Development of a quadruple qPCR assay for simultaneous detection of four common bovine pathogens

Bovine infectious diseases pose a significant threat to cattle health, causing widespread economic losses and profoundly impacting the well-being and productivity of affected herds. Among these, Bovine Herpesvirus 4 (BoHV4), Bovine Ephemeral Fever Virus (BEFV), Bovine Rotavirus (BRV), and Clostridium perfringens (CP) are four common pathogens responsible for a range of clinical manifestations in cattle. Notably, co-infections among these pathogens are relatively prevalent, contributing to the complexity and severity of disease outcomes in affected cattle. To simultaneously detect and differentiate these four pathogens in a single assay, we developed a TaqMan-based multiplex real-time PCR (qPCR) method containing four primer-probe sets, designed to target highly conserved or virulence-associated genes specific to each pathogen. The assay was optimized by adjusting primer-probe concentrations and annealing temperatures. Following optimization, a comprehensive evaluation was conducted to assess the analytical performance, including specificity, sensitivity, repeatability, and clinical applicability. The results demonstrated that the developed method exhibited no cross-reactivity with other bovine pathogens commonly encountered in clinical settings, achieved a detection limit of as few as 5 copies/μL for all four target pathogens, and showed coefficients of variation (CVs) below 2.26 % in repeatability tests. The method was applied to screen 1012 clinical samples collected from two commercial cattle farms in Jiangsu Province. The results revealed a positivity rate of 5.24 % (53/1012) for one or more of the four pathogens, with BRV, CP, BoHV4, and BEFV accounting for 3.66 %, 1.28 %, 0.30 %, and 0 % of the positive cases, respectively. Co-infections involving multiple pathogens were detected in 0.70 % (7/1012) of the samples. In conclusion, this study successfully developed a one-step multiplex qPCR assay for the simultaneous detection and differentiation of four common bovine pathogens. The assay provides a rapid, reliable, and cost-effective tool for bovine infectious disease surveillance and control. Its ability to detect mixed infections, combined with its high sensitivity and specificity, makes it particularly suitable for use in cattle farms, enabling rapid and accurate identification of pathogens to support disease management and control.