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[目的]构建马铃薯A病毒(Potato virusA,PVA)、烟草花叶病毒(Tobacco mosaicvirus,TMV)、马铃薯Y病毒(Potato virusY,PVY)3种马铃薯主要易受感染的病毒的多重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落copies/2μl,PVY为100copies/2μl,TMV为1000copies/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]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 study aimed to construct multiplex PCR (PCR) which is mainly susceptible to viruses of Potato virus A (PVA), Tobacco mosaic virus (TMV) and Potato virus Y (PVY) Detection method. [Method] According to the sequences of PVA, TMV and PVY published in GenBank, primers were designed and three kinds of multiplex PCR methods of potato virus were established. Three plasmids were amplified by PCR to amplify the target gene for verification of sensitivity. Specific tests were carried out using other potato viruses such as Potato virus X (PVX), Potato virus M (PVM), Potato virus S (PVS), Potato virus V (PVV), and Cucumber mosaic virus CMV. Virus diseased potato tubers were sampled for the test. [Results] The detection limit of three PCRs for detecting potato virus in the study was as follows: the detection limit was 100 copies / 2μl for PVA, 100copies / 2μl for PVY and 1000copies / 2μl for TMV, and PVX, PVM, PVV, PVS, CMV And other potato viruses did not cross-react with good specificity; seven samples of 11 potato tubers suspected to have viral disease were positive for the three viruses. [Conclusion] The multiplex PCR method of constructing potato PVA, TMV and PVY virus was successfully constructed, which laid a foundation for the application of potato virus detection technology.