3种甜樱桃病毒PNRSV、PDV及LChV-2的多重RT-PCR检测方法的建立与应用  

Development and Application of a Multiplex RT-PCR Assay for Detecting Three Sweet Cherry Virus Species

作　　者：宗晓娟[1] 王文文[1] 魏海蓉[1] 王甲威[1] 陈新[1] 徐丽[1] 刘庆忠[1] 

机构地区：[1]山东省果树研究所/山东省果树生物技术育种重点实验室,山东泰安271000

出　　处：《中国农业科学》2014年第6期1111-1118,共8页Scientia Agricultura Sinica

基　　金：农业部公益性行业(农业)科研专项(200903019;201203075);山东省现代农业产业技术体系水果创新团队专项基金

摘　　要：【目的】建立可同时检测李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)、李矮缩病毒(Prune dwarf virus,PDV)、樱桃小果病毒2(Little cherry virus-2,LChV-2)的多重RT-PCR检测方法。【方法】以复合感染3种病毒的甜樱桃病株叶片为材料,采用CTAB法提取样本总RNA,选用随机六聚体引物对植物样本总RNA进行反转录,所得cDNA作为多重RT-PCR的扩增模板。根据GenBank中PNRSV、PDV、LChV-2基因组序列共设计6对特异引物,分别通过单一RT-PCR和多重RT-PCR筛选出可用于同时检测3种甜樱桃病毒的引物组合。对多重RT-PCR的退火温度及循环数进行优化,以筛选出各引物组合的最适扩增条件。分别以单一感染PNRSV、PDV、LChV-2、复合感染3种病毒、单一感染樱桃病毒A(Cherry virus A,CVA)及甜樱桃无毒苗为样本,对多重RT-PCR引物的特异性进行分析。选取复合感染3种病毒的甜樱桃总RNA的反转录产物为初始模板,按照梯度稀释法依次将模板稀释为2、22、23、24、25倍,在相同PCR反应体系及反应条件下分别对各引物组合的灵敏度进行分析。多重RT-PCR的扩增条带经凝胶回收试剂盒回收纯化后连接至pMD18-T vector,克隆测序,以验证多重RT-PCR检测的准确性。并应用该方法对山东泰安地区甜樱桃生产园中间隔栽培的中国樱桃进行检测。【结果】筛选到2个可以应用的引物组合,组合1"PNRSV-S1/A1、PDV-S2/A2、LChV2-S1/A1"可分别特异性地扩增733、467、337 bp的片段。组合2"PNRSV-S1/A1、PDV-S3/A3、LChV2-S1/A1"可分别扩增得到733、265、337 bp的片段。扩增产物大小与预期相符。多重RT-PCR反应条件优化结果显示,在退火温度52℃、35个循环条件下,2个引物组合的检测效果均较为理想。特异性分析结果显示,2个引物组合均能特异性检测其各自的靶病毒。灵敏度分析结果显示,2个引物组合在cDNA的23×稀释液中仍能特异性扩增,但扩增条带的强度稍有差异,�[Objective] The objective of this study is to develop a multiplex RT-PCR protocol to detect 3 sweet cherry virusspecies Prunus necrotic ringspot virus （PNRSV）, Prune dwarf virus （PDV） and Little cherry virus-2 （LChV-2） simultaneously. [ Method] The leaves of the sweet cherry （Prunus avium Lindl.） infected by 3 virus species were selected as the experimental materials. Total RNA was extracted using CTAB extraction buffer. For cDNA synthesis, reverse transcription was carried out using random hexamer primer. Six pairs of primers were designed according to the genome sequences of PNRSV, PDV and LChV-2 which were published in GenBank. Single RT-PCR and multiplex RT-PCR were carried out, respectively, to select the primer groups which could be used in the multiplex virus detection. Annealing temperature and the number of the PCR cycles were evaluated to optimize the multiplex RT-PCR conditions. The specificity of the primers was analyzed by using the single-virus infected samples, complex-virus infected samples and virus-free leaf samples. To analyze the sensitivity of the multiples RT-PCR, the transcript which was prepared from the complex-virus infected samples was diluted to 2-fold series. All of the reactions were carried out in the same reaction buffer and under the same conditions. To confirm the accuracy of the multiplex RT-PCR, each amplified fragment was purified by DNA gel extraction kit, cloned into pMD18-T vector and sequenced. In this paper, the multiplex RT-PCR was also used to detect virus infection in Chinese cherry （P. pseudocerasus LindL） trees which are cultivated in the orchards in Taian, Shandong Province. [Result] Two primer groups were selected and could be used in the multiplex RT-PCR detection. Group 1 including primers ＇PNRSV-S1/A1, PDV-S2/A2 and LChV2-S1/AI＇ amplified the fragments of 733, 467 and 337 bp, respectively. Group 2 including ＇PNRSV-S1/A1, PDV-S3/A3, LChV2-S1/AI＇ amplified the fragments of 733, 265 and 337 bp, respectively. The PCR products were consis

关 键 词：李属坏死环斑病毒 李矮缩病毒 樱桃 果病毒-2 多重RT-PCR 病原检测 

分 类 号：S436.629[农业科学—农业昆虫与害虫防治]