacttattaaaatacg	120
121	atacaatgcaaggacgtttcactgaagaagttgaagttgtagatggcggattccgcgtga	180
181	atggtaaagaagttaaatcattcgaagaaccagatgcaagcaaattaccttggaaagatt	240
241	tagacattgatgttgtattagaatgtacaggtttctatacaagtgaagaaaaagcaaaag	300
301	ctcatatcgatgcaggcgctaaaaaagtattaatttcagcaccagctactggtgatgtta	360
361	aaacaatcgtttataacgtaaaccaagatactttagacagctctgacgtaatcgtttcag	420
421	gtgcttcttgtactacaaactctcttgctcctgtagcacaagttttaaatgacagctttg	480
481	gtttagttgaaggtttcatgactactatccatgcttatactggtgaccaaaatactcaag	540
541	atggtccacacagaaaaggtgacaaacgtcgtgctcgtgcagcagctgaaaacatcgttc	600
601	caaactcaactggtgcagctaaagcaattggtaaagtaattcctgaaatcgacggaaaat	660
661	tagacagcggcgctcaacgtgttcctgttgcaactggttcattagctgaattaacagttg	720
721	tattagataaagacgttacagttgacgaagtaaacgaagcaatgaaacaagcttctaacg	780
781	aatcattcggttacaacgaagacgaaatcgtatcttcagatgttgtaggtatgacattcg	840
841	gttcattatttgatgcaactcaaactcgtgtaatgactgtttcaggccgtcaattagtta	900
901	aagtagcagcttggtatgataacgaaatgtc

[0015] (6) Staphylococcus chromogenes

1	atggttttggtagaattggtcgtttagcattcagaagaattcaagacgtagaaaatattg	60	(SEQ ID NO:6)
61	aggttgtagctgtaaacgatttaacagacgacgatatgcttgcacatttattgaaatatg	120
121	acacaatgcaaggtcgttttactgaagaagtagatgtaattgatggtggtttccgcgtaa	180
181	atggtaaagaagtgaaatcattctctgaaccagaaccatcaaaattaccatggaaagatc	240
241	ttgacgtagatgttgttttagaatgtacaggtttctttacatcaaaagaaaaagcagaag	300
301	ctcacattgaagcaggtgctaaaaaagtattaatttctgcaccaggaactggcgatctta	360
361	aaacaatcgtatataatgtcaaccatgaagaattagacggttctgaaacagttgtatcag	420
421	gtgcttcttgtacaacaaactcattagcaccagtagcaaaaactttaaatgatgaatttg	480
481	gtatcgttgaaggtttaatgactacaattcacgcatacactggtgaccaaaatacacaag	540
541	actcaccacacagaaaaggtgacaaacgtcgtgcacgtgcagctgcagaaaacattattc	600
601	ctaactcaacaggtgctgcgaaagcaatcggtttagttatccctgaaattgatggaaaat	660
661	tagacggtggcgcacaacgtgtaccagtagcaacaggttcattaactgaattaacagttg	720
721	ttttagataaagaagtatcagtagaagacgttaacaatgcaatgaaaaatgcaacaaacg	780
781	aatcattcggttacactgaagacgaaattgtatcttcagatgttgtaggcatgactttcg	840
841	gagcattatttgatgcaactcaaacacgtgtaatgactgttggcgaccgtcaattagtta	900
901	aagtagcagcttggtatgataacgaaatgtc

[0016] (7) Staphylococcus cohni

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagatgtagaaggaatcg	60	(SEQ ID NO:7)
61	atgtagtagcagtaaacgacttaacagatgatgaaatgttagctcatttattaaaatatg	120
121	acactacacaaggtcgcttcacaggagaagttgaagttgaagaaaatggtttccgcgtta	180
181	acggattagaagttaaatcattttctgagccagatccaagtaaattaccttggggagact	240
241	tagatatcgatgttgtattagaatgtactggtttattcacagataaagaaaaagcagaag	300
301	cacacatcaatgcaggcgctaaaaaagtattaatttcagcaccagctaaaggtgacttaa	360
361	aaacaatcgtatacaacactaaccacgacactttagatggttcagaagatgttgtttcag	420
421	gtgcttcatgtactactaactcattagcaccagttgctaaagttttaaatggtgaattcg	480
481	gcttaatcgaaggtttcatgactacaatccacgcatatactggtgaccaaagcacacaag	540
541	atgcgcctcacagaaaaggtgacaaacgtcgtgcgcgtgcagctgctgaaaacattatcc	600
601	ctaactcaacaggtgctgctaaagctatcggcctagtaattcctgaaattgatggtaaat	660
661	tagacggtggagcacaacgtgtaccagttgcaactggttcattaactgaattaacagtag	720
721	tattagataaaaatgtaagtatcgaagacgtaaataacgcaatgaaaaatgcatctaacg	780
781	aatcattcggttatactgaagacgaaatcgtttcttctgacgtaatcggaatgacatacg	840
841	gttcattatttgatgcaactcaaacacgtgttatgactgttggagatcgtcaattagtta	900
901	aagtagcagcttggtatgataacgaaatgtc

[0017] (8) Staphylococcus delphini

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagatgtggaaaatatcg	60	(SEQ ID NO:8)
61	acgttgtagcagtaaacgatttaacagacgacgatatgcttgcacacttattaaaatatg	120
121	actcaacacaaggtcgttttactgaagaagtagaagtaattgacggtggtttccgtgtaa	180
181	atggtaaagaagtgaaatcattctctgaaccagaacctaaaaacttaccatggggcgagt	240
241	tagacatcgacgtggtattagaatgtactggtttcttcactgataaagaaaaagctgaag	300
301	cacacatcgaagcaggcgcgaaaaaagtattaatttctgcaccagctaaaggtgacctta	360
361	aaacagttgtatataacgttaaccacgaaattttagatggtactgaaacagttgtttctg	420
421	gtgcttcatgtacaacaaactcattagcacctgttgcaaaaactttacaagacaacttcg	480
481	gtatcgttgaaggtttaatgacaacaattcacgcttacactggtgaccaaaacacattag	540
541	acgcacctcacagaaaaggtgacaaacgtcgtgcgcgtgcagctgctgaaaacatcatcc	600
601	ctaactcaactggtgctgcgaaagcaatcggtttagttattcctgaaatcgatggtaaat	660
661	tagacggtggtgcacaacgtgttcctgtagcaacaggttcattaactgaattaactgtag	720
721	tattagaaaaagaagtctctgttgaagaagttaacaaagtcatgaaagaggcaactaacg	780
781	aatcattcggttacactgaagacgaaatcgtttcttcagacgttgttggtatgactttcg	840
841	gtgctttattcgacgcaactcaaacacgtgtaatgactgttggcgaccgtcaattagtta	900
901	aagttgcagcttggtatgataacgaaatgtc

[0018] (9) Staphylococcus epidermidis

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagacgtagaaggtttag	60	(SEQ ID NO:9)
61	aagtggttgcagtaaacgacttaactgatgacgatatgttagcacacttattaaaatatg	120
121	acactatgcaaggtcgcttcactggtgaagtagaagttatcgacggtggattccgtgtta	180
181	acggtaaagaagttaaatcattcgatgaaccagatgcaagcaaattaccttggaaagact	240
241	tagatatcgacgtagtattagaatgtactggtttctatactgacaaagataaagcacaag	300
301	ctcatgttgacgcaggtgctaaaaaagtattaatctcagctccagcaactggtgacttaa	360
361	aaacaatcgtttacaacactaaccacgatgaattagatggttctgaaacagtagtatcag	420
421	gtgcatcatgtactactaactcattagctcctgtagctaaagtgttaagtgatgagttcg	480
431	gtttagttgaaggtttaatgacaactatccacgcttacactggtgaccaaaatactcaag	540
541	atgctccacacagaagaggcgacaaacgtcgtgctcgtgctgcagcagaaaacatcatcc	600
601	ctaactcaactggtgctgctaaagcaatcggtaaagtaattcctgaaatcgacggaaaat	660
661	tagacggtggtgcacaacgtgttccagttgcaactggttcattaactgaattaacagttg	720
721	tattagaaaaagacgtaacagttgaacaagttaacgaagcaatgaaacaagcttctgacg	780
781	aatcattcggttacactgaagacgaaatcgtatcttctgatgtagttggtatgacttacg	840
841	gttcattattcgatgcaactcaaactcgtgtaatggctgttggtggtcgtcaattagtta	900
901	aagtagcagcttggtatgataacgaaatgtc

