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| Development and Preliminary Application of SYBR GreenⅠReal-time PCR Assay for Detection of Porcine deltacoronavirus |
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Abstract Abstract Porcine deltacoronavirus (PDCoV) is a new coronavirus that has been popular in China since the beginning of 2015. Development of a rapid and accurate detection method for the PDCoV is of great significance. A pair of primers were designed and synthesized according to the conserved region of the M gene sequence of PDCoV published in GenBank. The 260-bp fragment was successfully amplified and the recombinant plasmid of pMD18-T-PDCoV-M was constructed, and a SYBR GreenⅠreal-time RT-PCR was developed by optimization of the reacting conditions. The specificity, sensitivity and repeatability of the method were studied. The results demonstrated that the sensitivity of this assay was 54 copies/μL. In addition, the assay had good specificity for the PDCoV and had no cross-reaction with Transmissible gastroenteritis virus (TGEV), Pseudorabies virus (PRV), Porcine circovirus type 2 (PCV2), Porcine epidemic diarrhea virus (PEDV), and Porcine reproductive and respiratory syndrome virus (PRRSV). This SYBR Green Ⅰreal-time RT-PCR showed good repeatability, and the variations in intra- and inter-assays were both less than 1%. 198 clinical samples were tested for PDCoV by using this established method, and the positive rate was 20.71% (41/198). The established SYBR GreenⅠRT-PCR method for PDCoV provides a rapid and accurate diagnosis for PDCoV and provides a new technical means for the further study of the disease.
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Received: 17 November 2016
Published: 01 June 2017
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| Fund:Science and technology open cooperation project of Henan Province;National key research and development program |
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