葡萄卷叶伴随病毒2实时荧光定量RT-PCR技术的检测应用

通过引物筛选和体系优化建立了葡萄卷叶伴随病毒2 Grapevine leafroll-associated virus 2(GLRaV-2)的SYBR GreenⅠ染料法实时荧光定量RT-PCR检测技术。该技术标准曲线扩增效率为102.2%,决定系数为0.999,最低检出限可达10-3稀释梯度,是常规RT-PCR的100倍。对不同季节和不同部位葡萄样品的检出率普遍高于常规RT-PCR。春夏秋季样品检出率分别为67%、89%和86%,比常规RT-PCR检出率分别高42%、28%和17%。冬季休眠枝条检出率最高(100%),与常规RT-PCR相同。夏季老叶柄和卷须、秋季和冬季枝条等样品检测效果最好,检出率均为100%。对来自我国17个省38个品种的116份田间葡萄样品检测结果表明,qRT-PCR共检测到10个样品为阳性,检出率略高于常规RT-PCR。

Application of a real-time quantitative RT-PCR for detection of Grapevine leafroll-associated virus 2

A SYBR GreenⅠ based real-time quantitative RT-PCR(qRT-PCR) method for Grapevine leafroll-associated virus 2(GLRaV-2) was established. An excellent linear correlation(0.999) and amplification efficiency(102.2%) were obtained from the standard curve. The detection limit of the method was 10-3 dilution fold, which was 100 times higher than that of conventional RT-PCR. The method was subsequently used to detect grapevine samples in different seasons and different positions of plant. The detection efficiency of qRT-PCR for grapevine samples in most seasons and positions were generally higher than that of conventional RT-PCR. The detection rates of qRT-PCR for samples in spring, summer and autumn were 67%, 89% and 86%, which were 42%, 28% and 17% higher than those of conventional RT-PCR, respectively. For dormant branches in winter, the detection rates were the same for the two methods(100%). In general, the old petioles and tendrils in summer, branches in autumn and winter were the best materials for GLRaV-2 detection by qRT-PCR, with the detection rates of 100%. For field samples(belonging to 38 cultivars) from 17 provinces in China, 10 of 116 samples were detected to be positive by qRT-PCR, and the detection efficiency was higher than that of conventional RT-PCR.