Bertolini E, Olmos A, Martínez M C, Gorris M T, Cambra M
Departamento de Protección Vegetal y Biotecnología, Laboratorio de Virología e Inmunología, Instituto Valenciano de Investigaciones Agrarias, Apartado oficial, 46113 Moncada Valencia, Spain.
J Virol Methods. 2001 Jul;96(1):33-41. doi: 10.1016/s0166-0934(01)00313-5.
A single-step multiplex RT-PCR was developed for the simultaneous and colourimetric detection of six RNA viruses (Cucumber mosaic virus, Cherry leaf roll virus, strawberry latent ringspot virus, Arabis mosaic virus, Olive latent-1 virus and Olive latent-2 virus) which infect olive trees. Six compatible primer set for one-step RT-PCR amplification in a single closed-tube and 3' digoxigenin labelled probes were designed in optimal, specific and conserved regions. The method has been assessed with 195 Spanish field olive trees, suggesting that approximately 1.5% of the tested material was infected by Cucumber mosaic virus and 0.5% by Cherry leaf roll virus. This method saves time and reagent costs compared with monospecific RT-PCR which needs several reactions for the same number of tests. Using colourimetric detection, it is possible to analyse many samples, it increases sensitivity 10-fold, and whilst facilitating the interpretation of results, it avoids the use of gels and the toxic ethidium bromide. The method could be used routinely for sanitary and certification programmes.
开发了一种单步多重逆转录聚合酶链反应（RT-PCR），用于同时比色检测六种感染橄榄树的RNA病毒（黄瓜花叶病毒、樱桃叶卷曲病毒、草莓潜隐环斑病毒、南芥菜花叶病毒、油橄榄潜隐病毒1型和油橄榄潜隐病毒2型）。针对单管一步RT-PCR扩增设计了六组兼容引物，并在最佳、特异和保守区域设计了3'地高辛标记探针。该方法已对195棵西班牙田间橄榄树进行了评估，结果表明，约1.5%的测试材料感染了黄瓜花叶病毒，0.5%感染了樱桃叶卷曲病毒。与单特异性RT-PCR相比，该方法节省了时间和试剂成本，单特异性RT-PCR对相同数量的测试需要进行多次反应。使用比色检测，可以分析许多样品，灵敏度提高10倍，在便于结果解读的同时，避免了使用凝胶和有毒的溴化乙锭。该方法可常规用于卫生和认证计划。
