百合无症病毒实时荧光RT-PCR检测方法的建立

Detection of Lily symptomless virus by real-time RT-PCR

百合无症病毒(Lily symptomless virus,LSV)是侵染百合科植物的主要病毒之一。根据该病毒不同分离株外壳蛋白基因(coat protein,CP)的保守序列,设计特异性引物与荧光探针,建立了LSV的实时荧光RTPCR检测方法。利用该方法检测LSV的2个阳性样品,均能够得到典型扩增曲线,而在百合斑驳病毒、烟草环斑病毒、番茄环斑病毒等其他病毒阳性样品中没有典型的扩增曲线,表明了该方法的特异性。与普通RTPCR法相比,此方法灵敏度提高10倍。应用该方法,2012年6月至今,宁波出入境检验检疫局从荷兰进境的67批百合种球样品中检出53批百合无症病毒阳性,检出率为93%。

Lily symptomless virus （LSV） is one of the main viruses infecting Liliaceae plants.In the study, a real-time fluorescent RT-PCR method for LSV detection was established with specific primers and probe designed from the conservative sequences in coat protein genes of LSV isolates .Specificity study showed that typical amplification curves could be obtained for two LSV-positive samples , but not for Lily mottle virus, Tobacco ringspot virus and To-mato ringspot virus-positive samples .Sensitivity of this method was 10 times higher than that of regular RT-PCR. With this method, 53 batches of LSV were intercepted in 67 batches of lily imported from Holland since June 2012 by Ningbo Entry-Exit Inspection and Quarantine Bureau , and the detection rate was 93%.