Departmental Bulletin Paper Development of one-step real-time reverse transcriptase-PCR-based assays for the rapid and simultaneous detection of four viruses causing porcine diarrhea

Masuda, Tsuneyuki  ,  Tsuchiaka, Shinobu  ,  Ashiba, Tomoko  ,  Yamasato, Hiroshi  ,  Fukunari, Kazuhiro  ,  Omatsu, Tsutomu  ,  Furuya, Tetsuya  ,  Shirai, Junsuke  ,  Mizutani, Tetsuya  ,  Nagai, Makoto

64 ( 1 )  , pp.5 - 14 , 2016-02 , Graduate School of Veterinary Medicine, Hokkaido University
Porcine diarrhea caused by viruses is a major problem of the pig farming industry and can result in substantial losses of revenue. Thus, diagnosing the infectious agents is important to prevent and control diseases in pigs. We developed novel one-step real-time quantitative RT-PCR (qPCR) assays that can detect four porcine diarrheal viruses simultaneously: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine group A rotavirus (PRVA). The qPCR analysis takes only 75 minutes to detect the presence of the four viruses. The limits of detection of our new assays for PEDV, TGEV, PDCoV, and PRVA were 100, 10, 10 and 10 copies per reaction, respectively. The sensitivity of qPCR was 1-1000 times higher than that of published gel-based RT-PCR. We used our qPCR method to successfully diagnose clinical samples from infected pigs, and no false positive results were obtained. In conclusion, qPCR can drastically reduce the diagnostic time to detect viruses compared to currently employed methods. We predict that the qPCR assays will become a useful tool for detecting viral infections that cause diarrhea and other complications in pigs.

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