Abstract
This method was aimed targeting more Campylobacter species than conventional PCR-based identifications. They generally use species-specific primers focusing on clinically common species like C. jejuni, resulting in failure to recognize other species. We made the PCR-based identification more flexible using degenerate primers and DdeI- and MboI-separately used RFLP assay, which were designed on the basis of gyrB nucleotide sequence data of 14 Campylobacter species including C. jejuni, C. coli, and C. fetus. Ninety-four clinical isolates from patients with Campylobacter gastroenteritis and 13 biochemically identified C. fetus were used for its evaluation. In consequence, this method succeeded in identifying C. jejuni, C. coli, and C. fetus with tentative sensitivity (93.4–98.0%) and specificity (89.0–99.0%). According to our data-based analysis, the primers can possibly target other related species including Helicobacter and Arcobacter. This method may be a universal identification for Campylobacter and related organisms and would provide an alternative identification in clinical microbiology.
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References
Gilbert MJ, Kik M, Miller WG, Duim B, Wagenaar JA (2015) Campylobacter iguaniorum sp. nov., isolated from reptiles. Int J Syst Evol Microbiol 65:975–982. doi:10.1099/ijs.0.000048
Koziel M, O’Doherty P, Vandamme P, Corcoran GD, Sleator RD, Lucey B (2014) Campylobacter corcagiensis sp. nov., isolated from faeces of captive lion-tailed macaques (Macaca silenus). Int J Syst Evol Microbiol 64:2878–2883. doi:10.1099/ijs.0.063867-0
On SLW (2013) Isolation, identification and subtyping of Campylobacter: where to from here? J Microbiol Methods 95:3–7. doi:10.1016/j.mimet.2013.06.011
Kaakoush NO, Castaño-Rodríguez N, Mitchell HM, Man SM (2015) Global epidemiology of campylobacter infection. Clin Microbiol Rev 28:687–720. doi:10.1128/CMR.00006-15
Martiny D, Dediste A, Debruyne L, Vlaes L, Haddou NB, Vandamme P, Vandenberg O (2011) Accuracy of the API Campy system, the Vitek 2 Neisseria-Haemophilus card and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of Campylobacter and related organisms. Clin Microbiol Infect 17:1001–1006. doi:10.1111/j.1469-0691.2010.03328.x
Yamamoto S, Harayama S (1995) PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol 61:1104–1109
Gorkiewicz G, Feierl G, Schober C, Dieber F, Kofer J, Zechner R, Zechner EL (2003) Species-specific identification of campylobacters by partial 16S rRNA gene sequencing. J Clin Microbiol 41:2537–2546. doi:10.1128/JCM.41.6.2537-2546.2003
Gunther NW IV, Almond J, Yan X, Needleman DS (2011) GyrB versus 16s rRna sequencing for the identification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari. J Nucleic Acids Invest 2:39–42. doi:10.4081/jnai.2011.e7
Kawasaki S, Fratamico PM, Wesley IV, Kawamoto S (2008) Species-specific identification of campylobacters by PCR-restriction fragment length polymorphism and PCR targeting of the gyrase B gene. Appl Environ Microbiol 74:2529–2533. doi:10.1128/AEM.00975-07
Pitcher DG, Saunders NA, Owen RJ (1989) Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8:151–156. doi:10.1111/j.1472-765X.1989.tb00262.x
Fujimoto S, Allos BM, Misawa N, Patton CM, Blaser MJ (1997) Restriction fragment length polymorphism analysis and random amplified polymorphic DNA analysis of campylobacter jejuni strains isolated from patients with Guillain-Barré syndrome. J Infect Dis 176:1105–1108
Stonnet V, Guesdon J-L (1993) Campylobacter jejuni: specific oligonucleotides and DNA probes for use in polymerase chain reaction-based diagnosis. FEMS Immunol Med Microbiol 7:337–344. doi:10.1111/j.1574-695X.1993.tb00415.x
Stonnet V, Sicinschi L, Mégraud F, Guesdon JL (1995) Rapid detection of Campylobacter jejuni and Campylobacter coli isolated from clinical specimens using the polymerase chain reaction. Eur J Clin Microbiol Infect Dis 14:355–359. doi:10.1007/BF02116533
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. doi:10.1093/molbev/msw054
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791. doi:10.2307/2408678
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120. doi:10.1007/BF01731581
Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Bioinformatics 8:275–282. doi:10.1093/bioinformatics/8.3.275
On SLW, Jordan PJ (2003) Evaluation of 11 PCR assays for species-Level identification of Campylobacter jejuni and Campylobacter coli. J Clin Microbiol 41:330–336. doi:10.1128/JCM.41.1.330-336.2003
Acknowledgements
The authors thank the CRC corporation, Japan, to send clinical isolates to our laboratory.
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This work was supported by the Health Labour Sciences Research Grant (Research on Emerging and Re-emerging Infectious Diseases) from the Ministry of Health, Labour and Welfare, Japan.
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Since all the isolates used in this study were anonymously obtained and analyzed, ethical approval and informed consent were not required.
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Fukuda, H., Kojima, F. & Fujimoto, S. New Identification of Three or More Campylobacter Species on the Basis of a Degenerate PCR–RFLP Method Targeting gyrB Gene. Curr Microbiol 74, 1160–1168 (2017). https://doi.org/10.1007/s00284-017-1300-4
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DOI: https://doi.org/10.1007/s00284-017-1300-4