Genetic methods for speciating Campylobacter

A method of speciating Campylobacter by polymerase chain reaction, said method comprising:

• a) providing a standard sample mixture of DNA of each Campylobacter species (spp.):
• C. jejuni, C. coli, C. concisus, C. curvus, C. showae, C. mucosalis, C. fetus fetus, C. hyointestinalis, C. sputorum sputorum, C. helveticus, C. upsaliensis and C. lari or providing a test sample mixture suspected of containing DNA of any or all of said Campylobacter spp.;
• b) amplifying a target sequence of DNA of all of the Campylobacter spp. in said sample mixture, wherein said target sequence is the gyrB sequence of each Campylobacter spp. present in said standard or test sample mixture, by adding a mixture of the following species-specific primer sets complementary to the gyrB sequence of all of the following Campylobacter species;
• comprising, C. jejuni wherein said primer set consists of AGAATGGGTTTAACTCGTGTGATAAGT (SEQ ID NO:29) and TACCACGCA
• AAGGCAGTATAGCT (SEQ ID NO:
• 30), C. coli wherein said primer set consists of AAATGCTAGTGCTAGGGAAAAAGACTCT (SEQ ID NO:31) and TGAGGT TCAGGCACTTTTACACTTACTAC (SEQ ID NO:32), C. concisus wherein said primer set consists of AGCGGGCCTAACAAGAGTT ATTACA (SEQ ID NO:33) and TGTAAGCACGTCAAAAACCATCTTT (SEQ ID NO:
• 34), C. curvus wherein said primer set consists of CTGCCAAAGTAAGGAC GCAAGTATA (SEQ ID NO:35) and GGCAAGATCGCCTGAAATACG (SEQ ID NO:
• 36);
• C. showae wherein said primer set consists of AGGGTTTAAGCATA GGAACGCTG (SEQ ID NO:
• 37) and CACCAGATAAAGCTCGCTGATCG (SEQ ID NO:
• 38), C. mucosalis wherein said primer set consists of TGCGATTATGAA CAAGGCCCTA (SEQ ID NO:
• 39) and TCGCTTGAAACACACGGTCA (SEQ ID NO:
• 40), C. fetus fetus wherein said primer set consists of AGAGCTGGGCTTAC AAGAGCTATTACA (SEQ ID NO:
• 41) and GGTAAAATCGCTTGAAACGCTCTAT (SEQ ID NO:
• 42), C. hyointestinalis wherein said primer set consists of CGGTCA AAAGATGACTTTTGAAGTACTT (SEQ ID NO:
• 43) and GCTTCCCTGCCACGA GCT (SEQ ID NO:
• 44), C. sputorum sputorum wherein said primer set consists of AGCTTTACTTGCTGCAAGAGGAAGA (SEQ ID NO:
• 45) and AGGAAGCGTTCC AACAGAAAA GTT (SEQ ID NO:
• 46), C. helveticus wherein said primer set consists of CAATA ACATACGCACACCAGATGGA (SEQ ID NO:
• 47) and CAGGCACTTTAACGCTCACTATGG (SEQ ID NO:
• 48), C. upsaliensis wherein said primer set consists of GCTTACGCGTGTAATTACAAACTATGTC (SEQ ID NO:
• 49) and AATTGCCTTAGCCTCGATAGGG (SEQ ID NO:
• 50) and C. lari wherein said primer set consists of CTATGTTCGTCCTATAGTTTCTAAGGCTTC (SEQ ID NO:
• 51) and CCAGCACTATCACCCTCAACTAAATAA (SEQ ID NO:
• 52);
• and
• c) rapidly detecting the presence of amplification products of the target sequence of DNA of all Campylobacter spp. in said standard or test sample mixture and directly identifying the presence of all species and strains of Campylobacter spp. in said sample, wherein said amplification product of 493 bp identifies C. jejuni, said amplification product of 96 bp identifies C. coli, said amplification product of 217 bp identifies C. concisus, said amplification product of 108 bp identifies C. curvus, said amplification product of 86 bp identifies C. showae, said amplification product of 224 bp identifies C. mucosalis, said amplification product of 482 bp identifies C. fetus fetus, said amplification product of 108 bp identifies C. hyointestinalis, said amplification product of 94 bp identifies C. sputorum sputorum, said amplification product of 176 bp identifies C. helveticus, said amplification product of 250 bp identifies C. upsaliensis, said amplification product of 261 bp identifies C. lari.

