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.pdfVIP

烟草科技ITobacco Science Technology 病虫害防治IDise幽e Pest Control 2011 年第 10 期(总第 291 期) 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 张俊祺l ，刘相兵2 ，罗朝鹏1 ，杨军·』，宋纪真1 ，尹启生1 ，宋浩2 1.中国烟草总公司郑州烟草研究院，郑州高新技术产业开发区枫杨街2 号 450001 2. 湖南中烟工业有限责任公司，长沙市劳动中路426 号 410007 关键词:黄瓜花叶病毒;RT-PCR;检测试剂盒;特异性;稳定性;灵敏度 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，该试剂金具有很强的特异性、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。 中图分类号:5432.41 文献标iR码:A 文章编号: 1002 - 0861 (201 1) 10 - 0065 - 05 Development and Preliminary Application of One-step RT-PCR Kit for Detection of Tobacco CMV ZHANG JUN-QI( 1 ), LlU XIANG-BING (2) , LUO ZHAO-PENG ( 1 ), YANG JUN ( 1 ), SONG JI-ZHEN (1 ) , YIN QI-SHENG (1 ) , and SONG HAO (2 ) 1. Zhengzhou Tobacco Research Institute of CNTC , Zhengzhou 450001 , China 2. China Tobacco Hunan Industrial Co. , Ltd. , Changsha 410007 , China Keywords: Cucumber mosaic virus (CMV); RT-PCR; Detection kit; Specificity; Stability; Sensitivity Abstract: 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 CMV- infected 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 IOng magnitude level. The preliminary application results indicated that the detection results of infected leave