猪δ冠状病毒SYBR GreenⅠ荧光定量RT-PCR检测方法的建立及初步应用

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摘要摘要猪δ冠状病毒(Porcine deltacoronavirus, PDCoV)是一种新的引起仔猪腹泻的冠状病毒，2014年以来在我国各省开始流行，建立一种快速、准确的检测方法对于该病流行病学、致病机制的研究具有重要的意义。根据GenBank中公布的PDCoV M基因序列设计一对特异性引物，扩增M基因的特异性片段，构建pMD18-T-PDCoV-M重组质粒，以此为模板，SYBR Premix Ex Taq为荧光染料，通过对荧光定量RT-PCR(reverse transcription-PCR)扩增条件的优化，建立了针对PDCoV的荧光定量RT-PCR检测方法，并对所建立方法的特异性、灵敏性和稳定性进行了研究。结果显示，所建立的方法具有高度敏感性，最低检测限为54 拷贝/μL；应用该方法对猪传染性胃肠炎病毒(Transmissible gastroenteritis virus of swine, TGEV)、猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)和猪繁殖与呼吸综合症病毒(Porcine respiratory and reproductive syndrome virus, PRRSV)等其他常见猪病毒性病原检测均为阴性，表明所建立的方法具有较好的特异性；且具有较好的重复性，组内和组间变异系数均小于1%。对临床198份腹泻样品进行检测，阳性率为20.71%(41/198)。实验所建立的SYBR GreenⅠ荧光定量RT-PCR方法能快速、准确检测新发PDCoV，为该病的深入研究提供了新的技术手段。

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	张利卫
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	张君涛
	韦学雷
	兰培英
	胡慧

关键词 ：猪δ冠状病毒(PDCoV), M 基因, SYBR GreenⅠ, qRT-PCR

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.

Key words： Porcine deltacoronavirus (PDCoV) M gene SYBR GreenⅠ qRT-PCR

收稿日期: 2016-11-17 出版日期: 2017-06-01

基金资助:河南省科技开放合作项目;国家重点研发计划

通讯作者: 胡慧 E-mail: huhui2001@163.com

引用本文:

张利卫曹贝贝张云飞张君涛韦学雷兰培英胡慧. 猪δ冠状病毒SYBR GreenⅠ荧光定量RT-PCR检测方法的建立及初步应用[J]. , 2017, 25(6): 969-975.

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http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I6/969

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