[0019] (10) Staphylococcus equorum

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagacgtagaaggtattg	60	(SEQ ID NO:10)
61	acgtagtagcagttaacgatttaacagatgacgaaatgttagctcatttattaaaatatg	120
121	acactacacaaggtcgcttcacaggagaagttgaagtagaaaaagacggattccgtgtaa	180
181	atggacaagaagttaaatcattctcagaacctgaaccaagtaaattaccttggaaagatt	240
241	tagacatcgatgttgttttagaatgtactggtttcttcgctgataaagaaaaagcagaag	300
301	ctcacattgacgctggcgctaaaaaagtattaatctctgcaccagcaacaggcgacttaa	360
361	aaacaatcgtttataacactaaccacagtgaattagatggttcagaaacagttgtttcag	420
421	gtgcttcatgtactactaactcattagctccagtagctaaagttttaaatgacgacttcg	480
481	gcttagttgaaggtttcatgactactattcacgcatatactggtgaccaaagtactcaag	540
541	atgctccacacagaaaaggcgacaaacgccgtgcacgtgcagctgctgaaaacatcatcc	600
601	ctaactcaacaggtgctgctaaagcaattggtttagtaatccctgaaatcgatggtaaat	660
661	tagacggtggcgctcaacgtgttccagttgctactggttcattaactgaacttacagttg	720
721	tattagaaaaagacgtaagcgttgaagacgttaacgcagcaatgaaaaatgcttcagacg	780
781	aatcatttggttacactgaagacgaaatcgtttcttctgacgtaatcggtatgacttacg	840
841	gttcattattcgatgcaacgcaaactcgtgttatgacagttggagatcaccaattagtta	900
901	aaatagcagcttggtatgataacgaaatgtc

[0020] (11) Staphylococcus gallinarum

1	atggttttggtagaattggtcgtttagcattcagaagaattcaaaacgttgaaggaatcg	60	(SEQ ID NO:11)
61	acgtagtagcagtaaatgacttaacagatgacgaaatgttagctcacttattaaaatatg	120
121	atactatgcaaggtcgcttcactggagaagttgaagttgaaaaagacggtttccgtgtta	180
181	acggtcaagaagttaaatcattctctgagccagacccaagtaaattaccatggggtgact	240
241	tagacatcgatgtagtattagaatgtactggtttcttcgctgacaaaactaaagcagaag	300
301	ctcacatcaatgcaggtgctaaaaaagtattaatctcagctccagcaactggtgacttga	360
361	aaacaatcgttttcaacactaaccataacgaattagatggtacagaaacagttgtttcag	420
421	gtgcttcatgtactactaactcattagctccagtagctaaagtattaaatgatgactttg	480
481	gtttagttgaaggtcttatgactacaattcacgcttacactggtgaccaaaatacacaag	540
541	atgctccacatgctaaaggtgacaaacgccgtgctcgtgcagctgctgaaaatatcatcc	600
601	ctaactcaactggtgctgctaaagcaatcggtaaagttatccctgaaattgacggcaaat	660
661	tagacggtggtgcgcaacgtgtaccagttgctactggttctttaactgaattaacagttg	720
721	tattagaaaaagacgtaagcgttgaagacgttaacaatgcaatgaaaaacgcttcaaacg	780
781	aatcattcggttacactgaagacgaaatcgtatcttctgacgtagttggtatcacttacg	840
841	gtccattattcgatgcaacacaaactcgtgtaatgactgttggcgatcgtcaattagtta	900
901	aagttgcagcttggtatgataacgaaatgtc

[0021] (12) Staphylococcus haemolyticus

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagacgtaggaggtattg	60	(SEQ ID NO:12)
61	aagtagttgcagtaaacgacttaacagacgacgaaatgttagctcatttattaaaatatg	120
121	acactatgcaaggtcgtttcacaggagaagttgaagttgttgatggtggtttccgcgtaa	180
181	atggtaaagaagttaaatcatacgaagaaccagatgcaagcaaattaccttggggcgatt	240
241	tagatatcgacgtagtattagaatgtactggtttctatacagataaagaaaaagcagaag	300
301	cacacatcaatgcaggtgctaaaaaagtattaatctctgcaccagctcaaggtgatgtaa	360
361	aaacaatcgtattcaacactaaccacaatgacttagatggttcagaaacagttgtttcag	420
421	gtgcatcatgtactactaactcattagcaccagttgctaaagtattaagtgatgaatttg	480
481	gtttagttgaaggtttaatgacaactattcacgcatacactggtgaccaaatgactcaag	540
541	acggtccacataaaaaaggtgacaaacgtcgtgcacgtgcagcagctcaaaacatcgtac	600
601	ctaactcaacaggtgctgcaaaagcaatcggtaaagttattcctgaaatcgatggtaaat	660
661	tagacggtggtgctcaacgtgtaccagttgctacaggttcattaactgaagtgacagttg	720
721	tattagaaaaagacgttactgttgaagacgttaacaaagcaatgaaaaacgcttcaaacg	780
781	aatcatttggttacactgaagacgaaatcgtttcttctgacgtagttggcatgacttacg	840
841	gttcattattcgatgctactcaaactcgtgtaatgtctgttggtgaccgtcaattagtta	900
901	aagttgcagcttggtatgataacgaaatgtc

[0022] (13) Staphylococcus hominis

1	atggttttggtagaattggtcgtttagcattcagaagaattcaagacgtagaaggtattg	60	(SEQ ID NO:13)
61	aagtagttgcagtaaacgacttaacagacgacgaaatgttagctcatttattaaaatatg	120
121	acactatgcaaggtcgctttaacggagacgtagaagtagttgaaggtggtttccgtgtaa	180
181	atggtaaagaagttaaatcttttgaagaaccagatgcaagtaaattaccatggggcgatt	240
241	tagatatcgacgtagtattagaatgtactggtttctatacagaaaaagaaaaagctgaag	300
301	cacacattaatgcaggagctaaaaaagtattaatttctgctccagccaaaggtgatgtta	360
361	aaactatcgtatttaacacaaaccacaaagacttagatggatctgaaacagtagtatcag	420
421	gtgcttcatgtactacaaactcattagcaccagttgctaaagttttaaatgacgaatttg	480
481	gtattgttgaaggtttaatgacaactatccatgcttacactggtgaccaaatgactcaag	540
541	atggtccacacagaaaaggtgacaaacgtcgtgcacgtgcagcagcacaaaacatcgtac	600
601	ctaactcaacaggtgcagctaaagctatcggtaaagtaattcctgaaatcgatggtaaat	660
661	tagatggtggagcacaacgtgtaccagtagctactggttcattaactgaagtaacagttg	720
721	tattagaaaaagaagtaacagttgaagatgttaacaaagcaatgaaaaatgctgctgacg	780
781	aatcattcggttacactgaagatgaaatcgtatcatcagatgttgctggtatgaactttg	840
841	gttcattattcgatgcaactcaaactcgtgtcatctcagttggcgacaaacaattagtta	900
901	aagtagcagcttggtatgataacgaaatgtc

[0023] (14) Staphylococcus hyricus

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagacgtagaaaacattg	60	(SEQ ID NO:14)
61	aggtagtagctgtcaatgatttaactgacgacgacatgcttgcacatttattaaaatatg	120
121	acacgatgcaaggacgttttactgaagaagtagatgtaattgatggtggtttccgcgtaa	180
181	atggtaaagaagtgaaatcattctctgaaccagaaccatctaaattaccttggaaagact	240
241	tagaagtagatgttgttttagaatgtactggtttcttcacatctaaagaaaaagctgaag	300
301	cacacattgaagcaggcgctaaaaaagtcttaatttcagcaccaggtactggtgatctta	360
361	aaacaattgtatataacgttaaccatgaagaattagacggttcagaaacagttgtttcag	420
421	gtgcgtcttgtactacaaactcattagctccggtagcgaaaacattacacgatgaatttg	480
481	gtattgttgaaggtttaatgactacaattcacgcttatacaggtgaccaaaatacgcaag	540
541	actcacctcacagaaaaggtgacaaacgtcgtgcacgtgcagctgctgaaaacatcatcc	600
601	ctaactcaacaggtgctgcaaaagcaatcggtttagttattccagaaattgctggtaaat	660
661	tagatggtggcgcgcaacgtgtaccagttgctacaggttcattaacagaattaactgttg	720
721	ttttagaaaaagaagtatctgttgaagaagttaacaatgcaatgaaaaatgcaactaatg	780
781	aatcattcggttacactgaagatgaaatcgtctcttctgacgttgtaggtatgacgtttg	840
841	gtgcattattcgacgcaactcaaacacgcgttatgactgttggcgatcgtcaattagtta	900
901	aagttgcagcttggtatgataacgaaatgtc

