Multiplex nested reverse transcription-polymerase chain reaction in a single tube for sensitive and simultaneous detection of four RNA viruses and Pseudomonas savastanoi pv. savastanoi in olive trees
Multiplex nested reverse transcription-polymerase chain reaction in a single tube for sensitive and simultaneous detection of four RNA viruses and Pseudomonas savastanoi pv. savastanoi in olive trees
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DOI：
10.1094/phyto.2003.93.3.286
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发表时间：
2003-03-01
期刊：
影响因子：
3.2
通讯作者：
Cambra, M
中科院分区：
文献类型：
--
作者：
Bertolini, E;Olmos, A;Cambra, M
建立了一种同时检测黄瓜花叶病毒、樱桃卷叶病毒、草莓潜伏环斑病毒和阿拉伯花叶病毒4种核糖核酸病毒和细菌假单胞菌的多重套式逆转录-聚合酶链式反应方法。萨瓦斯塔尼。与传统的单特异性套式扩增相比，该方法首次实现了对植物病毒和细菌的RNA和DNA靶标的灵敏和同时检测，节省了时间，降低了污染风险，并降低了成本。该方法成功地与使用特定寡核苷酸探针的扩增的比色检测相结合，以简化常规检测。以15个不同品种的245株橄榄树为材料，采用多重逆转录-套式聚合酶链式反应结合比色检测技术对其进行分析。多重套式RT-PCR病毒检测使阳性树的识别率提高了8.1%。病毒在受侵染的树木中分布不均。用建立的多重套式方法和套式聚合酶链式反应在28.7%的分析树木中检测到细菌。这一强大的方法学可以应用于其他模型，在一次检测中检测几种病原体。
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.