Diaz-Lara Alfredo, Stevens Kristian, Klaassen Vicki, Golino Deborah, Al Rwahnih Maher
Department of Plant Pathology, University of California-Davis, Davis, CA 95616, USA.
Department of Evolution and Ecology, University of California-Davis, Davis, CA 95616, USA.
Plants (Basel). 2020 Feb 19;9(2):273. doi: 10.3390/plants9020273.
Viruses can cause economic losses in fruit trees, including spp., by reducing yield and marketable fruit. Given the genetic diversity of viruses, reliable diagnostic methods relying on PCR are critical in determining viral infection in fruit trees. This study evaluated the broad-range detection capacity of currently available real-time RT-PCR assays for -infecting viruses and developed new assays when current tests were inadequate or absent. Available assays for 15 different viruses were exhaustively evaluated in silico to determine their capacity to detect virus isolates deposited in GenBank. During this evaluation, several isolates deposited since the assay was designed exhibited nucleotide mismatches in relation to the existing assay's primer sequences. In cases where updating an existing assay was impractical, we performed a redesign with the dual goals of assay compactness and comprehensive inclusion of genetic diversity. The efficiency of each developed assay was determined by a standard curve. To validate the assay designs, we tested them against a comprehensive set of 87 positive and negative samples independently analyzed by high throughput sequencing. As a result, all the real-time RT-PCR assays described herein successfully detected the different viruses and their corresponding isolates. To further validate the new and updated assays a germplasm collection was surveyed. The sensitive and reliable detection methods described here will be used for the large-scale pathogen testing required to maintain the highest quality nursery stock.
病毒会导致果树出现经济损失，包括通过降低产量和影响果实的适销性，对包括[具体品种]在内的多种果树品种造成影响。鉴于病毒的遗传多样性，依赖聚合酶链反应（PCR）的可靠诊断方法对于确定果树中的病毒感染至关重要。本研究评估了当前可用的实时逆转录聚合酶链反应（RT-PCR）检测方法对[具体病毒名称]感染病毒的广泛检测能力，并在现有检测方法不足或缺乏时开发了新的检测方法。对15种不同病毒的现有检测方法进行了详尽的电子评估，以确定它们检测保存在基因库中的病毒分离株的能力。在评估过程中，自检测方法设计以来存入的几个分离株与现有检测方法的引物序列存在核苷酸错配。在无法更新现有检测方法的情况下，我们进行了重新设计，以实现检测方法紧凑性和全面涵盖遗传多样性这两个目标。通过标准曲线确定每种开发的检测方法的效率。为了验证检测方法的设计，我们针对通过高通量测序独立分析的一组全面的87个阳性和阴性[样本类型]样本对其进行了测试。结果，本文所述的所有实时RT-PCR检测方法均成功检测到了不同的病毒及其相应的分离株。为了进一步验证新的和更新的检测方法，我们对一个[种质资源名称]种质库进行了调查。这里描述的灵敏且可靠的检测方法将用于维持最高质量苗木所需的大规模病原体检测。