[0024] (15) Staphylococcus intermedius

1	atggttttggtagaattggtcgtttagcattccgtcgtattcaaaatgtggaaggaattg	60	(SEQ ID NO:15)
61	aagttgttgcaatcaatgacttaacagacgctaaaatgttagctcatctgttaaaatatg	120
121	atacaactcaaggccgttttgacggtgaagtagaagtacatgatggtttcttcaaagtaa	180
181	acggtaaagaagttaaagtattagctaaccgtaacccagaagaacttccatggggtgaac	240
241	taggagtagacatcgttcttgaatgtactggtttcttcacagcacaagacaaagctgaat	300
301	tacacattaaagctggcgctaaaaaagttgttatctccgctccagcaactggcgacatga	360
361	aaacaatcgtttacaatgtaaaccatgaaacattagacggaactgaaacagttatttctg	420
421	gtgcaagctgtactactaactgtttagctccaatggctaaagttttagaagacaaatttg	480
481	gtgttgttgaaggcctaatgactacaattcacgcatacactggtgaccaaaatacattag	540
541	acgctccacatccaaaaggtgacttccgtcgtgctcgtgctgctgcagaaaatatcatcc	600
601	ctaatacaactggtgctgcaaaagctatcggtgaagtattaccaagccttaaaggtaaat	660
661	tagacggagcagctcaacgtgttccagttccaactggttcccttactgaattagtaacag	720
721	ttcttaacaaaaaagttactgttgatgaagtaaatgcagctatggaagcagcttctgatc	780
781	cagaaacattcggttacactaatgacgcaatcgtttcttctgatatcaaaggtatgactt	840
841	tcggttctttatttgacgaaactcaaacaaaagttcttacagttggcgatcaacaattag	900
901	ttaaaactgcagcttggtatgataacgaaatgtc

[0025] (16) Staphylococcus kloosii

1	atggttttggtagaattggtcgtttagcatttagaagaattcaaaacgttgacggaatcg	60	(SEQ ID NO:16)
61	atgtagtagcagttaacgacttaacagatgacgaaatgttagcacacttattaaaatatg	120
121	acacaatgcaaggtcgtttcactggagaagttgaagttgaagaaaacggcttccgcgtaa	180
181	atggtcaagaagttaaatcattctctgaaccagatccaagtaaattaccatggggcgact	240
241	tagatatcgatgttgtcttagaatgtactggtttatttgctgataaagataaagcttcag	300
301	ctcatatcgatgcaggcgctaaaaaagttttaatttcagctccagctacaggcgacttaa	360
361	aaacaatcgtttacaacactaaccacaacgaattagacggttcagaatcagtagtatcag	420
421	gtgcttcatgtactactaactcattagctccagtagctaaagttttaaatgatgaattta	480
481	gtttagttgaaggtttaatgacaactatccacgcttacactggtgaccaaagcacacaag	540
541	atgctcctcacagaaaaggtgacaaacgtcgtgctcgtgcagcagcagaaaacatcatcc	600
601	ctaactcaacaggtgctgcaaaagcaatcggtttagttattcctgaaatcgacggaaaat	660
661	tagacggtggcgcacaacgtgttccagttgcaacaggttcattaactgaattaacagttg	720
721	tattagaaaaagacgtaagtgttgaagatgtaaacaacgcaatgaaaaatgcttcaaacg	780
781	aatcatttggttacactgaagacgaaatcgtttcttctgacgtaatcggtatgacttacg	840
841	gttcattattcgacgctacacaaactcgtgtaatgactgttggtgactcgtcaattagtt	900
901	aaagttgcagcttggtatgataacgaaatgtc

[0026] (17) Staphylococcus lentus

1	atggttttggtagaattggtcgtttagcatttagaagattacaagaagtagaaaatatcg	60	(SEQ ID NO:17)
61	aagtagtagcaatcaacgatttagcagatgacgctatgttagctcatttattaaaatacg	120
121	attctacacaaggtcgtttcaaagatgaagtagaagtaattgaaggcggattccgtgtaa	180
181	acggtaaagaaatcaaaactttcgaaaatcctaaccctaaagaattaccctggggagact	240
241	tagacatcgatgtagtattagaatgtactggtttcttcgctgataaagaaaaagctcatg	300
301	ctcacatcgccgcaggtgctaaaaaagtattaatttcagctccagcttcaggcgacttga	360
361	aaacaatcgtatacaatgttaaccatgatgaattagacggttcagaagaaatcgtatctg	420
421	gtgcatcttgtactactaactgtttagctccaatggctaaagtattaaatgatgaattcg	480
481	gtatcgttgaaggattaatgatgacaattcatgcttatactggtgaccaaaatacactag	540
541	atgctccacatgctaaaggtggcttccgtcgtgctcgtgcagcagctgaaaacatcgtac	600
601	ctaactcaactggtgcagctaaagcaattggcttagttatcccagaattaaaaggtaaat	660
661	tagatggatcagctcaacgtgttccagtagcaactggttcagtaactgaattaacagcag	720
721	tattagataaagaagtatctatagaagaaatcaacgaagcaatgaaaaatgctacaaatg	780
781	attcattcggatacactgaagacgaaatcgtttcttctgatgttattggcatcacttacg	840
841	gttcattattcgacgcaactcaaactcgtgtaatgacagttggagaccgtcaattagtta	900
901	aaactgcagcttggtatgataacgaaatgtc

[0027] (18) Staphylococcus lugdunesis

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagacgtagaaggcatcg	60	(SEQ ID NO:18)
61	aggtagtagcagtaaacgacttaacagatgatgatatgttagcgcatttattaaaatatg	120
121	acactatgcaaggtcgcttcactagcgaagttgaagttgttgatggtggtttccgtgtaa	180
181	atggtaaagaagttaaatcatttgaagaaccagacgcaagcaaattaccatggggtgacc	240
241	taggtgttgacgtggtattagaatgtactggattctatacagataaagaaaaagctgaag	300
301	cacacattcatgcaggtgctaaaaaagtattaatttctgcgccagctaaaggtgacgtta	360
361	aaactatcgtttacaacactaaccatagtgacttagacggttcagaaacagttgtatcag	420
421	gtgcttcatgtacaactaactctttagcaccagtagctaaagtaatcagcgatgaatttg	480
481	gtttagtagaaggtttaatgacaactattcatgcatacactggtgaccaaatgactcaag	540
541	atggtccacacagaaaaggtgacaaacgtcgtgctcgtgcagctgcacaaaacatcgtac	600
601	ctaactcaactggtgctgctaaagcaatcggtaaagttattcctgaaatcgatggtaaat	660
661	tagacggtggtgcgcaacgtgtacctgtagctacaggttcattaactgaattaactgtag	720
721	tattagaaaaacaagatgtaacagtagaacaagtaaacgaagcgatgaaaaaagcttcta	780
781	acgaatcattcggttacaatgaagatgaaattgtttcttctgacgtagttggtatgactt	840
841	acggttcattatttgatgcaacacaaactcgtgtaatgtcagttggtggccgtcaattag	900
901	ttaaagttgcagcttggtatgataacgaaatgtc

[0028] (19) Staphylococcus piscifermentans

1	atggttttggtagaattggtcgtttagcattcagaagaattcaagatgttgaaggtatcg	60	(SEQ ID NO:19)
61	atgtagtagcagtaaacgacttaacagatgatgaaatgttagcgcatttattaaaatacg	120
121	atacaatgcaaggacgtttcacagaagaagttgaagttgtagatggcggattccgtgtga	180
181	atggtaaagaagttaaatcattcgaagaaccagatgcaagcaaattaccttggaaagatt	240
241	tagacattgatgttgtattagaatgtactggtttctatacaagtgatgaaaaagctaaag	300
301	cacatatcgacgcaggtgctaaaaaagtattaatttctgctccagcaactggcgatgtta	360
361	aaacaatcgtttataacgtaaaccaagatactttagacagctctgatgttatcgtttcag	420
421	gtgcttcttgtactacaaactcacttgctccagtagcaaaagttttaaacgacagctttg	480
481	gtttagttgaaggtttcatgactactattcatgcttacactggtgaccaaaatactcaag	540
541	atggtccacacagaaaaggtgacaaacgtcgtgcacgtgcagcagctcaaaacatcgtac	600
601	caaactcaactggtgctgctaaagcaatcggtaaagtaatccctgaaattgacggtaaat	660
661	tagacggtggtgctcaacgtgttcctgttgcaactggttcattaacagaattaacagttg	720
721	tattagacaaagaagtttcagttgacgaagtaaacgaagcaatgaaacaagcttctaacg	780
781	aatcattcggttacaatgaagacgaaatcgtatcttctgacgtggttggtatgacattcg	840
841	gttcattattcgatgctacacaaactcgtgtgatgactgtatcaggtcgtcaattagtta	900
901	aagttgcagcttggtatgataacgaaatgtc

