单头灰飞虱体内两种水稻病毒的双重一步法 RT-PCR检测

Detection of two rice viruses in single planthopper( Laodelphax striatellus) with multiple one step RT-PCR

灰飞虱传播的水稻条纹病毒(Rice stripe virus,RSV)和水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)是危害我国水稻生产最主要的2种病毒,建立灰飞虱体内RSV和RBSDV快速、可靠的检测方法是水稻生产中的重要问题。本研究根据RSV RNA3和RBSDV S10序列分别设计了2个病毒的特异性引物,通过退火温度和PCR循环数等条件的优化,建立了灰飞虱体内RSV和RBSDV快速鉴定的双重一步法RT-PCR体系。对一步法和两步法RT-PCR检测结果进行比较,表明2种方法均能准确有效鉴定灰飞虱体内的2种病毒,且两步法的检测效果略好于一步法。而灵敏度实验表明一步法RT-PCR可以从0.005 ng·μL-1的灰飞虱RNA初始模板中准确检测到病毒,完全满足单头灰飞虱体内病毒检测的需要。应用本研究建立的双重一步法RT-PCR体系对200头获毒灰飞虱样品中的RSV和RBSDV进行了检测,结果进一步证实了此方法的稳定性和可靠性。

Rice stripe virus（RSV） and Rice black-streaked dwarf virus（RBSDV） are 2 important rice viruses which are transmitted by small brown planthopper （Laodelphax striatellus） and cause severe loss for rice production in China.Rapid and reliable detection of the 2 viruses in L.striatellus is a key issue for the control of these diseases . In this study, two sets of primers that are specific for RSV RNA3 and RBSDV S10 were designed, and multiple one step RT-PCR method was established for rapid detection of RSV and /or RBSDV in L.striatellus through optimized annealing temperature and cycles of PCR conditions .Comparison of one step and two step RT-PCR indicated that＆amp;nbsp;both methods can effectively detect RSV and/or RBSDV in L.striatellus and the result of two step RT-PCR was slightly better than that of one step .Serial dilutions assay showed that the sensitivity of one step RT-PCR method can detect the concentration as low as 0.005 ng·μL-1 , which is completely enough for the identification of viruses in single planthopper.Finally, multiple one step RT-PCR was applied to detect RSV and/or RBSDV in single insect for 200 planthoppers which further confirmed specific , sensitive and time-saving.