马铃薯病毒一步法多重RT-PCR检测技术的构建

Development of one-step multiplex RT-PCR for simultaneous detection of four potato viruses

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。

One-step multiplex reverse transcription polymerase chain reaction（M-RT-PCR） was established and optimized for the simultaneous detection of potato virus X,potato virus Y,potato virus A and potato leafroll virus.The primers for PVX（732 bp）,PVY（422 bp）,PVA（132 bp） and PLRV（336 bp） fragments were designed based on viral coat protein gene.The sensitivity of detection was about 7.8 pg/μL RNA.No fragments were obtained from PVM,PVS,AMV,TMV and PSTVd.The results showed that it was a specific,sensitive detecting method and quicker,simpler,more efficient and cheaper than two-step multiple RT-PCR.It was provided an effective method of the detection for potato virus.