关键词: 葡萄, 葡萄扇叶病毒, 实时荧光定量RT-PCR, 病毒浓度

Abstract: To establish high sensitivity and relative quantitative method of Grapevine fanleaf virus （GFLV），the SYBR Green RT-PCR real time fluorescence quantitative method of FL-HP for GFLV was established by using primer screen and system optimization. The results showed that a good linear correlation（R2 = 0.998）obtained from standard curve of cDNA，and the amplification efficiency was 100%. Sensitivity of real-time quantitative RT-PCR method was at least 100 times higher than that with conventional RT-PCR and 10 times higher than that with nested RT-PCR，respectively. Three-time repeats revealed that the coefficients of variation between the intra- and inter-assay were both within 2.59%，indicating a reproducibility detection method to GFLV. The stability test results showed that EF1α and GAPDH gene of grape has high stability. Real-time RT-PCR method was used to determine the content ofGFLV in 12 different grape plants and to detect a large number of grape samples（58）. There was noticeable differences among the content of GFLV in 12 field samples，the highest content can reach to 81 245.5 times than the lowest，in the 58 samples，the real-time fluorescent quantitative RT-PCR method can detect more GFLV isolates than DAS-ELASA and RT-nPCR.

Key words: grape, Grapevine fanleaf virus（GFLV）, quantitative RT-PCR, GFLV content