A method of speciating Campylobacter by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP), said method comprising:

• a) providing a standard sample mixture of DNA of each Campylobacter species (spp.):
• C. jejuni, C. coli, C. concisus, C. curvus, C. showae, C. mucosalis, C. fetus fetus, C. hyointestinalis, C. sputorum sputorum, C. helveticus, C. upsaliensis and C. lari or providing a test sample mixture suspected of containing DNA of any or all of said Campylobacterspp.;
• b) amplifying a target sequence of DNA of all of the Campylobacterspp. in said standard or test sample mixture, wherein said target sequence is the gyrB sequence of each Campylobacter spp. present in said standard or test sample mixture, by adding a mixture of the following primer sets to the standard or test sample mixture wherein the primer set mixture comprises oligonucleotide primers complementary to the gyrB sequence of all of the following Campylobacter species;
• comprising, C. jejuni wherein said primer set consists of CGTCAAGAATTTTCAGAAGGTAAAGTT ATC (SEQ ID NO:5) and TATAGCTTGGAAAGATCTTTCCCTACCTTG (SEQ ID NO:
• 6), C. coli wherein said primer set consists of CGCCAAGAATTTTCAGAAGGTAAAGTCATC (SEQ ID NO:7) and TATAGCTTGAAAAGATCTTTCTCTACCTTG (SEQ ID NO:8), C. concisus wherein said primer set consists of AGACAAG AATTTGCAAAAGGT ATCCCTCAA (SEQ ID NO:9) and AATCGCTTGAAAAGATCTTTCTCTTCCTTG (SEQ ID NO:
• 10), C. curvus wherein said primer set consists of AGGCAAGAATT TCAAAAAGGTATCCCGGTA (SEQ ID NO:11) and TATCGCCTGAAAAACTCT ATCACGTCCTTG (SEQ ID NO:
• 12), C. showae wherein said primer set consists of AGACAAGAATTTTCAAAAGGTATCCCTCAA (SEQ ID NO:
• 13) and GATCGC CTGAAATACGCGGTCGCGTCCTTG (SEQ ID NO:
• 14), C. mucosalis wherein said primer set consists of AGGCAAGAATTTGCAAAAGGAATTCCAGTA (SEQ ID NO:
• 15) and AATCGCTTGAAATACTCTATCGCGTCCTTG (SEQ ID NO:
• 16), C. fetus fetus wherein said primer set consists of CGTCAAGAGTTTTCAAAAG GAATACCCCAA (SEQ ID NO:
• 17) and AATCGCTTGAAACACACGGTCACG ACC CTG (SEQ ID NO:
• 18), C. hyointestinalis wherein said primer set consists of CGCCAAGAATTCGCCGAAGGCATACCTCAA (SEQ ID NO:
• 19) and AATCGCT TGAAACGCTCTATCACGACCTTG (SEQ ID NO:
• 20), C. sputorum sputorum wherein said primer set consists of AGACAAGAGTTTTCAAAAGGTGTTCCT ACA (SEQ ID NO:
• 21) and TATCGCTTGAAATGCTCTATCACGACCTTG (SEQ ID NO:
• 22), C. helveticus wherein said primer set consists of AGACAAGAATTT TCTAAAGGTCTAATTGCA (SEQ ID NO:
• 23) and GATGGCTTGAAAAACTCTAT TTCTACCTTG (SEQ ID NO:
• 24), C. upsaliensis wherein said primer set consists of CGCCAAGAATTTGCTAAAGGGCAAATAGCT (SEQ ID NO:
• 25) and AATCGC TTGAAAAGCCCTTTCACGCCCCTG (SEQ ID NO:26), and C. lari wherein said primer set consists of AGACAAGAATTTTCAGAAGGAAAAGTAACA (SEQ ID NO:
• 27) and AATAGCTTGAAAAGTTCTTTCTCTACCTTG (SEQ ID NO:
• 28);
• c) obtaining amplification products of the target sequence of DNA of all Campylobacter spp. in said standard or test sample mixture as an indication of the presence of Campylobacter species, wherein all of said Campylobacter spp. present in the standard or test sample mixture can be identified;
• d) digesting the DNA amplification products obtained by PCR with the restriction enzymes MboI and HindIII in combination, as a double digestion;
• e) analyzing the restriction fragment length polymorphisms resulting from said digesting step by gel electrophoresis;
• and
• f) identifying all species and strains of Campylobacter spp. in said standard or test sample mixture by their RFLP patterns, identification resulting from a comparison of said RFLP patterns to the standard RFLP patterns generated when a standard mixture known to contain gyrB DNA of all said species and strains of Campylobacter spp. is analyzed using said mixture consisting of all said oligonucleotide primers complementary to the gyrB sequence of all said Campylobacter spp.