莴苣花叶病毒RT-RPA检测方法的建立

Reverse transcription recombinase polymerase amplification assay for the detection of Lettuce mosic virus

根据莴苣花叶病毒(Lettuce mosic virus,LMV)的外壳蛋白保守位点设计引物,并构建片段大小为428 bp的内标质粒,利用引物和内标质粒对可侵染莴苣的5种病毒和与LMV同属的3种病毒进行反转录重组酶聚合酶扩增技术(Recombinase Polymerase Amplification,RPA)检测。结果表明,该方法特异性强,可从阳性样品RNA中扩增出大小为272 bp的目标片段,而其他7种病毒RNA、空白对照以及阴性对照均未扩增出条带;灵敏度高,能检测出稀释100倍的LMV RNA,与常规RT-PCR灵敏度一致;速度快,整个扩增过程只需要40 min,不需要特殊设备。本研究建立的RT-RPA检测方法可应用于LMV的快速检测。

A reverse transcription-recombinase polymerase amplification（RT-RPA） was developed based on the coat protein gene of Lettuce mosic virus（LMV）. Five viruses infecting lettuce and three potyviruses were selected for RT-RPA specificity analysis. The results showed that positive samples could amplify the target fragment of 272 bp,while the other 7 viruses,blank control and negative control did not show bands. The limit detection of RT-RPA for LMV was consistent with the conventional RT-PCR which can detect the diluted 100 fold of LMV RNA. Moreover,the whole process of RT-RPA only need 40 min without special equipment. The developed RT-RPA assay provide a rapid alternative tool for the detection of LMV.