烟草4种病毒的多重RT-PCR检测方法构建  

Simultaneous detection of four tobacco-infecting viruses by multiplex RT-PCR

作　　者：杨洋[1] 汪蝶 杨懿德 李林秋 鄢敏 杨辉[2] YANG Yang;WANG Die;YANG Yide;LI Linqiu;YAN Min;YANG Hui(Yibin Branch of Sichuan Provincial Tobacco Company,Yibin 644002,Sichuan,China;College of Agriculture,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川省烟草公司宜宾市公司,四川省宜宾市644002 [2]四川农业大学农学院,成都市611130

出　　处：《烟草科技》2020年第9期6-11,共6页Tobacco Science & Technology

基　　金：中国烟草总公司四川省公司科技项目“烤烟主要病毒病发生流行规律及综合防治技术研究与应用”(川烟科〔2017〕8号)。

摘　　要：为有效鉴定烟草的主要病毒,包括烟草普通花叶病毒(Tobacco mosaic virus,TMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、马铃薯Y病毒(Potato virus Y,PVY)和烟草脉带花叶病毒(Tobacco vein banding mosaic virus,TVBMV),建立了一种同时检测这4种病毒的多重RT-PCR检测方法。通过人工接种4种病毒试验,发现TVBMV在混合侵染中受TMV、CMV和PVY的拮抗作用。为提高4种病毒复合侵染下TVBMV的检测灵敏度,筛选出TVBMV中表达水平最高的PIPO基因,利用该基因结合TMV、CMV和PVY的CP基因,基于保守区域设计引物,并优化引物及模板的浓度,在一个PCR反应体系中稳定扩增获得大小分别为700、452、275和235 bp的产物。该方法实现了对多种病毒的同时检测,适用于目前大部分烟区田间病毒复合侵染的监控。In order to efficiently identify the main viruses,including Tobacco mosaic virus(TMV),Cucumber mosaic virus(CMV),Potato virus Y(PVY)and Tobacco vein banding mosaic virus(TVBMV),of tobacco,a multiplex RT-PCR approach for simultaneous detection of four viruses was established.Through inoculating the four viruses,the antagonism of TMV,CMV and PVY on TVBMV in mixed infection was discovered.In order to improve the detection sensitivity for TVBMV in mixed infection,PIPO with the highest expression level in TVBMV was screened out.By using the primers designed on the basis of the conservative sequences of PIPO gene of TVBMV and CP genes of TMV,CMV and PVY and optimizing the concentration of primers and templates,the fragments with sizes of 700,452,275 and 235 bp were amplified stably in one PCR reaction system.This method enables the simultaneous detection of the four viruses and is suitable for monitoring multiple virus infection in most tobacco growing areas.

关 键 词：烟草 病毒检测 多重PCR TMV CMV PVY TVBMV 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]