Viganó Felicita, Stevens Mark
Broom's Barn Research Centre, Higham, Bury St. Edmunds, Suffolk IP28 6NP, UK.
J Virol Methods. 2007 Dec;146(1-2):196-201. doi: 10.1016/j.jviromet.2007.06.018. Epub 2007 Aug 7.
A multiplex immunocapture-reverse transcription-polymerase chain reaction protocol (mIC-RT-PCR) was successfully developed to improve the detection of Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV) in plants and aphids in single and mixed infections. Viral particles from plant and aphid extracts were enriched by antibody-capture and lysed by heating to release the viral RNA. During the RT-PCR step, 5' end sequences specific to each virus were amplified and the products analysed by gel electrophoresis; the PCR products corresponding to BMYV and BChV were 440 and 348bp respectively. The test was evaluated on single aphids carrying BMYV, BChV or both viruses and the results demonstrated that the mIC-RT-PCR is specific and sensitive. Its sensitivity was found to be 10(5) times higher than the TAS-ELISA routinely used for detecting BMYV and BChV and 10(4) times better than RT-PCR when both viruses were present. Eliminating the antibody-capture step to simplify the technique did not affect the sensitivity of the test and a procedure using microtitre plates was developed to allow simultaneous processing of large numbers of samples.
成功开发了一种多重免疫捕获-逆转录-聚合酶链反应方案（mIC-RT-PCR），以提高对植物和蚜虫中甜菜轻度黄化病毒（BMYV）和甜菜褪绿病毒（BChV）单一感染和混合感染的检测。植物和蚜虫提取物中的病毒颗粒通过抗体捕获进行富集，并通过加热裂解以释放病毒RNA。在RT-PCR步骤中，扩增每种病毒特有的5'端序列，并通过凝胶电泳分析产物；对应于BMYV和BChV的PCR产物分别为440和348bp。对携带BMYV、BChV或两种病毒的单个蚜虫进行了该测试，结果表明mIC-RT-PCR具有特异性和敏感性。发现其灵敏度比常规用于检测BMYV和BChV的TAS-ELISA高10(5)倍，当两种病毒都存在时，比RT-PCR高10(4)倍。省去抗体捕获步骤以简化该技术不会影响测试的灵敏度，并且开发了一种使用微量滴定板的程序，以允许同时处理大量样品。