[0029] (20) Staphylococcus saprophyticus

1	atggttttggtagaattggtcgtttagcattcagaagaattcaaaacgttgacggaatcg	60	(SEQ ID NO:20)
61	acgtagtagcagtaaacgatttaacagatgacgaaatgttagctcatttattaaaatatg	120
121	atactatgcaaggacgcttcacaggagaagttgaagtagaaaaagacggtttccgcgtaa	180
181	acggacaagaagtaaaatcattctctgagcctgaaccaagtaaattaccttggaaagact	240
241	tagacatcgatgttgtattagaatgtactggtttcttcgctgataaagaaaaagcagaag	300
301	cacacatcaatgcaggtgctaaaaaagtattaatctctgctccagctacaggcgatttaa	360
361	aaacaatcgtttataatacaaaccaccaagaattagacggttcagaaactgttgtttcag	420
421	gtgcttcatgtactactaactcattagctcctgttgctaaagttttaaatgatgacttcg	480
481	gtttagtagaaggtttcatgactactatccacgcatacactggtgaccaaagcacactag	540
541	atgcaccacacagaaaaggcgacaaacgtcgtgcgcgtgcagctgctgaaaacatcatcc	600
601	ctaactcaactggtgctgctaaagcaattggcttagtaattcctgaaattgatggtaaat	660
661	tagatggaggagcgcaacgtgttcctgttgcaactggttcattaactgaattaacagttg	720
721	ttttagnnnnnnntgtaagcattgaagatgtaaatgctgcaatgaaaaatgcttcaaacg	780
781	aatcattcggttacacagaagacgaaatcgtatcttcagacgtaatcggtatgacttatg	840
841	gttcattatttgatgcaacacaaactcgtgtaatgactgttggcgaccgtcaattagtta	900
901	aagtagcagcttggtatgataacgaaatgtc

[0030] (21) Staphylococcus schleiferi

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagatgtagaaaacattg	60	(SEQ ID NO:21)
61	aggtcgtagctgtaaacgatttaacagatgacgatatgcttgcacatttattgaaatatg	120
121	acacaatgcaaggacgttttactgaagaagtggaagtaattgatggtggtttccgtgtga	180
181	atggtaaagaagcgaaatcattctctgaaccagaacctgctaaattaccttggggtgacc	240
241	ttggtgtggacgtagtattagaatgtactggtttcttcacagataaagaaaaagctgaag	300
301	cacacattcaagcaggcgctaaaaaagtattaatctcagcaccggctaaaggtgatctta	360
361	aaacaatcgtatataatgttaaccacgacgatttagatggttctgaaacagttgtttcag	420
421	gtgcatcatgtactacaaactcattagcacctgttgcaaaaactttacacgacgaattcg	480
481	gtattgttgaaggtttaatgactacaattcacgcatatactggtgaccaaaatacacaag	540
541	atgcacctcacagaaaaggtgacaaacgtcgtgcgcgtgctgctgctgaaaacattatcc	600
601	ctaactctacaggggcagcaaaagcaatcggtttagttattccagaaattgccggtaaat	660
661	tagacggtggtgcacaacgtgttccagttgcaactggttcattaacagaattaacagttg	720
721	ttttagataaagaagtgactgttgaagaagtaaacaaagtattgaaagcagcaactaacg	780
781	aaccattcggttacactgaagacgaaattgtttcttcagacgttgtaggtatgacttacg	840
841	gtgcattattcgatgcaactcaaactcgtgtaatgactgttggcgaccgtcaattagtta	900
901	aagttgcagcttggtatgataacgaaatgtc

[0031] (22) Staphylococcus sciuri

1	atggttttggtagaattggtcgtttagcatttagaagattacaagaagttgaaaatatcg	60	(SEQ ID NO:22)
61	aagtagtagcaatcaacgatttaacagatgacgcaatgttagctcatttattaaaatatg	120
121	attcaacacaaggtcgtttcaaagacgaagtagaagttatcgaaggcggattccgcgtaa	180
181	acggtagagaaatcaaaactttcgaaaatcctaatcctaaagaattaccatggggcgatt	240
241	tagatatcgatgtagtattagaatgtactggtttcttcgctgacaaagacaaagcttcag	300
301	ctcacatcgaagcaggtgctaaaaaagtattaatttcagctccagcatcaggtgacttaa	360
361	aaactatcgtttataacgttaaccatgacgaattagacggatctgaagaaatcgtttcag	420
421	gtgcatcttgtacaactaactgtttagctccaatggctaaagtattaaatgatgaattcg	480
481	gtatcgttgaaggtttaatgatgacaattcacgcttacactggtgaccaaaatactttag	540
541	atgctccacatgctaaaggtgacttccgtcgtgctcgtgcagctgctcaaaacatcgtgc	600
601	ctaactcaactggtgctgctaaagcaatcggtttagtaattccagaattaaaaggtaaat	660
661	tagatggatcagctcaacgtgttccagtagcaactggttcagtaacagaattaacagctg	720
721	tattggacaaagaagtttcagttgaagaaatcaatgcagcaatgaaaaatgctacaaatg	780
781	attcattcggttacactgaagacgaaatcgtatcttctgatatcattggtatcacttacg	840
841	gttcattatttgatgcaactcaaactcgtgttatgacagttggagatcgccaattagtta	900
901	aaactgcagcttggtatgataacgaaatgtc

[0032] (23) Staphylococcus simulans

1	atggttttggtagaattggtcgtttagcatttcagaagaattcaagatgttgaaggtatc	60	(SEQ ID NO:23)
61	gatgtagtagcagtaaacgacttaacagatgatgaaatgttagcacacttattaaaatac	120
121	gatacaatgcaaggacgtttcactgaagaagttgaagttgtagatggcggattccgcgtg	180
181	aatggtaaagaagttaaatcattcgaagaaccagatgcaagcaaattaccttggaaagat	240
241	ttagacattgatgtcgtattagaatgtactggtttctacactagcgacgaaaaagcacaa	300
301	gctcacattgacgcaggtgctaaaaaagtattaatctctgcaccagcaactggtgacgtt	360
361	aaaacaatcgtttataacgtaaaccaagatactttagacagctctgacgtaatcgtttca	420
421	ggtgcttcttgtactacaaactcacttgctccagtagcaaaagtattaaatgacagcttc	480
481	ggtttagtagaaggtttcatgactactatccacgcttacactggtgaccaaaatactcaa	540
541	gacggtccacacagaaaaggcgacaaacgtcgtgcacgtgcagcagctgaaaacatcgtt	600
601	cctaactcaactggtgctgctaaagcaatcggtaaagtaattcctgaaatcgacggaaaa	660
661	ttagacggtggcgctcaacgtgttcctgtagcaactggttcattaactgaattaacagtt	720
721	gtattagacaaagacgtaacaatcgaagaagtaaacgaagctatggaagcagcttctaac	780
781	gaatcattcggttacaacgaagacgaaatcgtatcttcagacgtagttggtatgacattc	840
841	ggttcattattcgatgcaactcaaactcgtgttatgactgtatctggtcgtcaattagtt	900
901	aaagttgcagcttggtatgataacgaaatgtc

[0033] (24) Staphylococcus xylosus

1	atggttttggtagaattggtcgtttagcatttagaagaattcaaaacgttgacggaattg	60	(SEQ ID NO:24)
61	acgtagtagcagtaaatgacttaacagatgacgaaatgttagcacatttattaaaatatg	120
121	acactatgcaaggacgcttcacaggagaagttgaagtagaaaatgacggtttccgtgtaa	180
181	acggacaagaagtaaaatcattctctgagccagacccaagcaaattaccttggaaagatt	240
241	tagacatcgatgttgtattagaatgtactggtttctacgctgataaagaaaaagcagaag	300
301	ctcacattaatgcaggtgctaaaaaagtattaatttcagctccagctactggtgatttaa	360
361	aaacaatcgtttataacacaaaccaccaagagttagatggtaaagaaacagttgtttcag	420
421	gtgcttcatgtacaactaactcattagctccagttgctaaagtattaaatgatgactttg	480
481	gtttagtagaaggtttcatgactacaattcatgcttacactggtgaccaaaatacacaag	540
541	atgcgccacacgctaaaggcgacaaacgtcgtgctcgtgcagctgctgaaaacattatcc	600
601	ctaactcaactggtgctgctaaagcaattggcctagttatccctgaaattgatggtaaat	660
661	tagacggtggagcgcaacgtgttcctgtagctactggttcgttaacagaattaacagttg	720
721	tattagacaaaaatgtaagtgttgaagacgttaatgctgcaatgaagaatgcttcaaacg	780
781	aatcattcggttacactgaagacgaaatcgtttcttctgatgtagttggtatgacttacg	840
841	gttcattattcgatgcaacacaaacacgtgttatgacagttggcgatcgccaattagtta	900
901	aagtagcagcttggtatgataacgaaatgtc

