关键词: 葡萄, 葡萄病毒A, 检测, 实时荧光定量RT-PCR, 常规RT-PCR

Abstract: To develop a rapid and highly sensitive method for Grapevine virus A（GVA）detection，a SYBR GreenⅠreal time fluorescence quantitative RT-PCR method（RT-qPCR）was established，and an excellent linear correlation（R2 = 0.999）and a high amplification efficiency（E = 99.2%）were obtained from standard curve of cDNA. The RT-qPCR method could be used to detect GVA specifically，and the sensitivity was 100-fold higher than conventional RT-PCR. Reproducibility test revealed that the coefficients of variation in the intra- and extra- assay were 0.16%–0.31% and 2.91%，respectively，indicating a good reproducibility. The RT-qPCR method could be used to detect a wide range of sample types，and the detection rates of samples from different seasons，cultivars and positions（young leaves，young petioles，old leaves，old petioles，tendrils and dormant branches）were generally higher than conventional RT-PCR. Comparison of the detection rates of samples in different seasons showed that samples in autumn and winter were best for detection，and except for young leaves，the detection rates of all samples in these two seasons were all 100%. The detection rates of samples in spring and summer were 10% to 100%. Comparison of the detection rates of samples in different positions showed that samples of old petioles and dormant branches were best for detection，for which the detection rates were all 100%. The detection rate for old leaves was 80% to 100%，and for samples from other positions was from 10% to 100%. In detection of field samples，the results of dormant branches were most consistent with conventional RT-PCR，but the detection rate of old tendrils in autumn by RT-qPCR was obviously higher than that by conventional RT-PCR.

Key words: grapevine, Grapevine virus A, detection, real-time fluorescent quantitative RT-PCR, conventional RT-PCR