PCR revisited: a case for revalidation of PCR assays for microorganisms using identification of Campylobacter species as an exemplar
Main Article Content
Keywords
Campylobacter jejuni, Campylobacter coli, identification, PCR, revalidation
Abstract
We re-examined the sensitivity and specificity of 31 PCR assays (including four commercially available and developed in-house methods) for the identification of Campylobacter species, with particular reference to taxa described since 2004, which are closely related to C. jejuni and C. coli, the pathogenic species of most interest. Each of the assays was used by at least one of the participating nine laboratories in eight countries. The sensitivity and specificity of these PCR assays examined varied considerably and ranged from 100% to 0% for sensitivity and 100% to 55% for specificity. None of the three assays examined for C. lari were successful in detecting all strains of this species, possibly reflecting its complex taxonomy. A number of assays for C. jejuni, C. coli, and a subgroup of enteropathogenic campylobacters, were found to yield false positive results for Campylobacter species described since PCR tests were reported, including C. cuniculorum, C. subantarcticus, C. peloridis and C. volucris. Our study supports the need for attention to detail in initial PCR assay design and evaluation, and also for on-going revalidation of laboratory assays to ensure that diagnoses are correct. Recommendations to guide the revalidation process are presented.
References
Abubakar, I., Irvine, L., Aldus, C.F., Wyatt, G.M., Fordham, R., Schelenz, S., Shepstone, L., Howe, A., Peck, M. and Hunter P.R., 2007. A systematic review of the clinical, public health and cost-effectiveness of rapid diagnostic tests for the detection and identification of bacterial intestinal pathogens in faeces and food. Health Technology Assessment 11: 1-216.
Debruyne, L., Broman, T., Bergström, S., Olsen, B., On, S.L.W. and Vandamme, P., 2010a. Campylobacter subantarcticus sp. nov., isolated from birds in the sub-Antarctic region. International Journal of Systematic and Evolutionary Microbiology 60: 815-819.
Debruyne, L., Broman, T., Bergström, S., Olsen, B., On, S.L.W. and Vandamme, P., 2010b. Campylobacter volucris sp. nov., isolated from black-headed gulls (Larus ridibundus). International Journal of Systematic and Evolutionary Microbiology 60: 1870-1875.
Debruyne, L., On, S.L.W., De Brandt, E. and Vandamme, P., 2009. Novel Campylobacter lari-like bacteria from humans and molluscs: description of Campylobacter peloridis sp. nov., Campylobacter lari subsp. concheus subsp. nov. and Campylobacter lari subsp. larisubsp. nov. International Journal of Systematic and Evolutionary Microbiology 59: 1126-1132.
Debruyne, L., Samyn, E., De Brandt, E., Vandenberg, O., Heyndrickx, M. and Vandamme, P., 2008. Comparative performance of different PCR assays for the identification of Campylobacter jejuni and Campylobacter coli. Research in Microbiology159: 88-93.
Duim, B., Wagenaar, J.A., Dijkstra, J.R., Goris, J., Endtz, H.P. and Vandamme, P.A., 2004. Identification of distinct Campylobacter lari genogroups by amplified fragment length polymorphism and protein electrophoretic profiles. Applied and Environmental Microbiology70: 18-24.
Englen, M.D. and Fedorka-Cray, P.J., 2002. Evaluation of a commercial diagnostic PCR for the identification of Campylobacter jejuni and Campylobacter coli. Letters in Applied Microbiology 35: 353-356.
Eyers, M., Chapelle, S., Van Camp, G., Goossens, H. and De Wachter, R., 1993. Discrimination among thermophilic Campylobacter species by polymerase chain reaction amplification of 23S rRNA gene fragments. Journal of Clinical Microbiology 31: 3340-3343 (Erratum, 1994. 32: 1623).
Foster, G., Holmes, B., Steigerwalt, A.G., Lawson, P.A., Thorne, P., Byrer, D.E., Ross, H.M., Xerry, J., Thompson, P.M. and Collins, M.D., 2004. Campylobacter insulaenigrae sp. nov., isolated from marine mammals. International Journal of Systematic and Evolutionary Microbiology 54: 2369-2673 (Erratum, 2005. 55: 981).
