TMV、CMV和PVY三重荧光定量PCR同步检测方法的建立  

Establishment of a Triplex Fluorescent Quantitative PCR for Simultaneous Detection of TMV,CMV and PVY

作　　者：白静科 牛龙龙 吴彦辉[2] 李建华[2] 李小杰[1] 李成军[1] 李淑君[1] BAI Jingke;NIU Longlong;WU Yanhui;LI Jianhua;LI Xiaojie;LI Chengjun;LI Shujun(Key Laboratory for Green Preservation&Control of Tobacco Diseases and Pests in Huanghuai Growing Area/Tobacco Research Institute,Henan Academy of Agricultural Sciences,Xuchang,Henan 461000,China;Xuchang Branch of Henan Provincial Tobacco Company,Xuchang,Henan 461000,China)

机构地区：[1]河南省农业科学院烟草研究所,烟草行业黄淮烟区烟草病虫害绿色防控重点实验室,河南许昌461000 [2]河南省烟草公司许昌市公司,河南许昌461000

出　　处：《中国烟草科学》2023年第5期55-61,共7页Chinese Tobacco Science

基　　金：河南省农业科学院自主创新项目(2023ZC023);河南省烟草公司重点项目(2023410000240021);许昌市烟草公司科技项目(2021411000240104)。

摘　　要：为建立同时检测烟草普通花叶病毒(TMV)、黄瓜花叶病毒(CMV)和马铃薯Y病毒(PVY)的多重荧光定量PCR方法,本研究根据GenBank中TMV、CMV及PVY的CP基因保守序列设计引物和探针,并对反应体系进行了优化,建立了可同时检测TMV、CMV和PVY的三重荧光定量PCR方法,并评价了该方法的敏感性、特异性、重复性及准确性。结果显示,建立的方法中TMV、CMV和PVY的检测下限分别为2.60×10^(1)、1.25×10^(1)、2.33×10^(1) copies/µL,检测灵敏度比普通PCR法提高10倍;该方法可特异性检测出TMV、CMV和PVY,但烟草蚀纹病毒(TEV)、烟草脉带花叶病毒(TVBMV)和番茄斑萎病毒(TSWV)无法检出且无交叉反应,批内与批间重复性试验变异系数均小于1.5%,24份样本检测结果中TMV、CMV和PVY的阳性检出率分别为75%、100%和41.67%,且三重荧光定量PCR方法与普通单重PCR方法得出一致的检测结果。结果表明本研究建立的三重荧光定量PCR检测方法具有特异性强、稳定性好、准确性高等优点,可为后续TMV、CMV和PVY的病害诊断与流行调查提供技术支持。To establish a multiplex fluorescent quantitative PCR for the detection of tobacco mosaic virus(TMV),cucumber mosaic virus(CMV)and potato virus Y(PVY),specific primers and probes were designed based on the conserved CP gene sequences of TMV,CMV and PVY,respectively.And the reaction system was optimized,subsequently the sensitivity,specificity,repeatability and accuracy were evaluated.Results showed that the detection limit for TMV,CMV and PVY was 2.60×10^(1),1.25×10^(1),2.33×10^(1) copies/µL,respectively.The detection sensitivity was 10 times higher than the conventional PCR method.No cross-reaction was found with tobacco etch virus(TEV),tobacco vein banding mosaic virus(TVBMV)and tomato spotted wilt virus(TSWV).The coefficient of variation between intra-assay and inter-assay were both under 1.5%.By using the triple fluorescent quantitative PCR,24 samples were detected and the positive rate of TMV,CMV and PVY was 75%,100%,41.67%,respectively,which were consistent with the single-PCR.In conclusion,a specific,stable and accurate triple fluorescent quantitative PCR was established and could be used for diagnosis and epidemiological investigation of TMV,CMV and PVY.

关 键 词：烟草普通花叶病毒 黄瓜花叶病毒 马铃薯Y病毒 三重荧光定量PCR 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]