Abstract A single step Direct real-time quantitative reverse transcription quantitative polymerase chain reaction (DRT-qPCR) was applied and optimized for the detection of Grapevine rupestris stem-pitting associated virus (GRSPaV) in grapevine. A probe and primer set were designed from conserved genomic regions from eight GRSPaV accessions from GenBank and used with the Direct Plant Extraction Buffer (DiPEB) system in place of total nucleic acid extraction using commercial kits to identify 14 grapevine accessions and field grapevine isolates for the presence of GRSPaV. Optimal detection of GRSPaV was obtained using foliar tissue macerated first in ELISA extraction buffer that was further diluted in DiPEB buffer (1:9 v/v). Middle-aged grape leaves on young canes gave acceptably low Cq (quantification cycle) values, with no significant differences detected between bisected half leaves, refrigerated and frozen leaves, and frozen leaf macerates made in ELISA buffer. Cq values of macerates derived from cambial tissue were comparable to those of leaf tissue macerates. The DRT-qPCR was shown to be a reproducible, specific test capable of detection of as little as 4.59 pg of viral RNA. This is the first study to use this simplified method for the detection of viruses that infect grapevine. Because this system does not require the use of a commercial nucleic acid extraction kit, it provides savings in cost and time allowing higher throughput testing.

中文翻译：

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直接实时定量逆转录聚合酶反应 (DRT-qPCR) 检测葡萄藤茎点相关病毒 (GRSPaV) 检测的优化

摘要 单步直接实时定量逆转录定量聚合酶链反应 (DRT-qPCR) 被应用和优化用于检测葡萄中的葡萄树茎点相关病毒 (GRSPaV)。从来自 GenBank 的 8 个 GRSPaV 种质的保守基因组区域设计探针和引物组，并与直接植物提取缓冲液 (DiPEB) 系统一起使用商业试剂盒代替总核酸提取，以鉴定 14 个葡萄种质和田间葡萄分离株，用于GRSPaV 的存在。使用首先在 ELISA 提取缓冲液中浸渍的叶组织获得 GRSPaV 的最佳检测，然后在 DiPEB 缓冲液 (1:9 v/v) 中进一步稀释。幼藤上的中年葡萄叶给出了可接受的低 Cq（量化周期）值，在对分的半叶、冷藏和冷冻叶以及在 ELISA 缓冲液中制备的冷冻叶浸渍液之间没有检测到显着差异。来自形成层组织的浸渍物的 Cq 值与叶组织浸渍物的 Cq 值相当。DRT-qPCR 被证明是一种可重复的特异性测试，能够检测低至 4.59 pg 的病毒 RNA。这是第一项使用这种简化方法检测感染葡萄藤的病毒的研究。由于该系统不需要使用商业核酸提取试剂盒，因此可以节省成本和时间，从而实现更高通量的测试。DRT-qPCR 被证明是一种可重复的特异性测试，能够检测低至 4.59 pg 的病毒 RNA。这是第一项使用这种简化方法检测感染葡萄藤的病毒的研究。由于该系统不需要使用商业核酸提取试剂盒，因此可以节省成本和时间，从而实现更高通量的测试。DRT-qPCR 被证明是一种可重复的特异性测试，能够检测低至 4.59 pg 的病毒 RNA。这是第一项使用这种简化方法检测感染葡萄藤的病毒的研究。由于该系统不需要使用商业核酸提取试剂盒，因此可以节省成本和时间，从而实现更高通量的测试。