甘薯羽状斑驳病毒的RT-PCR检测技术构建  

Construction of RT-PCR Detection Technique for Sweet Potato Feathery Mottle Virus

作　　者：肖海峻[1] 孟利前[1] 朱建晨[1] 张进 李庞博 黄广学[1] Xiao Haijun;Meng Liqian;Zhu Jianchen;Zhang Jin;Li Pangbo;Huang Guangxue(Beijing Vocational College of Agriculture, Beijing, 102442)

机构地区：[1]北京农业职业学院

出　　处：《分子植物育种》2019年第13期4302-4306,共5页Molecular Plant Breeding

基　　金：北京农业职业学院博士基金项目;北京市农委项目共同资助

摘　　要：随着现代分子生物学技术的发展,反转录聚合酶链式反应技术(reverse transcription-polymerase chain reaction, RT-PCR)在甘薯病毒检测上的应用越来越广泛,其有灵敏度高、特异性强等优点,采用该技术可检测出甘薯组织中含量极低的病毒RNA。甘薯羽状斑驳病毒(sweet potato feathery mottle virus, SPFMV)是导致甘薯发生病害的主要病毒之一,为建立甘薯SPFMV快速检测技术,更好地防止该病毒的发生,本研究根据NCBI GenBank中收录的甘薯羽状斑驳病毒外壳蛋白(CP)基因核苷酸序列的保守区域设计了4对特异性引物,使用天根生化科技(北京)有限公司生产的植物总RNA提取试剂盒、FastQuant cDNA第一链合成试剂盒和2×Taq PCR MasterMix试剂盒,完成特异性引物的筛选并设置不同的退火温度对PCR扩增体系进行优化,确定了甘薯羽状斑驳病毒的RT-PCR检测技术体系。该方法的建立旨在为生产上甘薯SPFMV病毒的快速检测提供技术支持。With the development of modern molecular biological technology, reverse transcription-polymerase chain reaction has been widely used in the detection of sweet potato viruses. Little RNA viruses from sweet potato tissue can be detected by RT-PCR which is with such advantage as more sensitive and specificity. SPFMV is one of the main viruses which reduce in sweet potato viruses. In order to build a rapid detection technology and better prevent the occurrence of the virus, four primers were designed and synthesized according to the included nucleotide sequence of coat protein gene of sweet potato feather mottle virus(SPFMV) in NCBI GenBank. The PCR amplification system was optimized by selecting specific primers and setting different annealing temperatures using the total RNA extraction kit, FastQuant cDNA first strand synthesis kit and 2 ×Taq PCR MasterMix kit produced by tiangen biochemical technology(Beijing) Co. LTD. The RT-PCR detection system for the PCR virus was determined. The method was established to provide technical support for the rapid detection of SPFMV virus in sweet potato.

关 键 词：甘薯(Ipomona batatas) 羽状斑驳病毒 RT-PCR检测技术 

分 类 号：S435.313[农业科学—农业昆虫与害虫防治]