牛轮状病毒实时荧光定量PCR检测方法的建立

发布时间：2018-01-22 20:23

本文关键词： 牛轮状病毒 VP基因实时荧光定量PCR　出处：《中国兽医科学》2017年06期 　论文类型：期刊论文

【摘要】：为建立快速特异的定量检测牛轮状病毒的荧光定量PCR方法,本研究从牛轮状病毒NCDV株DNA中扩增的VP6基因片段(211 bp)并克隆于pMD19-T载体(p MD19-T-VP6)作为重组质粒标准品,建立了SYBR Green I荧光定量PCR检测方法。结果表明,VP6片段长211 bp,经鉴定所构建的重组质粒成功;用该质粒测得real-time PCR的最低检测量为15.39 copies/L。用该方法检测猪传染性胃肠炎病毒、牛细小病毒、猪流行性腹泻病毒及伪狂犬病病毒的结果均为阴性,表明其具有良好的特异性。重复性试验结果表明,批内和批间重复变异系数均小于3%,表明该方法具有良好的重复性。以上结果表明,本研究建立的基于VP6基因的牛轮状病毒qRT-PCR检测方法具有特异、灵敏、快速等特点,可用于牛轮状病毒的早期诊断。
[Abstract]:To establish a rapid and specific PCR method for quantitative detection of bovine rotavirus. In this study, the fragment of VP6 gene from bovine rotavirus NCDV strain DNA was amplified and cloned into pMD19-T vector pMD19-T-VP6. As a recombinant plasmid standard. A fluorescence quantitative PCR assay for SYBR Green I was established. The results showed that the fragment length of VP6 was 211bp. the recombinant plasmid was identified successfully. The lowest detection of real-time PCR by this plasmid was 15.39 copias / L. The method was used to detect porcine infectious gastroenteritis virus and bovine parvovirus. The results of porcine epidemic diarrhea virus and pseudorabies virus were negative, which indicated that they had good specificity. The repeatability test showed that the coefficient of variation was less than 3%. The results showed that the qRT-PCR detection method based on VP6 gene was specific, sensitive and rapid. It can be used for the early diagnosis of bovine rotavirus.
【作者单位】：东北农业大学动物医学学院;黑龙江省疾病控制中心;
【基金】：“十三五”国家重点研发计划项目(2016YFD0500904)
【分类号】：S852.653
【正文快照】： 牛轮状病毒(bovine rotaviens,BRV),属于呼肠孤病毒科(reoviridae)轮状病毒属(rotavirus)成员。牛轮状病毒感染多发生在15~45日龄的犊牛,其临床症状表现为精神沉郁、食欲废绝、腹泻和脱水[1-2]。Corresponding author:QIAO Xin-yuan,E-mail:qiaoxinyuan@126.com轮状病毒粒子核

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