Patent Publication Number: US-2003232337-A1

Title: Detection of 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.,  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 &amp; 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.