采用 18S rRNA 作为内参，利用多重 RT-PCR 检测侵染番茄的黄瓜花叶病毒亚组和烟草花叶病毒。
Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting Tomato using 18S rRNA as an internal control.
机构信息
Institute of Bioengineering, Zhejiang Sci-Tech University, Hangzhou, China.
出版信息
Acta Biochim Biophys Sin (Shanghai). 2011 Jun;43(6):465-71. doi: 10.1093/abbs/gmr031. Epub 2011 Apr 28.
A multiplex reverse-transcription polymerase chain reaction (RT-PCR) protocol was developed for simultaneous detection and discrimination of subgroups of Cucumber mosaic virus (CMV), including its satellite RNA, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV), using 18S rRNA as an internal control. Species- and subgroups-specific primers designed to differentiate CMV subgroups I and II, ToMV and TMV, were assessed using the cDNA clones of viral genomes, CMV satellite RNA and 18S rRNA gene from tomato (Solanum lycopersicum L.) or tobacco (Nicotiana tobacum). Using total RNA extracted from artificial mixture of tomato leaf tissues infected by each virus, the reaction components and cycling parameters were optimized and a multiplex RT-PCR procedure was established. Six fragments of 704, 593, 512, 421, 385, 255 bp, specific to CMV subgroup II, CMV subgroup I, ToMV, TMV, satellite RNA and 18S rRNA, respectively, were simultaneously amplified. The sensitivity of the multiplex RT-PCR method for detecting CMV was 100 times higher than that of double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA). This method was successfully used for field detection. Among 141 samples collected from East China through tomato growth seasons, 106 single infections with one of the above isolates were detected and 13 mixed infections were found. The results showed the potential use of this method for investigating the epidemiology of viral diseases infecting tomato.
建立了一种多重反转录聚合酶链反应（RT-PCR）方案，用于同时检测和区分黄瓜花叶病毒（CMV）的亚组，包括其卫星 RNA、烟草花叶病毒（ToMV）和烟草花叶病毒（TMV），并以内源 18S rRNA 作为对照。使用番茄（Solanum lycopersicum L.）或烟草（Nicotiana tobacum）的病毒基因组 cDNA 克隆、CMV 卫星 RNA 和 18S rRNA 基因，评估了设计用于区分 CMV 亚组 I 和 II、ToMV 和 TMV 的种特异性和亚组特异性引物。使用从受每种病毒感染的番茄叶片组织的人工混合物中提取的总 RNA，优化了反应成分和循环参数，并建立了多重 RT-PCR 程序。分别针对 CMV 亚组 II、CMV 亚组 I、ToMV、TMV、卫星 RNA 和 18S rRNA 的 704、593、512、421、385 和 255 bp 的 6 个片段得到了同时扩增。与双抗体夹心酶联免疫吸附测定（DAS-ELISA）相比，该多重 RT-PCR 方法检测 CMV 的灵敏度提高了 100 倍。该方法成功用于田间检测。在中国东部番茄生长季节采集的 141 个样本中，检测到 106 个单一感染上述分离株的样本，发现 13 个混合感染的样本。结果表明，该方法有可能用于调查感染番茄的病毒病的流行病学。
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