Development and application of a multiplex PCR assay for detection of pathogenic Clostridium species

Clostridium perfringens ( C. perfringens ), Clostridium novyi ( C. novyi ), Clostridium septicum ( C. septicum ), and Clostridium chauvoei ( C. chauvoei ) are the major pathogenic Clostridium species causing clostridial diseases in cattle and sheep. The diseases they cause are characterized by acute onset, high mortality, and similar clinical manifestations and pathological lesions that are difficult to distinguish, posing a substantial threat to livestock health and production efficiency. Conventional diagnostic methods for these pathogens are generally time–consuming and labor–intensive, and fail to reliably distinguish co‑detections. There is therefore an urgent demand for a rapid and robust method for simultaneous detection and differentiation of these dominant pathogenic Clostridium species. In this study, we developed a quadruplex polymerase chain reaction (PCR) assay for the simultaneous differential detection of the four target pathogens. Species–specific primers were designed targeting the plc gene of C. perfringen s, the fliC gene of C. novyi , the α–toxin gene of C. septicum , and the cctA gene of C. chauvoei . Key reaction parameters, including annealing temperature, primer concentrations, and amplification cycle number, were systematically optimized via gradient PCR and single–factor tests. The specificity, sensitivity, and repeatability of the assay were comprehensively validated using nucleic acid samples, after which the optimized assay was applied to 206 clinical fecal samples of bovine origin. Under optimal conditions (annealing temperature of 55 °C; primer concentrations of 0.2, 0.2, 0.2, and 0.4 µmol/L for the four respective targets; 30 amplification cycles), the assay demonstrated high specificity, showing no cross–reactivity against common intestinal pathogenic microorganisms, including Clostridium tetani ( C. tetani ), Escherichia coli ( E. coli ), Staphylococcus aureus ( S. aureus ), Salmonella spp., bovine enterovirus (BEV), bovine astrovirus (BAstV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV) and peste des petits ruminants virus (PPRV). The limits of detection for recombinant plasmids were 10², 10³, 10⁴, and 10³ copies/µL for C. perfringens , C. novyi , C. septicum , and C. chauvoei , respectively. Both intra–assay and inter–assay repeatability were excellent. Among the 206 field–collected samples, the positivity rates of the four pathogens were 0%, 2%, 12%, and 9%, and all results showed 100% concordance with singleplex PCR and Sanger sequencing. This highly specific, sensitive, and reproducible quadruplex PCR method enables rapid simultaneous detection and differentiation of four major pathogenic Clostridium species, providing a reliable and efficient tool for clinical pathogen identification and epidemiological surveillance of clostridial infections. However, the assay detects only four target species and relies on a single sample type (anal/fecal swabs) collected from one geographic region, which may lead to underestimation of the carriage rates of certain clostridial species. Future studies will expand the target panel and specimen types and incorporate quantitative or point–of–care platforms to improve field applicability.