Institute of Environmental Science and Research (ESR), 2011. Foodborne disease in New Zealand 2010. ERS, Christchurch, New Zealand, 119 pp.
Kawasaki, S., Fratamico, P.M., Wesley, I.V. and Kawamoto, S., 2008. Species-specific identification of campylobacters by PCR-restriction fragment length polymorphism and PCR targeting of the gyrase B gene. Applied and Environmental Microbiology74: 2529-2533.
Kehl, S.C. and Kumar, S., 2009. Utilization of nucleic acid amplification assays for the detection of respiratory viruses. Clinical Laboratory Medicine 29: 661-671.
Linton, D., Lawson, A.J., Owen, R.J. and Stanley, J., 1997. PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples. Journal of Clinical Microbiology 35: 2568-2572.
Linton, D., Owen, R.J. and Stanley, J., 1996. Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals. Research in Microbiology147: 707-718.
Man, S.M., 2011. The clinical importance of emerging Campylobacter species. Nature Reviews in Gastroenterology and Hepatology 25: 669-685.
Menard, A.F., Dachet, F., Prouzet-Mauleon, V., Oleastro, M. and Megraud, F., 2005. Development of a real-time fluorescence resonance energy transfer PCR to identify the main pathogenic Campylobacter spp. Clinical Microbiology and Infection 11: 281-287.
Miller, W.G., On, S.L.W., Wang, G., Fontanoz, S., Lastovica, A.J. and Mandrell, R., 2005. An extended multilocus sequence typing system (MLST) for Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus. Journal of Clinical Microbiology 43: 2315-2329.
On, S.L.W., 2005. Taxonomy, phylogeny, and methods for the identification of Campylobacter species. In: Ketley, J.M. and Konkel, M.E. (eds.) Campylobacter: molecular and cellular biology. Horizon Bioscience, Norfolk, UK, pp. 13-42.
On, S.L.W. and Jordan, P.J., 2003. Evaluation of 11 PCR assays for species-level identification of Campylobacter jejuni and Campylobacter coli. Journal of Clinical Microbiology 41: 330-336.
On, S.L.W., McCarthy, N., Miller, W.G. and Gilpin, B.J., 2008. Molecular epidemiology of Campylobacter species. In: Nachamkin, I., Szymanski, C.M. and Blaser, M.J. (eds.) Campylobacter (3rd Ed.). ASM Press, Washington D.C., USA, pp. 191-211.
Petersen, R.F., Harrington, C.S., Kortegaard, H.E. and On, S.L.W., 2007. A PCR-DGGE method for detection and identification of Campylobacter, Helicobacter, Arcobacter and related Epsilobacteria and its application to saliva samples from humans and domestic pets. Journal of Applied Microbiology 103: 2601-2615.
Stucki, U., Frey, J., Nicolet, J. and Burnens, A.P., 1995. Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein. Journal of Clinical Microbiology 33: 855-859.
Van Camp, G., Fierens, H., Vandamme, P., Goossens, H., Huyghebaert, A. and De Wachter, R., 1993. Identification of enteropathogenic Campylobacter species by oligonucleotide probes and polymerase chain reaction based on 16S rRNA genes. Systematic and Applied Microbiology 16: 30-36.
Vandamme, P., Van Doorn, L.J., Al Rashid, S.T., Quint, W.G.V., Van der Plas, J., Chan, V.L. and On, S.L.W., 1997. Campylobacter hyoileiAlderton et al. 1995 and Campylobacter coli Véron and Chatelain 1973 are subjective synonyms. International Journal of Systematic and Evolutionary Microbiology 47: 1055-1060.
Wang, G., Clark, C.G., Taylor, T.M., Pucknell, C., Barton, C., Price, L., Woodward, D.L. and Rodgers, D., 2002. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus subsp. fetus. Journal of Clinical Microbiology 40: 4744-4747.
Wong, T.L., Devane, M.L., Hudson, J.A., Scholes, P., Savill, M.G. and Klena J.D., 2004. Validation of a PCR method for Campylobacter detection on poultry packs. British Food Journal 106: 642-650.
Zanoni, R.G., Debruyne, L., Rossi, M., Revez, J. and Vandamme, P., 2009. Campylobacter cuniculorum sp. nov., from rabbits. International Journal of Systematic and Evolutionary Microbiology 59: 1666-1671.