摘要
根据葡萄扇叶病毒(Grapevine fanleaf virus,GFLV)RNA2序列设计4对特异性引物,通过引物筛选、体系优化,建立了以FL-HP1-F/R为引物的GFLV的SYBR GreenⅠ实时荧光定量RT-PCR方法。该方法标准曲线循环阈值与模板浓度呈良好的线性关系,扩增效率为100%,相关性系数为0.998,灵敏性分别是常规RT-PCR和巢式RT-PCR的100倍和10倍。重复性试验表明组内和组间变异系数均小于2.59%,显示该方法具有较好的检测稳定性。内参基因的稳定性测试结果表明葡萄EF1和GAPDH基因具有较好的试验稳定性。利用建立的荧光定量方法测定12个葡萄样品中GFLV含量,并检测58个葡萄样品,结果表明不同葡萄样品中GFLV含量差异较大,最高含量是最低含量的81 245.5倍,与ELISA和巢式RT-PCR相比,实时荧光定量方法能检测出更多的GFLV分离物。
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（R-2 = 0.998）obtained from standard curve of c DNA,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 of GFLV 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.
出处
《园艺学报》
CAS
CSCD
北大核心
2016年第3期538-548,共11页
Acta Horticulturae Sinica
基金
国家现代农业产业技术体系建设专项资金项目(CARS-30-bc-3)
