Development of a multiplex TaqMan probe-based real-time PCR for discrimination of variant and classical porcine epidemic diarrhea virus

Since October 2010, porcine diarrhea outbreaks have occurred widely, resulting in major losses in suckling piglets in China. A variant porcine epidemic diarrhea virus (PEDV), characterized by base deletion and insertion in the S gene, compared to classical PEDV CV777, was shown to be responsible for this outbreak. In this study, a multiplex TaqMan probe-based real-time PCR was developed for detecting PEDV and differentiating the variant from classical PEDV, by using two sets of primers and probes based on the S gene of PEDV. The limits of detection of both variant and classical PEDV were 5 × 102 DNA copies. Specificity was determined using eight other viral pathogens of swine. Reproducibility was evaluated using standard dilutions, with coefficients of variation <1.4%. Standard dilutions included in each test allowed quantification of the amount of PEDV. Among 42 intestinal samples from pigs with severe watery diarrhea, 36 variant PEDV and three classical PEDV samples were detected, with viral loads of 102-108 copies/μl and 103-105 copies/μl, respectively, which suggested that the variant PEDV was prevalent in China. The multiplex TaqMan probe-based real-time PCR should be a useful tool for quantifying viral load, detecting PEDV, and differentiating variant from classical PEDV.