Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of porcine epidemic diarrhea virus, , and .

Introduction: PEDV, and , are highly contagious diarrheal pathogens that have caused significant harm to the global swine industry. Co-infections with multiple pathogens are common, making it challenging to identify the actual causative agents depending only on clinical information. It is crucial to develop a reliable method to simultaneously detect and differentiate these pathogens.

Methods: Based on the conserved regions of the M gene of PEDV, NADH oxidase gene of , and the 16S rDNA gene of , specific probes and primers for the multiplex real-time PCR assay were designed. The concentrations of primers and probes were optimized using a matrix method.

Results: The approach demonstrated high specificity and no cross-reactivity with major pathogens related to diarrheal diseases. It showed high sensitivity with a detection limit of 10 copies/μL for and , and 100 copies/μL for PEDV, respectively. It also demonstrated high reproducibility and stability with low coefficients of variation. Results from the multiplex real-time PCR method were in complete agreement with the commercial singleplex real-time PCR kit for detecting PEDV, and . Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for , and 98.6% for . The co-infection rates were 16.85% for PEDV + , 31.46% for PEDV + , 57.86% for  + , and 16.85% for PEDV +  + , respectively.

Discussion: The new multiplex real-time PCR method can simultaneously differentiate PEDV, and , making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.