La Notte P, Minafra A, Saldarelli P
Dipartimento di Protezione delle Piante, Università degli Studi, Bari, Italy.
J Virol Methods. 1997 Jun;66(1):103-8. doi: 10.1016/s0166-0934(97)00038-4.
Specific amplification of genomic fragments of grapevine trichovirus A (GVA), grapevine trichovirus B (GVB) and grapevine leafroll-associated closterovirus 3 (GLRaV3) was obtained by reverse transcription-PCR on total nucleic acid solubilized from small pieces of charged nylon membrane, on which a drop of crude infected grapevine sap was spotted (spot-PCR). A thermal treatment (95 degrees for 10 degrees) of spotted sap in a buffered solution improved the release of viral template. Consistent amplification was obtained with three viruses from fragments of the same respective blots up to 1 month after spotting, while a detection threshold limit comparable with standard PCR techniques was found for this method. Duplex PCR (i.e. amplification of different viruses from a mixed-infected grapevine source) was also found to be effective, since GVA and GLRaV3 were amplified by a mixture of specific primers in the same reaction. This rapid and easy sampling technique, using leaf petioles to express crude sap, may have a wide field application for screening grapevine viruses.
通过逆转录聚合酶链反应（RT-PCR），从小片带电尼龙膜上溶解的总核酸中，特异性扩增葡萄毛形病毒A（GVA）、葡萄毛形病毒B（GVB）和葡萄卷叶相关病毒3（GLRaV3）的基因组片段。在带电尼龙膜上点一滴粗制的感染葡萄汁液（斑点PCR）。在缓冲溶液中对斑点汁液进行热处理（95℃，10分钟）可改善病毒模板的释放。在点样后长达1个月的时间里，从相同印迹的片段中对三种病毒进行了一致的扩增，同时发现该方法的检测阈值与标准PCR技术相当。双链PCR（即从混合感染的葡萄来源中扩增不同病毒）也被证明是有效的，因为在同一反应中，GVA和GLRaV3通过特异性引物混合物进行了扩增。这种使用叶柄表达粗汁液的快速简便的采样技术，可能在葡萄病毒筛查中具有广泛的应用前景。
