SYBR GreenⅠ实时荧光定量RT-PCR检测水泡性口炎病毒方法的建立  

Establishment of SYBR GreenⅠreal-time fluorescence quantitative RT-PCR for detection of vesicular stomatitis virus

作　　者：李嘉阳 赵佳男 秦彤[1] 季芳 李刚[1,2] 王承民 LI Jia-yang;ZHAO Jia-nan;QIN Tong;JI Fang;LI Gang;WANG Cheng-min(Beijing Institute of Animal Sciences and Veterinary Medicine,Chinese Academy of Agricultural Sciences/Beijing Scientific Observation Experimental Station for Veterinary Drug and Diagnosis,Ministry of Agriculture and Rural Affairs of PRC,Beijing 100193,China;Institute of Zoology,Guangdong Academy of Sciences/Guangdong Key Laboratory of Animal Protection and Resource Utilization/Guangdong Public Laboratory of Wildlife Protection and Utilization,Guangzhou 510260,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所农村农业部兽用药物与诊断技术北京科学观测试验站,北京100193 [2]广东省科学院动物研究所广东省动物保护与资源利用重点实验室广东省野生动物保护与利用公共实验室,广东广州510260 [3]中国农业大学动物医学院,北京100193

出　　处：《中国兽医科学》2022年第2期152-161,共10页Chinese Veterinary Science

基　　金：“十三五”国家重点研发计划项目(2016YFD0501102);国家自然科学基金面上项目(31472203);广东省“银龄专项”(2020A1313030136)。

摘　　要：本研究旨在建立一种快速、灵敏的用于诊断水泡性口炎的SYBR GreenⅠ实时荧光定量RT-PCR检测方法。通过RT-PCR分别扩增印第安纳型水泡性口炎病毒(VSV-IN)和新泽西型水泡性口炎病毒(VSVNJ)的G基因,然后将其连接到克隆载体pMD19-T上,构建质粒标准品pMD-VSV-IN-G和pMD-VSV-NJG。根据GenBank中VSV-IN和VSV-NJ的G基因保守序列设计引物,利用SYBR GreenⅠ法进行实时荧光定量RT-PCR,建立标准曲线,并进行特异性、敏感性和重复性试验。结果,在拷贝数6.82~6.82×10^(8)copies/μL范围内,C;值与质粒拷贝数的对数值呈良好的线性关系,pMD-VSV-IN-G标准曲线的R;值为0.99812,p MD-VSV-NJ-G标准曲线的R^(2)值为0.99748;检测其他病毒均无特异性扩增曲线,特异性良好。pMD-VSVIN-G的检测灵敏度为6.82 copies/μL,pMD-VSV-NJ-G的检测灵敏度也为6.82 copies/μL,是普通RTPCR方法的10倍。用该方法对实验室保存的VSV-IN和VSV-NJ各5份攻毒小鼠样品进行检测,检测结果与普通RT-PCR检测一致。上述结果表明,本检测方法的建立对快速、灵敏鉴别诊断IN型和NJ型水泡性口炎病毒具有重要的应用价值。The study is to establish a rapid and sensitive SYBR Green I real-time fluorescent quantitative RT-PCR method for the diagnosis of vesicular stomatitis.In this study,G genes of vesicular stomatitis virus(IN serotype and ND serotype)were amplified by RT-PCR and ligated into p MD19-T cloning vector to construct standard plasmids.According to the conserved sequences of G gene of vesicular stomatitis virus in Gen Bank,primers were designed.SYBR Green I method was used for real-time fluorescent quantitative PCR.The specificity test,sensitivity test and repeatability test were carried out.The results showed that there were good linear relationships between Ctvalue and logarithm of plasmid copy number in the range of 0.99812 for VSV-IN standard curve and 0.99748 for VSV-NJ standard curve at the copy number of 6.82—6.82×10^(8)copies/μL,respectively.No other viruses had specific amplification curve.The sensitivities of the method were 6.82 copies/μL for VSV-IN and 6.82 copies/μL for VSV-NJ,respectively.The method was used to detect VSV infected mice in the laboratory,and the results were consistent with those of RT-PCR.The establishment of this detection method is of great significance for rapid and sensitive diagnosis of vesicular stomatitis virus.

关 键 词：印第安纳型水泡性口炎病毒(VSV-IN) 新泽西型水泡性口炎病毒(VSV-NJ) RT-PCR 实时荧光定量PCR SYBR GreenⅠ 

分 类 号：S852.659.5[农业科学—基础兽医学]