Simple Summary
Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis are important pathogenic species within the genus Yersinia. Currently available detection methods are generally limited to identifying only one or two of these pathogens in a single reaction. In this study, we developed a multiplex TaqMan assay capable of simultaneously detecting all three pathogens in a single tube. The assay demonstrated high sensitivity, specificity, reproducibility, and good clinical applicability. This method improves the efficiency of Yersinia detection and provides a practical tool for clinical diagnosis and surveillance.
Abstract
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.