Simultaneous detection of five pig viruses associated with enteric disease in pigs using EvaGreen real-time PCR combined with melting curve analysis

In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube. Five distinct melt peaks were obtained with different melting temperature (Tm) value corresponding to each of the five viruses. This method was highly sensitive to detect and distinguish TGEV, RVA, RVC, PEDV and PCV2 with the limits of detection ranging from 5 to 50 copies/μL. The intra-assay and inter-assay reproducibility were good with coefficient of variation of Tm and cycle threshold values less than 0.32% and 2.86%, respectively. Testing of 90 field samples by the single and multiplex real-time PCR assays demonstrated a concordance of 91.1%. Thus, the EvaGreen multiplex real-time PCR is a rapid, sensitive and low-cost diagnostic tool for differential detection and routine surveillance of TGEV, RVA, RVC, PEDV and PCV2 in pigs.