Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay

article i nfo Phenotypicdifferentiation between Campylobacter fetus (C. fetus)subspeciesfetus and C. fetussubspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length poly- morphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identifica- tion, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking. The aim of this study was to evaluate the most practical and routinely implementable published PCR assays designed for C. fetus species and subspecies identification. The sensitivity and specifi city of the assays were calcu- lated by using an extensively characterized and diverse collection of C. fetus strains. AFLP and MLST identification were used as reference. Two PCR assays were able to identify C. fetus strains correctly at species level. The C. fetus speciesidentification target, gene nahE, of one PCR assay was used to develop a real-timePCR assay with 100% sen- sitivity and 100% specificity, but the development of a subspecies venerealis specific real-time PCR (ISCfe1) failed due to sequence variation of the target insertion sequence and prevalence in otherCampylobacter species. None of the published PCR assays was able to identify C. fetus strains correctly at subspecies level. Molecular analysis by AFLP or MLST is still recommended to identify C. fetus isolates at subspecies level.