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A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types
REN Fang, ZHANG Zun-ping, FAN Xu-dong, HU Guo-jun, ZHANG Meng-yan, DONG Ya-feng
2020, 19 (7): 1834-1841.   DOI: 10.1016/S2095-3119(19)62784-X
Abstract137)      PDF in ScienceDirect      
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%). 
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Prevalence and genetic diversity of grapevine fabavirus isolates from different grapevine cultivars and regions in China
FAN Xu-dong, ZHANG meng-yan, ZHANG Zun-ping, REN Fang, HU Guo-jun, DONG Ya-feng
2020, 19 (3): 768-774.   DOI: 10.1016/S2095-3119(19)62677-8
Abstract129)      PDF in ScienceDirect      
A total of 288 grapevine samples of 61 different grapevine cultivars, collected from 22 provinces and regions, were analyzed by reverse transcription-PCR (RT-PCR) for the presence of grapevine fabavirus (GFabV).  PCR detection results showed the incidences of GFabV were 12.8% (30/235) and 48.1% (25/52) in the asymptomatic and symptomatic vines, respectively.  The genetic diversity of GFabV isolates was analyzed based on partial nucleotide and encoded amino acid sequences of the RNA1 and RNA2 polyprotein genes.  Phylogenetic analyses of the RNA1 and RNA2 gene sequences divided the GFabV isolates into five well-defined groups.  Groups 1, 2, and 4 comprised only Chinese isolates.  This article represents the first report for the prevalence and genetic diversity of GFabV in grapevines grown in China.
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