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Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis
Published on: June 17, 2019
Development of a Multiplex TaqMan Real-Time PCR Assay for the Simultaneous Detection and Differentiation of Three
Xue Yang1, Xin Lei1, Yongjun Luo2
1Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China.
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.
Insights
A new multiplex qPCR assay enables simultaneous detection of three pathogenic Yersinia species. This method offers improved sensitivity and specificity for Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica detection.
Area of Science:
- Molecular Biology
- Microbiology
- Infectious Diseases
Background:
- Three pathogenic Yersinia species (Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica) cause significant human infections.
- Existing qPCR methods often detect only one or two Yersinia species per reaction, limiting comprehensive surveillance.
- Multiplex assays are more common for broader bacterial genera, highlighting a need for multi-Yersinia detection.
Purpose of the Study:
- To develop a multiplex TaqMan qPCR assay for simultaneous detection of Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica.
- To establish a sensitive and specific molecular tool for identifying these three key pathogenic Yersinia species.
Main Methods:
- Designed species-specific primer and probe sets targeting the inv gene (Y. pseudotuberculosis), caf1 gene (Y. pestis), and foxA gene (Y. enterocolitica).
- Optimized reaction conditions and evaluated assay performance including standard curve slopes, correlation coefficients (R²), and amplification efficiencies.
- Determined limits of detection (LOD) and assessed specificity against non-target bacterial species. Validated assay on 173 wild rodent spleen samples.
Main Results:
- The multiplex qPCR assay demonstrated high efficiency (109-115%) and excellent linearity (R² 0.993-0.996).
- Achieved low LODs: 5 × 10² copies/μL for inv (FAM), 1 × 10¹ copies/μL for caf1 (ROX), and 1 × 10¹ copies/μL for foxA (CY5).
- Assay showed no cross-reactivity with tested non-Yersinia bacteria and detected Y. enterocolitica in 1.73% of wild rodent samples.
Conclusions:
- The developed multiplex qPCR assay is a sensitive and specific tool for simultaneous detection of three pathogenic Yersinia species.
- This assay offers improved detection efficiency compared to conventional PCR, with 10- to 100-fold higher sensitivity.
- The assay holds significant potential for enhancing clinical diagnostics and epidemiological surveillance of Yersinia infections.
