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

摘要:

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。

Abstract:

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.