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A novel duplex TaqMan probe-based real-time RT-qPCR for detecting and differentiating classical and variant porcine epidemic diarrhea viruses

Author:
Su, Yunfang, Liu, Yunchao, Chen, Yumei, Xing, Guangxu, Hao, Huifang, Wei, Qiang, Liang, Yue, Xie, Weitao, Li, Dongliang, Huang, Huimin, Deng, Ruiguang, Zhang, Gaiping
Source:
Molecular and cellular probes 2017
ISSN:
0890-8508
Subject:
Porcine epidemic diarrhea virus, diarrhea, genes, genotype, mixed infection, pathogens, piglets, plasmids, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, viral load, China
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 × 102 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 102–108, 102–103, and 104 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.
Agid:
5858859