(en)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.

1.ApplicationNumber: US-16475802-A
1.PublishNumber: US-2003232337-A1
2.Date Publish: 20031218
3.Inventor: LIU LU-YIENG
LEE KAN-HUNG
TERNG HARN-JING

4.Inventor Harmonized: LIU LU-YIENG(TW)
LEE KAN-HUNG(TW)
TERNG HARN-JING(TW)

5.Country: US
6.Claims:
(en)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.

7.Description:
(en)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., 32 P 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 ˜1×10 10 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 MgCl 2 (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 dH 2 O. 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

[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 (10 8 -10 10 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 4.1×10 5 -1.7×10 8 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.