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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。
In order to establish a rapid and specific quantitative PCR method for the detection of bovine rotavirus, a VP2 gene fragment (211 bp) amplified from the DNA of bovine rotavirus NCDV strain was cloned into the pMD19-T vector -VP6) as a recombinant plasmid standard, established SYBR Green I fluorescence quantitative PCR detection method. The results showed that the VP6 fragment was 211 bp in length and the recombinant plasmid was identified successfully. The lowest detection level of real-time PCR using this plasmid was 15.39 copies / L. The results of this method for the detection of swine transmissible gastroenteritis virus, bovine parvovirus, porcine epidemic diarrhea virus and pseudorabies virus were all negative, indicating that they have good specificity. Repeatability test results showed that the intra-and inter-assay repeated variation coefficients were less than 3%, indicating that the method has good repeatability. The above results show that the VP6 gene-based qRT-PCR assay established in this study is specific, sensitive and rapid and can be used for the early diagnosis of bovine rotavirus.