Dual priming oligonucleotide (DPO)-based real-time RT-PCR assay for accurate differentiation of four major viruses causing porcine viral diarrhea

Publication date: Available online 12 August 2019Source: Molecular and Cellular ProbesAuthor(s): Shuo Jia, Baohua Feng, Zhuo Wang, Yingying Ma, Xuwen Gao, Yanping Jiang, Wen Cui, Xinyuan Qiao, Lijie Tang, Yijing Li, Li Wang, Yigang XuAbstractCurrently in China, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are the major causes of porcine viral diarrhea, and mixed infections in clinics are common, resulting in significant economic losses in pig industry. Here, a dual priming oligonucleotide (DPO)-based multiplex real-time SYBR Green RT-PCR assay were developed for accurately differentiating PEDV, TGEV, PoRV, and PDCoV in clinical specimens targeting the N gene of TGEV, PEDV, and PDCoV, and the VP7 gene of PoRV. Results showed that the DPO primer allowed a wider annealing temperature range (40–65 °C) and had a higher priming specificity compared to conventional primer, in which more than 3 nucleotides in the 3′- or 5′-segment of DPO primer mismatched with DNA template, PCR amplification efficiency would decrease substantially or extension would not proceed. DPO-based multiplex real-time RT-PCR method had analytical detection limit of 8.63 × 102 copies/μL, 1.92 × 102 copies/μL, 1.74 × 102 copies/μL, and 1.76 × 102 copies/μL for PEDV, TGEV, PoRV, and PDCoV in clinical specimens, respectively. A total of 672 clinical specimens of piglets with diarrh...
Source: Molecular and Cellular Probes - Category: Molecular Biology Source Type: research