痘苗病毒SYBR GreenⅠ荧光定量PCR检测方法的建立与应用  

Establishment and application of SYBR GreenⅠreal-time PCR method for detection of vaccinia virus

作　　者：汪伟[1,2] 杜倩 韩知晓 辛佳亮 孔子荣 闭璟珊 曹亮 孙文超[3,4] 胡传活 郑敏[2] WANG Wei;DU Qian;HAN Zhi-xiao;XIN Jia-liang;KONG Zi-rong;BI Jing-shan;CAO Liang;SUN Wen-chao;HU Chuan-huo;ZHENG Min(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Guangxi Center for Animal Disease Prevention and Control,Nanning 530000,China;Institute of Mlitary Veterinary,The A cade my of Military Sciences,Changchun 130122,China;Institute of Virology-,Wenzhou University,Wenzhou 325035,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]广西壮族自治区动物疫病预防控制中心,广西南宁530000 [3]军事科学院军事兽医研究所,吉林长春130122 [4]温州大学病毒学研究所,浙江温州325035

出　　处：《中国兽医科学》2020年第4期430-436,共7页Chinese Veterinary Science

基　　金：广西自然科学基金项目(2012GXNSFAA053073);浙江省青年基金项目(LQ19C180001);温州市基础性科研项目(N20190005,N20180010)。

摘　　要：为实现痘苗病毒的快速检测,根据痘苗病毒TA27L基因设计引物,经各项条件优化后,建立痘苗病毒SYBR GreenⅠ荧光定量PCR检测方法,同时将PCR扩增的TA27L基因克隆至pMD18-T载体,将测序正确的重组质粒作为标准品进行敏感性、特异性和重复性试验。结果显示,所建立方法的Ct值与标准品在7.58×10^9~7.58×10^2copies/μL范围内呈良好线性关系,R^2为0.997,斜率为-3.175,检测下限为75.8copies且具有良好的特异性。临床样本检测结果表明该方法较普通PCR方法的检出率更高。结果表明,建立的痘苗病毒SYBR GreenⅠ荧光定量PCR检测方法为痘苗病毒及其相关基因重组毒株的生物学特性研究提供了必要的技术手段。To achieve rapid detection of vaccinia virus,the specific primers were designed using vaccinia virus TA27L gene.Vaccinia virus SYBR Green Ⅰ real-time PCR method was established,after optimization of various conditions and the TA27L gene amplified by PCR was cloned into pMD18-T vector,then the correct recombinant plasmid was used as standards for sensitivity,specificity and reproducibility testing.The test results showed that the Ctvalue showed a good linear relationship with the standard in the range of 7.58×10^9 to 7.58×10^2 copies/μL and the correlation was 0.997 with a slope of -3.175.The detection limit of this method was 75.8 copies,and it has good specificity.The clinical samples were tested and the results showed that the method established in this study had a higher detection rate than the common PCR method. The SYBR GreenⅠreal-time PCR assay was established,which provides the necessary technical means for the study of the biological characteristics of vaccinia virus and its related recombinant strains.

关 键 词：痘苗病毒 SYBR GreenⅠ 荧光定量PCR 

分 类 号：S852.659.1[农业科学—基础兽医学]