A two-group quadruplex real-time quantitative PCR assay for molecular patho-serotyping of Yersinia enterocolitica

Abstract Yersinia enterocolitica is one of the most important foodborne pathogens with significant impact on public health. It can be divided into six biotypes and approximately 60 O-serotypes, with O:3, O:9, O:8 and O:5,27 being predominantly associated with human yersiniosis. We present a two-group quadruplex real-time quantitative PCR (RT‒qPCR) for the patho-serotyping of Y. enterocolitica. The design of primers and probes within group 1 was based on sero-specific genes within the O antigen gene cluster, and those within group 2 were selected from the virulence markers and the restriction modification system to distinguish O:5,27 from O:5. The specificity was tested using reference strains, and was confirmed by a comparison with those obtained by a previous multiplex PCR. The limit of detection is 0.1 ng, or 104 copies μl−1 of genomic DNA, and the standard curves exhibited high linearity and correlation coefficients, demonstrating our assay's robustness. Among the 81 isolates used to evaluate the reproducibility, the results for 76 were consistent between the two approaches, indicating that the sensitivity of our RT‒qPCR is 100%, and the positive predictive value is 94%. Our assay can serve as a tool for identifying sources of Y. enterocolitica contamination and for epidemiological monitoring of this bacterium.