Real-time quantitative PCR based sensitive detection and genotype discrimination of Pepino mosaic virus
Real-time quantitative PCR based sensitive detection and genotype discrimination of Pepino mosaic virus
复制标题
DOI：
10.1016/j.jviromet.2009.07.008
复制
发表时间：
2009-12-01
影响因子：
3.1
通讯作者：
Ravnikar, Maja
中科院分区：
文献类型：
--
作者：
Gutierrez-Aguirre, Ion;Mehle, Natasa;Ravnikar, Maja
近十年来，一种由番茄花叶病毒（PepMV）引起的新型病毒病一直威胁着全球番茄产业。可靠的检测对帮助疾病控制至关重要。方法必须既灵敏又能检测已鉴定的不同基因型的范围。本文描述了五种新的反转录实时定量PCR （RT-qPCR）检测方法的发展，这些方法可以检测所有已知的PepMV基因型。对秘鲁、欧洲番茄、Ch2和US1 PepMV基因型的检测结果进行了评估，并对两步和一步RT-qPCR检测格式进行了优化。一步RT-qPCR检测PepMV欧洲番茄基因型颗粒的灵敏度比ELISA至少高2个数量级。这种方法在5000个未感染的种子中只检测到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。欧洲番茄-秘鲁番茄、Ch2和US1基因型组合均被成功区分。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在流行病学研究或评估植物对病毒感染的抗性的研究中非常有用。(C) 2009 Elsevier B.V.版权所有
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. (C) 2009 Elsevier B.V. All rights reserved.