Development of a Multiplex TaqMan Real-Time PCR Assay for the Simultaneous Detection and Differentiation of Three Pathogenic Yersinia Species.

Three pathogenic species of the genus Yersinia, including Plague-associated Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, are commonly associated with human infection. Current qPCR detection methods are mainly limited to the identification of one or two Yersinia species in a single reaction tube, while multiplex assays for multiple genera have been more commonly reported. Therefore, the present study aimed to establish a multiplex TaqMan qPCR assay for the simultaneous detection of these three pathogenic Yersinia species. Primer and probe sets were designed based on the inv gene for Y. pseudotuberculosis, the caf1 gene for Y. pestis, and the foxA gene for Y. enterocolitica. Under the optimized reaction conditions, the standard curve slopes for the caf1, inv, and foxA genes were -3.046, -2.968, and -2.948, respectively. The correlation coefficients (R2) ranged from 0.993 to 0.996, while the amplification efficiencies ranged from 109% to 115%. The limits of detection (LOD) were determined to be 5 × 102 copies/μL for inv (FAM), 1 × 101 copies/μL for caf1 (ROX), and 1 × 101 copies/μL for foxA (CY5). The sensitivity of the multiplex qPCR assay was 10- to 100-fold higher than that of conventional PCR, depending on the target. Specificity experiments demonstrated that no cross-reactivity was observed with non-target bacteria, including Francisella tularensis, Brucella spp., Vibrio cholerae, Salmonella Typhi, and Shigella spp. The intra-assay coefficients of variation (CVs) ranged from 0.13% to 0.79%, whereas the inter-assay CVs ranged from 0.62% to 2.61%. Among 173 spleen samples collected from wild rodents, no positive signal for Y. pestis or Y. pseudotuberculosis was detected. In contrast, Y. enterocolitica was detected in three samples (1.73%, 3/173). In conclusion, the multiplex qPCR assay developed in this study provides a sensitive and specific tool for the simultaneous detection of three pathogenic Yersinia species and has the potential to improve detection efficiency in clinical and epidemiological investigations.