[0034] (25) Staphylococcus caseolyticus

1	atggttttggtagaattggtcgtttattagaacagcatgcacgggtaaaagcattggatg	60	(SEQ ID NO:25)
61	cacaatatccagaaattgatatttattcaggtgttgagatggatatattagcagatggcg	120
121	aaatggattatagcaatgacgtgctagcacagcttgactattgtattggtgctattcatc	180
181	agtcgttgaatcaaagtgaagatgagattatgaaacgcctgatcaatgcctgtaataatc	240
241	catacattagacatattgctcatccaacgggaaggttgattggtcgccgtaatggttatc	300
301	atgtaaacatgccgaaactcatcgaaactgcacagaagacaaataccatccttgaaatca	360
361	atgcacatccgatgcgccttgatttatcgagcgacgtattaaagcaatatccagatatta	420
421	aacttgtgatcaacacagacgcgcgtgcaatcgatcagcttgatttaatgaaatatggtg	480
481	tgggtacagcaataaaaggccatgtgaaaaaagaacaggtaataaacactttaccgcgta	540
541	aggattttaaatcttggatacagaatgggaagtaattatatgaataaaaaagcattaaat	600
601	atattagaatataacaaaattattgagcgtgttgacgcatttactcaaaatgaactttca	660
661	agcaaaaagtgaggatgacacancctatcagcgacaaagctgaaatagatagcatgcttg	720
721	cacagcttggtatgataacgaaatgtc

[0035] (26) Staphylococcus aureus

1	atggttttggtagaattggtcgtttagcattcagaagaattcaagaagtagaaggtcttg	60	(SEQ ID NO:26)
61	aagttgtagcagtaaacgacttaacagatgacgacatgttagcgcatttattaaaatatg	120
121	acactatgcaaggtcgtttcacaggtgaagtagaggtagttgatggtggtttccgcgtaa	180
181	atggtaaagaagttaaatcattcagtgaaccagatgcaagcaaattaccttggaaagact	240
241	taaatatcgatgtagtgttagaatgtactggtttctacactgataaagataaagcacaag	300
301	ctcatattgaagcaggcgctaaaaaagtattaatctcagcaccagctactggtgacttaa	360
361	aaacaatcgtattcaacactaaccaccaagagttagacggttctgaaacagttgtttcag	420
421	gtgcttcatgtactacaaactcattagcaccagttgctaaagttttaaacgatgactttg	480
481	gtttagttgaaggtttaatgactacaattcacgcttatacaggtgatcaaaatacacaag	540
541	acgcacctcacagaaaaggtgacaaacgtcgtgctcgtgcagcagcagaaaacatcatcc	600
601	ctaactcaacaggtgctgctaaagctatcggtaaagttattcctgaaatcgatggtaaat	660
661	tagatggtggtgcacaacgtgttcctgtagctacaggttcattaactgaattaacagtag	720
721	tattagaaaaacaagacgtaacagttgaacaagttaacgaagctatgaaaaatgcttcaa	780
781	acgaatcattcggttacactgaagacgaaatcgtttcttcagacgttgtaggtatgactt	840
841	acggttcattatttgacgctacacaaactcgtgtaatgtcagttggcgaccgtcaattag	900
901	ttaaagttgcagcttggtatgataacgaaatgtc

[0036] (27) Staphylococcus warneri

1	atggttttggtagaattggtcgtttagcatttagaagaattcaagacgtagaaggtttag	60	(SEQ ID NO:27)
61	aagtagttgcagtaaacgacttaactgatgacgatatgttagcacacttattaaaatatg	120
121	acactatgcaaggtcgcttcactggtgaagtagaagttatcgacggtggattccgtgtta	180
181	acggtaaggaagttaaatcattcgatgaaccagatgcaagcaaattaccttggaaagact	240
241	tagatatcgacgtagtattagaatgtactggtttctatactgacaaagataaagcacaag	300
301	ctcatgatgacgcaggtgctaaaaaagtattaatctcagctccagcaactggtgacttaa	360
361	aaacaatcgtttacaacactaaccacgatgaattagatggttctgaaacagtagtatcag	420
421	gtgcatcatgtactactaactcattagctcctgtagctaaagtgttaagtgatgagttcg	480
481	gtttagttgaaggtttaatgacaactatccacgcttacactggtgaccaaaatactcaag	540
541	atgctccacacagaaaaggcgacaaacgtcgtgctcgtgctgcagcagaaaacatcatcc	600
601	ctaactcaactggtgctgctaaagcaatcggtaaagtaattcctgaaatcgacggaaaat	660
661	tagacggtggtgcacaacgtgttccagttgcaactggttcattaactgaattaacagtag	720
721	tattagaaaaagacgtaacagttgaacaagttaacgaagcaatgaaacaagcttctgacg	780
781	aatcattcggttacactgaagacgaaatcgtatcttctgatgtagttggtatgacttacg	840
841	gttcattattcgatgcaactcaaactcgtgtaatggctgttggtggtcgtcaattagtta	900
901	aagtagcagcttggtatgataacgaaatgtc

[0037] SEQ ID Nos: 1-25 correspond to NCBI GenBank Accession Nos. AF495474-495498, respectively; SEQ ID NO:26 corresponds to nucleotides 1867-2800 of NCBI GenBank Accession No. AJ133520; and SEQ ID NO:27 corresponds to nucleotides 32-962 of NCBI GenBank Accession No. AY024363.

[0038] The present invention provides a method for detecting Staphylococcus spp. Specifically, a nucleic acid template from a sample suspected of containing Staphylococcus spp. is amplified with a pair of Staphylococcus spp.-specific primers. The amplification product, if any, is detected by either gel electrophoresis and staining, or by probe hybridization. Detection of an expected amplification product indicates the presence of Staphylococcus spp. in the sample.

[0039] The nucleic acid template can be DNA (e.g., a genomic fragment or a restriction fragment) or RNA, in a purified or unpurified form. A nucleic acid template can be obtained from a human or an animal (e.g., a specimen).

[0040] The present invention features Staphylococcus spp.-specific primers containing oligo-nucleotides selected from the gap gene region described above. One primer contains an oligo-nucleotide selected from a member of the group consisting of SEQ ID NOs:28-52 (corresponding to SEQ ID NOs: 1-25 but excluding the first 26 and the last 25 nucleotides); the other primer contains an oligo-nucleotide selected from a sequence complementary to the member. Typically, a primer is 14-40 nucleotides in length (PCR Application Manual, Boehringer Mannheim, 1995, page 37). Non-Staphylococcus sequences can be added to the 5′-end of a primer. An example of a non-Staphylococcus sequence is a sequence containing a restriction site, which can be used to facilitate cloning of the amplification product.

[0041] The present invention also features Staphylococcus-specific probes chosen from the gap gene region described above, i.e., SEQ ID NOs: 1-27 and their complimentary sequences. These probes can be used for detecting Staphylococcus spp. by hybridizing to an unamplified Staphylococcus nucleic acid or an Staphylococcus nucleic acid amplified with the above-described primer pairs. SEQ ID NOs: 1-27 and their complimentary sequences are examples of such probes.

[0042] The probes can be immobilized on the surface of a solid support, such as a membrane (a nylon membrane or a nitrocellulose membrane), a glass, or a plastic polymer. Immobilization of probes to a membrane can be achieved by baking at 80° C. or UV cross-linking. The probes can also be covalently linked to a material (e.g., poly-lysine) coated on the surface of a glass. In addition, a novel method of immobilizing probes on a plastic polymer has recently been developed. See U.S. application Ser. No. 09/906,207. Alternatively, the probes can be synthesized de novo at precise positions on a solid substrate. See Schena et al., 1995, Science 270: 467; Kozal et al., 1996, Nature Medicine 2(7): 753; Cheng et al., 1996, Nucleic Acids Res. 24(2): 380; Lipshutz et al., 1995, BioTechniques 19(3): 442; Pease et al., 1994, Proc. Natl. Acad. Sci. USA 91: 5022; Fodor et al., 1993, Nature 364: 555; and Fodor et al., WO 92/10092.

