利用RT-PCR检测库尔勒香梨苹果茎痘病毒的研究  

RT-PCR detection of Apple stem pitting virus in Kuerla pear

作　　者：刘连科[1] 牛建新[1] 王小兵[1] 覃伟铭[1] 

机构地区：[1]石河子大学农学院园艺系,石河子832003

出　　处：《植物病理学报》2003年第3期286-288,共3页Acta Phytopathologica Sinica

基　　金：国家自然科学基金资助项目 (30 0 60 0 53) ;教育部科学技术研究重点项目 (0 2 1 80 )

摘　　要：以库尔勒香梨新鲜、冷藏、冷冻叶片和皮层为材料 ,对提取双链RNA (dsRNA)的 2种方法和提取总RNA的 3种方法进行了分析比较 ,并对总RNA的提取方法进行了改进 ,获得了纯度较高、完整性较好的dsRNA和总RNA ,在此基础上进行了反转录 (RT)和PCR扩增。在国内首次完成了对苹果茎痘病毒 (ASPV)的RT PCR检测 ,建立了ASPV有效RT PCR反应体系。用此体系扩增到ASPV一个长约 316bp的片段。实验表明以dsRNA和总RNA为模板均能成功进行RT PCR检测 ,且dsRNA优于总RNA。Two methods for double strand RNA(dsRNA) and three methods for total RNA were used to extract the RNAs from fresh, refrigerated, freezing leaf and bark of Kuerla pear infected with Apple stem pitting virus (ASPV). The methods of total RNA extraction was improved by adding 1%CTAB, 2%PVP, 2% 2 mercaptoethanol to extraction buffer, and low concentration ethanol was used to deposit amylose. dsRNA and total RNA of high purity and integrity were obtained. Using them as template, reverse transcription polymerase chain reaction (RT PCR) was performed.This is the first report in China that RT PCR was used to detect ASPV in Kuerla pear. PCR amplification conditions were optimized. A 316 bp fragment of ASPV was obtained based on the optimum system. The results indicated that RT PCR can be performed well using total RNA or dsRNA as template, and dsRNA is superior to total RNA.

关 键 词：RT—PCR检测 库尔勒香梨 苹果 茎痘病毒 纯度 反转录 PCR扩增 

分 类 号：S436.612[农业科学—农业昆虫与害虫防治] S436.611[农业科学—植物保护]