Phytopathology. 2003 Mar;93(3):286-92. doi: 10.1094/PHYTO.2003.93.3.286.
ABSTRACT A multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) in a single closed tube was developed for the simultaneous detection of four RNA viruses: Cucumber mosaic virus, Cherry leaf roll virus, Strawberry latent ringspot virus, and Arabis mosaic virus, and the bacterium Pseudomonas savastanoi pv. savastanoi. The method enabled, for the first time, the sensitive and simultaneous detection of RNA and DNA targets from plant viruses and a bacterium, saving time, decreasing risks of contamination, and reducing costs compared with conventional monospecific nested amplifications. The method was successfully coupled with colorimetric detection of amplicons using specific oligoprobes to simplify routine detection. Two hundred forty-five olive trees from 15 different cultivars were analyzed by multiplex RT-nested PCR coupled with colorimetric detection. Multiplex nested RT-PCR for viral detection increased the identification of positive trees by 8.1%. An uneven distribution of the viruses was observed in the infected trees. The bacterium was detected in 28.7% of the analyzed trees by the developed multiplex nested method and by a nested PCR previously developed. This powerful methodology could be applied to other models for the detection of several pathogens in a single assay.
摘要 本研究开发了一种在单个封闭管中进行的多重巢式逆转录聚合酶链反应（RT-PCR），用于同时检测四种 RNA 病毒：黄瓜花叶病毒、樱桃叶卷叶病毒、草莓潜隐环斑病毒和胡萝卜花斑病毒，以及细菌丁香假单胞菌 pv. savastanoi。该方法首次实现了从植物病毒和细菌中敏感且同时检测 RNA 和 DNA 靶标，与传统的单特异性巢式扩增相比，节省了时间、降低了污染风险并降低了成本。该方法与使用特定寡探针对扩增子进行比色检测成功结合，简化了常规检测。对来自 15 个不同品种的 245 棵橄榄树进行了多重 RT-巢式 PCR 结合比色检测分析。用于病毒检测的多重巢式 RT-PCR 将阳性树的鉴定率提高了 8.1%。在感染的树木中观察到病毒的分布不均匀。通过开发的多重巢式方法和之前开发的巢式 PCR 检测到 28.7%的分析树木中存在细菌。这种强大的方法学可以应用于其他模型，用于在单次检测中检测多种病原体。
