一步法RT-PCR和两步法RT-PCR对马铃薯病毒诊断研究的比较

本研究采用试剂盒和Total试剂两种方法从马铃薯叶片和茎秆中提取总RNA，同时设计了一步法RT-PCR和两步法RT-PCR的反应体系，依据马铃薯X病毒、马铃薯Y病毒、马铃薯S病毒、马铃薯A病毒以及马铃薯卷叶病毒的基因组RNA保守序列设计特异性引物对，并运用两种RT-PCR反应体系对染病的马铃薯进行病毒检测，结果与报道的这五种马铃薯病毒核苷酸序列进行BLAST比对, 表明扩增的五种马铃薯病毒靶带的序列与靶序列完全一致。两种诊断方法对马铃薯病毒核酸进行浓度检测，结果证明，两步法RT-PCR具有较高灵敏性。但这两种方法均可快速、简便、有效地诊断马铃薯病毒。

Research on the Detection of Potato Viruses by One-step RT-PCR and Two-step RT-PCR

In order to establish a technique using RT-PCR to detect potato viruses, which was rapid, accurate and sensitive, the RNAprep Plant Kit and RNAiso Plus RNA-release methods were developed for extracting potato viruses （PVX, PVY, PVS, PVA, PLRV） from potato stems and leaves. The primers for the viruses were designed. The one step RT-PCR and two step RT-PCR were used to detect the five virus and the amplification fragment were 620 bp（PVX）, 550 bp（PVY）, 435 bp（PVS）, 300 bp（PVA） and 222 bp（PLRV）. The plasmids were extracted and sequenced, compared with those in NCBI BLAST; the result showed that the sequences of amplification products were correct. Serial dilutions were used to detect the sensitivity of one and two-step RT-PCR testing system. One-step RT-PCR testing system was also can used to detect many potato viruses with the optimized conditions but the sensitivity was inferior to two-step RT-PCR.