单头灰飞虱体内两种水稻病毒的双重一步法RT PCR检测

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。

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

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 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．