TaqMan-probe-based multiplex real-time qPCR assay for simultaneous detection of Duck Tembusu virus, Novel duck reovirus, and Duck circovirus.

Duck Tembusu virus (DTMUV), Novel duck reovirus (NDRV), and Duck circovirus (DuCV) are the primary epidemic pathogens threatening China's waterfowl farming industry. The immunosuppressive characteristics of these viruses readily facilitate mixed infections between pathogens, thereby increasing the difficulty of clinical diagnosis and disease prevention and control. To achieve rapid simultaneous detection of these three pathogens, this study designed specific primers and probes based on conserved viral regions, successfully establishing a TaqMan multiplex real-time quantitative PCR assay. The results demonstrate that the method established in this study exhibits excellent specificity and shows no cross-reactivity with other pathogens in waterfowl. The limit of detection (LOD) of this method for the three pathogens was 101 copies/μL, representing a 100-fold improvement in detection sensitivity compared to conventional PCR assay. Repeatability experiments demonstrate that this method exhibits excellent stability and reproducibility. Utilizing this method, we analyzed 120 clinical samples collected from diseased ducks across different regions of Jiangxi Province, China. Compared with conventional PCR, the detection rates for the three viruses were substantially higher, and co-infections with these viruses were also identified among the samples. In summary, the method established in this study offers distinct advantages of high specificity, sensitivity, and reproducibility. It enables rapid identification of these three pathogens in a single reaction, providing an efficient and viable technical approach for the rapid diagnosis of mixed viral infections in waterfowl farming.