[0043] A target Staphylococcus nucleic acid (e.g., an amplification product described above) can be detected by binding it to an immobilized probe. To facilitate the detection, a labeled amplification product can be generated with a labeled amplification primer. Alternatively, the labeling can be done, chemically or enzymatically, after amplification. Examples of labeling reagents include, but are not limited to, a fluorescent molecule (e.g., fluorescein and rhodamine), a radioactive isotope (e.g., 32P and 125I), a colorimetric reagent, and a chemiluminescent reagent. Biotin and digoxgenin are frequently used for calorimetric detection on a membrane or a plastic polymer. Fluorescent labels, such as Cy3 and Cy5, are widely used for detection on a glass. In addition, artificial tagging tails (e.g., a protein or its antibody) can be conjugated to the 5′-end of the primers or either end of the probes. See Stetsenko and Gait, 2000, J. Org. Chem. 65(16): 4900.

[0044] The specificity of the Staphylococcus spp. detection method of this invention is unexpectedly high. A probe derived from the gap gene region of Staphylococcus intermedius (67-71% identical to 25 other Staphylococcus spp.) detected only genomic DNA from Staphylococcus spp., but not that from other bacteria such as Salmonella spp., E. coli, Shigella spp., Enterobacter aerogenes, Citrobacterfreundii, Klebsiella pneumoniae, Listeria monocytogenes, Vibrio parahaemolyticus, Bacillus cereus, and Streptococcus aglactiae (see Example 2 below). Most unexpected is the ability of a probe derived from a Staphylococcus species to discriminate this Staphylococcus species from other Staphylococcus species having as high as 94% homology in the gap gene region (see Example 1 below).

[0045] Also within the scope of this invention is use of Staphylococcus spp.-specific sequences described above in combination with other species-specific nucleic acid sequences for simultaneously identification of multiple microorganisms.

[0046] The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

EXAMPLE 1

Typing Staphylococcus spp. by Probe Hybridization

[0047] 1. Bacterial Strains

[0048] Twenty-seven Staphlylococci strains were obtained from Culture Collection and Research Center (CCRC), Hsin-Chu, Taiwan. The bacterial strains and their registration numbers are listed in TABLE 1.

TABLE 1
LIST OF STAPHYLOCOCCUS STRAINS
No.	Bacterial Strain	CCRC No.	ATCC No.
1	Staphylococcus arlettae	13975	43957
2	Staphylococcus auricularis	13912	33753
3	Staphylococcus caprae	13911	35538
4	Staphylococcus capitis	12161	27840
5	Staphylococcus carnosus	12922	—
6	Staphylococcus chromogenes	12924	43764
7	Staphylococcus cohni	12155	29974
8	Staphylococcus delphini	15270	49171
9	Staphylococcus epidermidis	10783	155
10	Staphylococcus equorum	13974	43958
11	Staphylococcus gallinarum	13913	35539
12	Staphylococcus haemolyticus	12923	29970
13	Staphylococcus hominis	12156	27844
14	Staphylococcus hyricus	12925	11249
15	Staphylococcus intermedius	15235	49051
16	Staphylococcus kloosii	13973	43959
17	Staphylococcus lentus	12926	29070
18	Staphylococcus lugdunesis	13971	43809
19	Staphylococcus piscifermentans	15314	51337
20	Staphylococcus saprophyticus	10786	15305
21	Staphylococcus schleiferi	13972	43808
22	Staphylococcus sciuri	12972	29062
23	Staphylococcus simulans	10778	11631
24	Staphylococcus xylosus	12930	29971
25	Staphylococcus caseolyticus		13548
26	Staphylococcus aureus	10780	12600
27	Staphylococcus warneri	12929	27836

[0049] 2. Cultivation of Bacterial Strains

[0050] One loopful of each test strain was plated on Luria-Bertani agar (LB; 0.5% yeast extract, 1% trypton, 0.5% NaCl, 1.5-2% agar) and incubated for overnight (14 hr) at 37° C. to recover the bacterial vitality. A single colony was picked for each strain, inoculated into 3 ml sterilized LB broth, and incubated for overnight at 37° C. with shaking at 150-180 rpm. The cell density of each bacterial strain was up to ˜1×1010 cells/ml.

[0051] 3. Preparation of Bacterial Total Genomic DNA

[0052] Total genomic DNA was prepared from 1 ml of bacterial overnight culture. Cells were harvested by centrifugation at 6,000×g for 5 min and discarding the culture supernatant. Cell pellets were resuspended in 50 μl STET buffer (0.1M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 5% Triton X-100) containing 5 μl lysozyme (10 mg/ml). The cells were lysed after incubation for 15 min at 37° C. followed by 10 min-boiling. The DNA-containing supernatant were roughly separated from cell debris after 5-min centrifugation, extrated with phenol/chloroform (1:1), and centrifuged at 10,000×g for 10 min. The upper layer of the extraction mixture, ca. 40 μl, was transferred to a new eppendorf tube and used as a DNA template for amplification.

[0053] 4. Amplification of Bacterial Genomic DNA with Genus-Specific Oligo-Nucleotide Primers

[0054] Partial gap gene sequences of 27 different Staphylococcus species (TABLE 1) were analyzed by amplification using a primer pair (Yugueros et al., 2000, J. Clinical Microbiology 38: 4351-4355) located in the conserved region of the gap gene and subsequent sequence analysis. The primers used were GF-1 (5′-atggttttggtagaattggtcgttta-3′) and GR-2 (5′-gacatttcgttatcataccaagctg-3′), both of which were synthesized by GENASIA SCIENTIFICS INC. This pair of primers has been shown to be specific for Staphylococcus (Yugueros et al., 2000, J. Clinical Microbiology 38: 4351-4355).

[0055] The amplification reaction mixture (50 μl) contained 5 μl 10× Taq DNA polymerase buffer (Promega, Madison, Wis., USA), 5 μl 25 mM MgCl2 (Promega), 5 μl 2.5 mM dNTPs (Promega), 1 μl 20 μM of each oligo-nucleotide primer, 1 μl of extracted total genomic DNA, 1 U of Taq DNA polymerase (Promega), and sterilized dH2O. Amplification was carried out using GeneAmpg® PCR System 2400 (Perkin-Elmer) as followed: 95° C. for 2 min; 30 cycles of 95° C. for 30 sec, 55° C. for 30 sec, 72° C. for 30 sec; and a final extension of 72° C. for 6 min.

[0056] 5. Cloning and Sequencing of Staphylococcus spp. Partial Gap Gene Fragments

[0057] Each of the amplified partial gap gene fragments (ca. 933 bp) was cloned into a pGEM-T Easy vector system (Promega, Madison, Wis., USA), which was transformed into an E. coli bacterial host.

[0058] Three E. coli transformants were selected from each transformation, and the plasmid DNA containing the amplified Staphylococcus spp. gap gene fragment was isolated using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The presence of the amplified partial gap gene fragment was confirmed first by restriction enzyme digestion and then by sequencing using two primers, T7 promoter primer and SP6 promoter primer (Promega, Madison, Wis., USA). Twenty-five of these gene fragments are newly identified sequences; 2 of them have been previously published (i.e., Staphylococcus aureus gap gene fragment, NCBI GenBank Accession No. AJ133520, nucleotides 1867-2800; and Staphylococcus warneri gap gene fragment, NCBI GenBank Accession No. AY024363, nucleotides 32-962). The plasmids containing the 27 Staphylococcus spp. partial gap gene fragments are listed in TABLE 2 below.

TABLE 2
PLASMIDS CARRYING PARTIAL STAPHYLOCOCCUS
SPP. GAP GENE FRAGMENTS
	No.	Strains	Plasmid Name
	1	Staphylococcus aureus	pGAP-A1-1
	2	Staphylococcus arlettae	pGAP-A1-34
	3	Staphylococcus auricularia	pGAP-A2-2
	4	Staphylococcus carnusus	pGAP-A2-5
	5	Staphylococcus caprae	pGAP-A1-29
	6	Staphylococcus capitis	pGAP-A1-46
	7	Staphylococcus caseolyticus	pGAP-A2-37
	8	Staphylococcus hominis	pGAP-A1-4
	9	Staphylococcus cohni	pGAP-A1-7
	10	Staphylococcus chromogenes	pGAP-A2-31
	11	Staphylococcus delphini	pGAP-A2-19
	12	Staphylococcus epidermidis	pGAP-A1-32
	13	Staphylococcus equorum	pGAP-A2-16
	14	Staphylococcus gallinarum	pGAP-A2-5
	15	Staphylococcus haemolyticus	pGAP-A1-41
	16	Staphylococcus hyricus	pGAP-A2-27
	17	Staphylococcus intermedius	pGAP-A2-38
	18	Staphylococcus kloosii	pGAP-A2-14
	19	Staphylococcus lentus	pGAP-A1-17
	20	Staphylococcus lugdunesis	pGAP-A2-9
	21	Staphylococcus piscifermentans	pGAP-A2-20
	22	Staphylococcus saprophyticus	pGAP-A1-38
	23	Staphylococcus sciuri	pGAP-A1-50
	24	Staphylococcus schleifer	pGAP-A2-11
	25	Staphylococcus simulans	pGAP-A1-22
	26	Staphylococcus warneri	pGAP-A1-19
	27	Staphylococcus xylosus	pGAP-A1-24

[0059] 6. Sequence Alignment of Amplified Partial Gap Gene Fragments

[0060] Classification of homology degree of these bacteria is based on sequence analysis results obtained by using the phylogenetic tree and sequence pair distance program (Lesegene, DNAstar Inc., Wis., USA) and the BLAST sequence alignment program (NCBI GenBank).

