马铃薯3种病毒的多重PCR检测方法构建

Establishment of Multiplex PCR for Three Vrius of Potato

[目的]构建马铃薯A病毒(Potato virus A,PVA)、烟草花叶病毒(Tobacco mosaic virus,TMV)、马铃薯Y病毒(Potato virus Y,PVY)3种马铃薯主要易受感染的病毒多重PRC(multiplex PCR)检测方法。[方法]根据GenBank公布的PVA、TMV、PVY基因序列分别设计引物,建立3种马铃薯病毒的多重PCR方法;通过构建3种病毒PCR扩增目的基因的质粒用于制备标准品进行敏感性验证,利用PVX(Potato virus X)、PVM(Potato virus M)、PVS(Potato virus S)、PVV(Potato virus V),CMV(Cucumber mosaic virus)等其他马铃薯病毒进行特异性试验;并对11份疑似发生病毒病的马铃薯块茎采集样品进行了检测。[结果]研究中构建的马铃薯3种病毒多重PCR检测方法,最低检测下限:PVA为100 copies/2μl,PVY为100 copies/2μl,TMV为1 000 copies/2μl,与PVX、PVM、PVV、PVS、CMV等其他马铃薯病毒无交叉反应,具有良好的特异性;11份疑似发生病毒病的马铃薯块茎中7份样品检出3种病毒感染阳性。[结论]该研究成功构建了马铃薯PVA、TMV、PVY病毒的多重PCR方法,为马铃薯病毒检测技术应用奠定一定基础。

[Objective] The aim was to establish the multiplex PCR method for three vrius of potato： PVA（potato virus A）,TMV（Tobacco mosaic virus） and PVY（potato virus Y）.[Method] According to the PVA,TMV and PVY sequences available in GenBank,pairs of primer were designed for establishing a multiplex PCR method,and constructing recombinant plasmid of target genes by PCR amplified of three viruses as reference standard simple to be used in sensitivity test;PVX（Potato virus X）,PVM（Potato virus M）,PVS（Potato virus S）,PVV（Potato virus V） and CMV（Cucumber mosaic virus） were used to carry out the specificity test and detection of 11 samples which were suspected of virus infected.[Result] The detection limit for PVA,TMV and PVY was 14 copies/ml,14 copies/ml and 14 copies/ml,respecitively.No cross-reactivity was observed with other viruses.Seven of 11 samples were infected by three viruses.[Conclusion] The mulitiplex PCR for PVA,TMV,PVY three viruses of potato was established successfully,which had provided basis for the detection technology of potato vrius.