A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types

摘要

To develop a rapid and high-sensitivity method for detection of grapevine virus E(GVE),a SYBR Green based real-time fluorescence quantitative RT-PCR method(RT-qPCR)was established.This method could be used to detect GVE specifically,and the sensitivity was about 100 times greater than conventional RT-PCR.An excellent linear correlation(R=0.997)and a high amplification efficiency(E=97.5%)were obtained from the standard curve of this method.Reproducibility tests revealed that the coefficients of variation in the intra-and inter-assay results were 0.31-1.03%and 0.82--262%,respectively,indicating a good reproduiblity.The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types.The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR.The detection rates in spring,summer,autumn and winter increased gradually.Samples in autumn and winter were best for detection,and the detection rates of most samples were 80-100%,which were 10 to 40%higher than conventional RT-PCR.In general,old petioles and branches were the best tissues for GVE detection.The detection rates of these samples in each season were all 100%,which were 20 to 40%higher than conventional RT-PCR.The second highest rates were in the old leaf,with detection rates for RT-qPCR of 80-100%in all seasons,which were 20 to 40%higher than conventional RT-PCR.GVE could be difficultly detected in young leaves by conventional RT-PCR,and the detection rates were only 0-50%,while by RT-qPCR the rates could increase to 0--80%.A total of 33 out of 363 samples(belonging to 68 cultivars)from 20 regions in China were detected to be positive by RT-qPCR(9.1%),which was more than twice the rate of the conventional RT-PCR(3.9%).