Journal of Integrative Agriculture (Jul 2020)
A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types
Abstract
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 (R2=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 0.82–2.62%, respectively, indicating a good reproducibility. 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%).
