Department of Biotechnology and Systems Biology, National Institute of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia.
J Virol Methods. 2009 Dec;162(1-2):46-55. doi: 10.1016/j.jviromet.2009.07.008. Epub 2009 Jul 28.
Over the last decade a new virus disease caused by Pepino mosaic virus (PepMV) has been threatening the tomato industry worldwide. Reliable detection is vitally important to aid disease control. Methods must be both sensitive and capable of detecting the range of distinct genotypes that have been identified. The development of five new reverse transcription real-time quantitative PCR (RT-qPCR) assays is described, which allow the detection of all known PepMV genotypes. The performance of the assays was evaluated on Peruvian, European tomato, Ch2 and US1 PepMV genotypes and optimised for both two- and one-step RT-qPCR detection formats. One-step RT-qPCR detected PepMV European tomato genotype particles at least two orders of magnitude more sensitively than ELISA. The method detected as little as one naturally infected seed among 5000 uninfected seeds. The genotype-specificity of the five assays was compared using PepMV isolates representing all of the different genotypes. The following genotype combinations were all discriminated successfully: European tomato-Peruvian, Ch2, and US1. In addition to its application for diagnostic purposes, the genotype-specificity and the quantitative potential of the method, makes it very useful for epidemiological studies or for studies evaluating resistance of plants to virus infection.
在过去的十年中，一种由 Pepino 花叶病毒（PepMV）引起的新病毒疾病一直在威胁着全球的番茄产业。可靠的检测对于帮助控制疾病至关重要。方法必须既敏感又能够检测到已确定的不同基因型。本文描述了五种新的逆转录实时定量 PCR（RT-qPCR）检测方法的开发，这些方法可用于检测所有已知的 PepMV 基因型。对秘鲁、欧洲番茄、Ch2 和 US1 PepMV 基因型进行了检测方法的性能评估，并对两步和一步 RT-qPCR 检测方法进行了优化。一步法 RT-qPCR 比 ELISA 至少灵敏两个数量级，能够检测到每 5000 粒未感染种子中一个自然感染的种子。使用代表所有不同基因型的 PepMV 分离株比较了五种检测方法的基因型特异性。成功区分了以下基因型组合：欧洲番茄-秘鲁、Ch2 和 US1。除了用于诊断目的外，该方法的基因型特异性和定量潜力使其非常适用于流行病学研究或评估植物对病毒感染的抗性的研究。
