水稻黑条矮缩病毒RT-LAMP快速检测方法的建立

Development of a RT-LAMP Assay for Rapid Detection of Rice black-streaked dwarf virus

【目的】建立一种快速、灵敏的逆转录环介导等温扩增方法(RT-LAMP)检测寄主植物和传毒介体体内的水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)。【方法】合成4条针对RBSDV S10核苷酸序列6个位点的特异性引物。分别对引物浓度、MgSO4浓度、反应温度和时间进行优化。将感病水稻总RNA梯度稀释后进行灵敏性检验并与RT-PCR比较分析。选择RBSDV和南方水稻黑条矮缩病毒(SRBSDV)验证该方法的特异性。用本RT-LAMP方法检测田间病株。【结果】RT-LAMP检测方法可排除SRBSDV的干扰而特异地检测植物和飞虱体内的RBSDV,与RT-PCR灵敏性基本一致。检测结果易于判定。【结论】RT-LAMP检测方法适合寄主植物和介体体内RBSDV的快速检测。

【Objective】The objective of this study is to develop a reverse transcription loop-mediated isothermal amplification（RT-LAMP） assay for rapid and sensitive detection of Rice black-streaked dwarf virus（RBSDV） from host plants and insect vector.【Method】Four primers matching a total of 6 sequences of the S10 of RBSDV were synthesized for the RT-LAMP assay.The concentration of the primer and MgSO4 were optimized,and the best temperature and reaction time for detecting the virus were found,respectively.Detection sensitivity comparisons were performed between the RT-LAMP and RT-PCR assay using a single extraction of total RNA from RBSDV-infected rice leaves which was serially diluted in ten-fold increments in DEPC-treated water.In order to determine the specificity of the RT-LAMP assay,total RNA of rice leaves infected with RBSDV or Southern rice black-streaked dwarf virus（SRBSDV） was applied separately to the reaction system.RBSDV-infected rice plants collected from rice field were detected.【Result】 This method demonstrated a high degree of specificity for RBSDV,which can distinguish RBSDV from SRBSDV.The method was also proved to be extremely sensitive,which was as much as the RT-PCR for RBSDV detection.The detection of amplified products was easily monitored.【Conclusion】The RT-LAMP assay is suitable for rapid detection of RBSDV in host plant and vectors.