Objective   To establish a multiplex polymerase chain reaction (PCR) method to differentiate C fetus subsp fetus cff ,C fetus subsp venerealis cfv and C fetus subsp testudinum cft.

  Methods  The specific primers targetting C fetus,cfv and cft were designed and the reaction conditions were optimized. The multiplex PCR system was also evaluated by using 38 strains (including 18 Campylobacter fetus strains and 20 non-Campylobacter fetus strains) and further applied to 53 fecal samples from reptile animals.

  Results  The multiplex PCR assay with 3 primer pairs could yield specific fragments: only one fragment of 359 bp for cff, two fragments of 359 bp and 156 bp for cfv, two fragments of 359 bp and 266 bp for cft. The amplification conditions were also optimized and the extending temperature was 58 ℃. The optimal concentration of primers “MG3f and Cf359r”, “ISC2mf and ISC2mr”,“CoA266f and CoA266r” were 0.1 μmol/L , 0.2 μmol/L and 0.2 μmol/L respectively. The sensitivity of the assay for detecting cff, cfv and cft was 0.40 ng/μL、0.39 ng/μL and 0.47 ng/μL respectively. The analytical specificity was 100% by examination of 18 Campylobacter fetus strains and 20 non-Campylobacter fetus strains. In 53 fecal samples from reptile animals, one was PCR postive for cft and the suspective colonies were also identified as cft.

  Conclusion  The established multiplex PCR assay can differentiate three subspecies simutaneously and provide technical support for rapid identification, epidemiological investigation and tracing infection source.