烟草黄瓜花叶病毒一步法RT-PCR检测试剂盒的研制与初步应用

摘要

为快速准确地检测烟草黄瓜花叶病毒(CMV),根据黄瓜花叶病毒基因组序列相对保守区域设计了一对特异性引物,整合了反转录和PCR反应所需酶和缓冲液,通过反应条件的优化,建立了基于一步法RT-PCR检测烟叶感染CMV的方法,在此基础上组装了检测试剂盒.进一步分析了该试剂盒的特异性、稳定性和检测灵敏度.结果表明,该试剂盒具有很强的特异性、良好的重复性和较高的灵敏度(模板RNA浓度最低达到10 ng量级).初步应用结果显示,在多个烟区采集的病叶样品检测结果均与ELISA检测结果一致,能够成功检测出CMV.%To detect tobacco CMV quickly and accurately, a pair of specific primers was designed according to the relatively conservative region in the CMV genomic sequence, and enzymes and buffers needed for reverse transcription and PCR were integrated. By optimizing reaction conditions, a method for detecting CMVinfected tobacco based on one-step RT-PCR was developed and a detection kit was assembled. The specificity, stability and sensitivity of the kit were analyzed. The results showed that the kit featured strong specificity, good repeatability and higher sensitivity. The lowest RNA concentration of template reached lOng magnitude level. The preliminary application results indicated that the detection results of infected leaves sampled from several areas agreed with the results of ELISA test, the kit could detect CMV successfully.