Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of porcine epidemic diarrhea virus, Brachyspira hyodysenteriae, and Lawsonia intracellularis

Introduction PEDV, Brachyspira hyodysenteriae, and Lawsonia intracellularis , 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 B. hyodysenteriae , and the 16S rDNA gene of L. intracellularis , 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 B. hyodysenteriae and L. intracellularis , 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, B. hyodysenteriae and L. intracellularis . Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for B. hyodysenteriae , and 98.6% for L. intracellularis . The co-infection rates were 16.85% for PEDV + B. hyodysenteriae , 31.46% for PEDV + L. intracellularis , 57.86% for B. hyodysenteriae + L. intracellularis , and 16.85% for PEDV + B. hyodysenteriae + L. intracellularis , respectively. Discussion The new multiplex real-time PCR method can simultaneously differentiate PEDV, B. hyodysenteriae and L. intracellularis , making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.