A novel duplex TaqMan probe-based real-time RT-qPCR for detecting and differentiating classical and variant porcine epidemic diarrhea viruses. (February 2018) - Su, Yunfang
Abstract: Two different genotypes of porcine epidemic diarrhea virus (PEDV), the classical and variant strains, are classified by multiple insertions and deletions in their S genes. It is critical to detect and differentiate two genotypes in the pork industry to prevent PEDV outbreaks. In the present study, a novel duplex TaqMan RT-PCR was developed for detecting and differentiating PEDV strains in China. There was no cross-amplification between the two probes when using standard recombinant plasmids, and the specificity was further confirmed by using other seven non-PEDV swine pathogens. The minimum copies required for the detection of both classical and variant PEDV were 4.8 × 10 2 DNA copies/reaction. The repeatability of TaqMan RT-PCR was evaluated using standard recombinant plasmids and gave coefficients of variation 0.19–4.93. In recent 5 years, 79 clinical samples were collected from piglets with severe diarrhea in the Central China. Among these clinical samples, 51 were confirmed as PEDV positive by conventional RT-PCR, whereas 63 variant PEDV, 3 co-infections and 1 classical PEDV were confirmed by this duplex TaqMan RT-PCR, with viral loads of 10 2 –10 8, 10 2 –10 3, and 10 4 copies/reaction, respectively. Therefore, the duplex TaqMan RT-PCR could be a useful method for detecting and differentiating variant and classical PEDV strains. The results showed that variant PEDV was prevalent in clinical samples in central China. Moreover, in this study, co-infection by PEDV strains was detected for the first time and might help explain the emergence of the novel recombinant PEDV in recent years. Highlights: A novel duplex TaqMan probe-based real-time RT-PCR has been developed for detection and differentiation of the classical and variant porcine epidemic diarrhea viruses (PEDV) which could be used as a useful clinical diagnostic tool for detecting and differentiating classical and variant PEDV. Variant PEDV was prevalent in central China. For the first time co-infection by classical and variant PEDV strains has been detected and might help explain the emergence of the novel recombinant PEDV in recent years. The PEDV N gene-based real-time RT-PCR was more sensitive than the duplex TaqMan probe-based real-time RT-PCR, thus, it is suggested to use it as a screening PCR for a primary test and then the duplex TaqMan real-time RT-PCR for differentiating PEDV types.