犬轮状病毒荧光定量RT-PCR检测方法的建立  

Construction of real-time fluorescence quantitative RT-PCR method for detecting canine rotavirus

作　　者：魏锁成[1] 何丽[1] 

机构地区：[1]西北民族大学动物医学研究所,甘肃兰州730030

出　　处：《中国动物检疫》2009年第5期38-41,共4页China Animal Health Inspection

基　　金：国家民委2002年度重点资助项目(MW2002-ZD-010);甘肃省2007年度科技支撑项目(编号0708NKCA079)

摘　　要：目的:建立轮状病毒快速准确的检测方法,为研发诊断试剂盒和疫苗奠定基础。方法:用Primer5软件设计VP7引物,从采集的腹泻犬粪便样品抽提病毒RNA,用反转录聚合酶链式反应(RT-PCR)技术,将RNA反转录为cDNA,并进行PCR扩增,对扩增产物进行非变性聚丙烯酰胺凝胶电泳和测序,与代表株的VP7进行同源性比较。结果:从腹泻病犬粪便样品中抽提到了轮状病毒RNA,反转录后扩增产物为51bp的基因片段,经核苷酸序列分析,其PCR序列与犬轮状病毒VP7基因编码源序列的同源性为86.3%。结论:正确克隆了轮状病毒主要保护性抗VP7基因中抗原表位区,建立了犬轮状病毒实时定量诊断方法,为研制实时定量诊断试剂盒奠定了基础。Objective： To establish a fast and accurate detection method for canine rotavirus and to provide foundation for developing the diagnostic kit and DNA vaccine. Methods： VP7 primer was designed with Primer 5 software, the rotavirus RNA was extracted feces samples, RNA reverse transcripted into cDNA, and amplified by using PCR, the amplified products were electrophorised with polyacrylamide gel and the gene sequences measured. Its sequence homology was compared with those of rotavirus strains VP7. The real-time fluorescence quantitative PCR assay was carried out through the fluorescent probe （SyB dyes）to obtain rotavirus copies. Results： The rotavirus RNA had been extracted from stool samples of diarrhea dogs, RT-PCR amplified products possessed 51 bp gene fragments, their PCR sequence homology was 86.3% of dogs rotavirus VP7 gene coding sequence; fluorescence value were detected and rotavirus copies got. Conclusions： The rotavirus RNA were extracted, reverse transcripted and amplified successfully by using RT-PCR technology, the virus VP7 sequences were analyzed through fluorescence quantitative PCR. The fluorescence quantitative RT-PCR detection method for canine rotavirus was established preliminarily.

关 键 词：轮状病毒 VP7 RT-PCR 实时荧光定量PCR 

分 类 号：S852.655[农业科学—基础兽医学]