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根据马铃薯病毒PVX、PVY、PVA、PLRV的CP基因序列设计4对特异性引物,通过对试剂浓度和反应条件进行优化,建立了能够同步检测PVX、PVY、PVA、PLRV的一步法多重RT-PCR检测方法。该方法对PVX、PVY、PVA、PLRV扩增出的靶带大小分别为732、422、132和336 bp,凝胶电泳易辨别区分。病毒RNA最低检测限度为7.8 pg/μL,对PVM、PVS、AMV、TMV及PSTVd的扩增为阴性。研究结果表明,该方法特异、灵敏,比两步法多重RT-PCR检测更加快速、简便,提高了检测效率,降低检测成本,为马铃薯病毒的高效检测提供了有效手段。
Four pairs of specific primers were designed according to the CP gene sequences of the PVX, PVY, PVA and PLRV of the potato virus. One-step multiplex RT-PCR to detect PVX, PVY, PVA and PLRV simultaneously was established by optimizing the reagent concentration and reaction conditions Detection method. The target bands of PVX, PVY, PVA and PLRV amplified by this method were 732, 422, 132 and 336 bp, respectively, and gel electrophoresis was easily distinguished. The detection limit of viral RNA was 7.8 pg / μL, which was negative for PVM, PVS, AMV, TMV and PSTVd. The results showed that the method was specific and sensitive, and was more rapid and simpler than the two-step multiplex RT-PCR, which improved the detection efficiency and reduced the detection cost, which provided an effective method for the efficient detection of the potato virus.