[0061] The homology degree among 26 of the Staphylococcus spp. is in the range of 68-99%. One exception is the strain Staphylococcus caseolyticus, which shows only 22-27% homology to the other 26 Staphylococcus spp. This strain has been previously misidentified as Micrococcus caseolyticus (Schleifer et al., 1982, J. Systematic Bacteriology 32: 15-20). DNA-DNA hybridization data and other physiological and biochemical data have been used to reclassify this bacterium. Thirteen pairs of Staphylococcus spp. (TABLE 3) have a homology degree over 90%. In particular, pair no. 1 (i.e., Staphylococcus epidermidis-Staphylococcus warneri) shows 99% homology, and the other 12 pairs have a homology degree up to 94%.

TABLE 3
HOMOLOGY DEGREES FOR THIRTEEN
PAIRS OF STAPHYLOCOCCUS SPP.
Pair			Homology
No.	Bacterial Strain-1	Bacterial Strain-2	Degree*
1	Staphylococcus	Staphylococcus	99.5%
	epidermidis	warneri
2	Staphylococcus	Staphylococcus	94%
	capitis	caprae
3	Staphylococcus	Staphylococcus	94.3%
	camusus	hyricus
4	Staphylococcus	Staphylococcus	93.8%
	camusus	simulans
5	Staphylococcus	Staphylococcus	92.3%
	saprophyticus	xylosus
6	Staphylococcus	Staphylococcus	92.3%
	lentus	sciuri
7	Staphylococcus	Staphylococcus	91.3%
	wareri	capitis
8	Staphylococcus	Staphylococcus	91.2%
	hyricus	chromogenes
9	Staphylococcus	Staphylococcus	91.1%
	epidermidis	capitis
10	Staphylococcus	Staphylococcus	90.5%
	saprophyticus	equorum
11	Staphylococcus	Staphylococcus	90.5%
	arlettae	kloosii
12	Staphylococcus	Staphylococcus	90.3%
	caprae	wareri
13	Staphylococcus	Staphylococcus	90.1%
	caprae	epidermis
*Degree of homology between two species is determined by using either the DNA sequence analyzing software Lesegene (i.e., pair nos 3-13) or GenBank BLAST (i.e., pair nos 1 and 2).

[0062] 7. Preparation of Hybridization Probes and Simulative Target DNA

[0063] Both hybridization probes and simulative target DNA were generated by the amplifying the gap gene of three bacteria, Staphylococcus aureus, Staphylococcus caprae, and Staphylococcus capitis, using the primer pair GF-1 and GR-2. The amplified partial gap gene fragments from Staphylococcus caprae and from Staphylococcus capitis are 94% identical, while each of them shows 89.2% homology to the Staphylococcus aureus gap gene fragment. The amplification products (around 930 bp) obtained by using biotin-labeled primers were used as simulative target DNA, while the amplification products obtained by using unlabeled primers were used as hybridization probes. The amplification was carried out as described above, except that around 100 ng purified plasmid DNA was used as DNA template instead of bacterial genomic DNA. The amplified products were extracted with phenol/chloroform (v/v=1/1) and precipitated with ethanol to remove excess primers, dNTPs and the enzyme.

[0064] 8. Hybridization

[0065] The three hybridization probes were dissolved in a probe solution (DR. Probsol, DR. Chip Biotechnology Inc.) to a final concentration of 20 ng/μl, spotted and immobilized on a solid support (DR. Chip Biotechnology Inc.).

[0066] Four microliters of each biotin-labeled simulative target DNA (stock concentration: 40 ng/μl) were mixed with 500 μl hybridization buffer (Dr. Hyb™ buffer, DR. Chip Biotechnology Inc.). The DNA mixture was boiled for 5 min, chilled, applied to the solid support, and incubated at 80° C. for 1 hr with shaking.

[0067] Three wash steps were carried out to eliminate unspecific binding. The solid support was first washed with a wash buffer (DR. Wash from DR. Chip Biotechnology Inc., Taiwan) for at least three times at room temperature. A stringent wash was then performed with the same wash buffer at 80° C. for 30 min with shaking. The solid support was finally washed with the same wash buffer for at least three times at room temperature again.

[0068] Biotin-specific calorimetric detection was performed by incubating the solid support in a Blocking Reagent (Roche) containing alkaline phosphatase-conjugated streptavidin (Promega). The solid support was subsequently washed three times with the wash buffer, and incubated with NBT/BCIP solution (Roche) diluted with a detection buffer in a ratio recommended by the supplier for about 10 minutes in dark.

[0069] Unexpectedly, the Staphylococcus aureus target DNA only hybridized to the probe originated from the same bacterial species, but not to the probes originated from the other two Staphylococcus species. Similarly, the Staphylococcus caprae and Staphylococcus capitis target DNAs only hybridized to the corresponding probe. This result indicates that this method can be used to differentiate Staphylococcus spp. strains with very high homology degree (e.g., 94%).

EXAMPLE 2

Detecting Staphylococcus Genus by Probe Hybridization

[0070] 1. Bacterial Strains

[0071] Eighty food-borne bacterial strains listed in TABLE 4 were used in this study. Group I contains 10 Staphylococcus spp.; Group II contains another 10 Staphylococcus spp.; Group III contains 10 Salmonella spp.; Group IV contains 30 E. coli strains, including non-pathogenic strains and pathogenic strains such as ETEC, EIEC, EPEC, EAggEC and EHEC; and Group V contains other bacterial strains, including 11 pathogenic strains (i.e., 4 Shigella spp.; 3 coliform bacterial strains Enterobacter aerogenes, Citrobacterfreundii, and Klebsiella pneumoniae; 3 other food-borne pathogenic bacterial strains Listeria monocytogenes, Vibrio parahaemolyticus, and Bacillus cereus; and 1 infectious bacterial strain causing cattle mastitis, Streptococcus agalactiae). These bacterial strains were obtained from different sources, i.e., Culture Collection and Research Center (CCRC), Hsin-Chu, Taiwan; American Type Culture Collection (ATCC), Rockville, Md., USA; United States Department of Agriculture (USDA), Washington, D.C., USA; Department of Food Science, National Chung-Hsing University (NCHU; Taichung, Taiwan, R.O.C.); Pingtung University of Technology (PT), Pingtung, Taiwan; and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; Braunschweig, Germany).

