葡萄病毒A实时荧光定量RT-PCR检测技术的建立及应用

Development and Application of a Quantitative RT-PCR Approach for Detection of Grapevine virus A

根据文献报道和GenBank已登录序列设计引物建立了葡萄病毒A(Grapevine virus A,GVA)SYBRGreenⅠ染料法实时荧光定量RT-PCR检测技术体系。该技术标准曲线循环阈值与模板浓度呈现良好线性关系,扩增效率为99.2%,决定系数为0.999,可特异性检测GVA,灵敏度高(达常规RT-PCR的100倍),重复性好。对不同季节、不同品种以及不同部位葡萄样品(嫩叶、嫩叶柄、老叶、老叶柄、卷须和休眠枝条)中GVA的检出率普遍高于常规RT-PCR。不同季节间比较,对秋、冬季样品检测效果最好,除嫩叶外其余部位检出率均达100%,春夏季检出率为10%～100%。不同部位间比较,老叶柄和休眠枝条检测效果最好,检出率均达100%,其次为老叶(80%～100%),其余部位样品检出率为10%～100%。在大量田间样品检测中,休眠枝条样品检测结果与常规RT-PCR一致,而秋季老叶柄样品检出率明显高于常规RT-PCR。

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(R 2 = 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.