不对称PCR制备单链探针检测登革Ⅱ型病毒复制型RNA和复制中间体RNA  

DETECTION OF DENGUE VIRUS REPLICATIVE INTERMEDIATE AND REPLICATIVE FORM RNA WITH SINGLE STRAND DNA HYBRIDIZATION PROBES GENERATED BY ASYMMETRIC PCR

作　　者：郭晓霞[1] 赵彤言[1] 董言德[1] 蒋书楠[2] 陆宝麟 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071 [2]西南农业大学植保学院,重庆400716

出　　处：《寄生虫与医学昆虫学报》2003年第3期157-162,共6页Acta Parasitologica et Medica Entomologica Sinica

基　　金：国家自然科学基金资助项目 (批准号 :39970 6 6 5 )

摘　　要：登革病毒是具包膜的单股正链RNA虫媒病毒 ,病毒的复制过程发生在感染细胞胞浆 ,复制型 (RF)RNA是病毒半保留复制的循环模板 ,复制中间体 (RI)RNA的合成则是病毒复制所必需的。经RT PCR获得的DNA模板进行不对称PCR扩增 ,当限制性引物终浓度为 2 5 0nmol L ,两引物比例为 1 0 0∶1时 ,即得到不对称PCR的预计单链和双链DNA产物。此单链产物用于标记探针进行核酸杂交。Dengue(DEN)virus is an enveloped arbovirus with a single stranded sense RNA genome.The replicative procedure of the virus occurred in cytoplasm of infected cells.Replicative form RNA (RF RNA) can be used as a recycling template for semiconservative replication;the synthesis of the replicative intermediate RNA(RI RNA) is indispensable for the virus replication.The asymmetric PCR was employed to amplify a DNA template which was got by reverse transcription and polymerase chain reaction(RT PCR).When the final concentration of the restricted primer was 250nmol and the ration of the two primers was on 1∶100.the expected products of single and double stranded DNAs could be obtained.The single stranded DNA can be used for labeling the probes in nucleic acid hybridization.The results revealed that this method is useful for the detection of RI RNA and RF RNA.

关 键 词：不对称PCR 单链探针 检测 登革Ⅱ型病毒 复制型RNA 复制中间体RNA 

分 类 号：R373[医药卫生—病原生物学]