TABLE 4
BACTERIAL STRAINS
Group	Strains	No.	Source & Number
Group I	Staphylococcus	1	CCRC 13975	ATCC 43957
Staphylococcus	arlettae
	Staphylococcus	1	CCRC 13912	ATCC 33753
	auricularia
	Staphylococcus	1	CCRC 12924	ATCC 43764
	chromogenes
	Staphylococcus	1	CCRC 10783	ATCC 155
	epidermidis
	Staphylococcus	1	CCRC 13913	ATCC 35539
	gallinarum
	Staphylococcus	1	CCRC 12156	ATCC 27844
	hominis
	Staphylococcus	1	CCRC 12926	ATCC 29070
	lentus
	Staphylococcus	1	CCRC 15314	ATCC 51337
	piscifermentans
	Staphylococcus	1	CCRC 10778	ATCC 11631
	simulans
	Staphylococcus	1	CCRC 13972	ATCC 43808
	schleiferi
	Staphylococcus	1	CCRC 12930	ATCC 29971
	xylosus
	Staphylococcus	1	CCRC 12161	ATCC 49324
	capitis
	Staphylococcus	1	CCRC 12922	DSM 20501
	carnusus
Group II	Staphylococcus	1	CCRC 12155	ATCC 29974
Staphylococcus	cohni
	Staphylococcus	1	CCRC 13974	ATCC 43953
	equorum
	Staphylococcus	1	CCRC 12923	ATCC 29970
	haemolyticus
	Staphylococcus	1	CCRC 12925	ATCC 11249
	hyricus
	Staphylococcus	1	CCRC 13973	ATCC 43959
	kloosii
	Staphylococcus	1	CCRC 13971	ATCC 43809
	lugdunesis
	Staphylococcus	1	CCRC 10786	ATCC 15305
	saprophyticus
	Staphylococcus	1	CCRC 12927	ATCC 29062
	sciuri
	Staphylococcus	1	CCRC 12929	ATCG 27336
	warneri
	Staphylococcus	1	CCRC 15270	ATCC 49171
	delphini
	Staphylococcus	1	CCRC 15263	ATCC 13543
	caseolyticus
Group III	Salmonella	1	CCRC 14875	ATCC 9992V
Salmonella	typhi
spp.	Salmonella	1	CCRC 10747	ATCC 14028
	typhiurium
	Salmonella	1	CCRC 15450	ATCC 6956
	salamae
	Salmonella	1	CCRC 14878	ATCC 9150
	paratyphi A
	Salmonella	1	CCRC 15454	ATCC 23201
	california
	Salmonella	1	CCRC 10744	ATCC 13076
	enteritidis
	Salmonella	1	CCRC 15455	ATCC 19128
	etterbeele
	Salmonella	1	CCRC 15453	ATCC 15782
	postsdam
	Salmonella	1	NCHU	USDA
	aberdeen
	Salmonella	1	NCHU	USDA
	albany
	Salmonella	1	NCHU	USDA
	amger
	Salmonella	1	NCHU	PT
	anatum
Group IV	EAggEC	4	NCHU	—
E. coli	ETEC	4	NCHU	—
	EHEC	5	NCHU	—
	EPEC	5	NCHU	—
	EIEC	3	NCHU	—
	Non-	9	NCHU	—
	pathogenic
Group V	Streptococcus	1	CCRC10787	ATCC13813
Other strains	agalactea
	Bacillus cereus	1	CCRC11827	ATCC25428
	Shigella	1	CCRC13983	ATCC13313
	dysenteria
	Shigella boydii	1	CCRC15961	ATCC8704
	Shigella	1	CCRC10772	ATCC12022
	flexneri
	Shigella sonnei	1	CCRC10773	ATCC9290
	Vibrio para-	1	CCRC10806	ATCC17802
	haemolyticus
	Listeria	1	CCRC14930	—
	monocytogenes
	Enterobacter	1	CCRC10370	ATCC13048
	aerogenes
	Citrobacter	1	CCRC12291	ATCC8090
	freundii
	Klebsiella	1	CCRC15627	—
	pneumoniae
Positive control	Staphylococcus	1	CCRC10780	ATCC12600
	aureus
strains	Staphylococcus	1	CCRC13911	ATCC35538
	caprae
	Staphylococcus	1	CCRC15235	ATCC49051
	intermedius

[0072] 2. Cultivation of Bacterial Strains

[0073] One loop of each test strain was plated on Luria-Bertani agar (LB; 0.5% yeast extract, 1% trypton, 0.5% NaCl, 1.5-2% agar) and incubated for overnight (14 hr) at 37° C. A single colony was picked for each strain and inoculated into 10 ml sterilized LB broth. Bacterial cultures were incubated for overnight at 37° C. with shaking at 120-150 rpm.

[0074] 3. Preparation of Bacterial Mixtures and Extraction of Genomic DNA

[0075] One milliliter suspension (108-1010 cells) was taken from each bacterial culture and mixed in one tube according to the groups listed in TABLE 1. QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used for extraction of total genomic DNA from 1 ml mixed culture and for further purification. Quantification of extracted genomic DNA was carried out by spectrophotometric method (V-530, Jasco, Jascon International Co., LTD, Japan). The final concentration of the genomic DNA prepared from each bacterial mixture was adjusted to 5 μg/μl.

[0076] 4. Immobilization of DNA on a Solid Support

[0077] One microliter (5 μg/μl) of prepared DNA (equivalent to a DNA extract from 4.1×105-1.7×108 cells for each bacterial strain), including mixed genomic DNA of each bacterial group, genomic DNA of a certain bacteria species, and control plasmid DNA, was dissolved in a probe solution (DR. Probsol, DR. Chip Biotechnology Inc., Taiwan), denatured at 95° C. for 5 min, and chilled immediately on ice. The pretreated DNA solution was dotted and cross-linked on a nylon membrane (Hybond™-N, Amersham Pharmacia Biotech) of 5 cm×5 cm.

[0078] 5. Preparation of Biotin-Labeled Detection Probes

[0079] Biotin-labeled probes were generated as described above. Plasmid pGAP-A2-38 containing the Staphylococcus intermedius gap gene fragment was used as a DNA template for amplification. Among the 26 Staphylococcus spp. (Staphylococcus caseolyticus excluded), Staphylococcus intermedius shows the lowest homology (67%-71%) to other species.

[0080] 6. Hybridization

[0081] The biotin-labeled detection probe was dissolved in 50 ml hybridization buffer (Dr. Hyb™ buffer, DR. Chip Biotechnology Inc.) to a final concentration of 0.1 nM, denatured at 95° C. for 5 min, and immediately chilled on ice for 5 min. This pretreated detection probe solution was then hybridized to DNA spotted on a nylon membrane for 12 hr at 50° C. The nylon membrane was then washed with 50 ml wash buffer (DR. Wash from DR. Chip Biotechnology Inc., Taiwan) twice at 50° C. for 5 min.

[0082] Unexpectedly, positive signals were detected at spots containing the mixed genomic DNA of Group I and II bacteria, the genomic DNA of three Staphylococcus spp. (i.e., Staphylococcus aureus, Staphylococcus caprae, and Staphylococcus intermedius), and positive controls. No signal was detected at spots containing the mixed genomic DNA of Group III, IV, or V bacteria. This result indicates that the Staphylococcus spp. gap gene fragment can be used to differentiate Staphylococcus from other bacterial genera.

OTHER EMBODIMENTS

[0083] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0084] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

Claims

1. A novel nucleic acid comprising an oligo-nucleotide selected from a member of the group consisting of SEQ ID NOs: 1-25 and sequences complementary to SEQ ID NOs:1-25, wherein the nucleic acid is 10-1000 nucleotides in length.

2. The nucleic acid of claim 1, wherein the nucleic acid is 10-500 nucleotides in length.

3. The nucleic acid of claim 2, wherein the nucleic acid is 10-200 nucleotides in length.

4. The nucleic acid of claim 3, wherein the nucleic acid is 10-50 nucleotides in length.

5. The nucleic acid of claim 4, wherein the nucleic acid is 10-20 nucleotides in length.

6. The nucleic acid of claim 1, wherein the oligo-nucleotide is a member of the group consisting of SEQ ID NOs:1-25 and sequences complementary to SEQ ID NOs:1-25.

7. A pair of amplification primers, comprising a first primer containing a first oligo-nucleotide selected from a member of the group consisting of SEQ ID NOs:28-52, and a second primer containing a second oligo-nucleotide selected from a sequence complementary to the member, wherein each primer is 14-40 nucleotides in length.

8. The pair of primers of claim 7, wherein each primer is 14-30 nucleotides in length.

9. The pair of primers of claim 8, wherein each primer is 14-20 nucleotides in length.

10. A method of detecting a target Staphylococcus species, comprising: providing a sample having a nucleic acid from an unknown microorganism; amplifying the nucleic acid with a pair of primers containing a first primer including a first oligo-nucleotide selected from a member of the group consisting of SEQ ID NOs:28-52 and a second primer including a second oligo-nucleotide selected from a sequence complementary to the member, each primer being 14-40 nucleotides in length; and detecting an amplification product; whereby detection of the amplification product indicates the presence of the target Staphylococcus species.

11. The method of claim 10, wherein each primer is 14-30 nucleotides in length.

12. The method of claim 11, wherein each primer is 14-20 nucleotides in length.

13. The method of claim 10, wherein the detecting step includes hybridizing the amplification product to a nucleic acid probe that is 10-1000 nucleotides in length and contains a sequence selected from a member of the group consisting of SEQ ID NOs: 1-27 and sequences complementary to SEQ ID NOs:1-27.

14. The method of claim 13, wherein the nucleic acid probe is 10-500 nucleotides in length.

15. The method of claim 14, wherein the nucleic acid probe is 10-200 nucleotides in length.

16. The method of claim 15, wherein the nucleic acid probe is 10-50 nucleotides in length.

17. The method of claim 16, wherein the nucleic acid probe is 10-20 nucleotides in length.

18. The method of claim 17, wherein the nucleic acid probe is a member of the group consisting of SEQ ID NOs:1-27 and sequences complementary to SEQ ID NOs:1-27.