套式逆转录聚合酶链反应在黄热病毒快速检测中的应用  

Application of nested ploymerase chain reaction in detection of Yellow Fever virus

作　　者：任瑞文[1] 郝丽[1] 方美玉[1] 程刚锋[1] 洪文艳[1] 刘建伟[1] 田小东[1] 林立辉[1] 

机构地区：[1]广州军区联勤部军事医学研究所微生物研究室,510507

出　　处：《中华检验医学杂志》2005年第4期397-399,共3页Chinese Journal of Laboratory Medicine

基　　金：全军医学科研"十五"计划重大课题资助项目(01Z014);广东省自然科学基金项目(04001618)

摘　　要：目的建立灵敏、特异的套式逆转录聚合酶联反应(RTPCR)快速检测黄热病毒方法。方法参照黄热病毒标准株D17序列设计套式引物,并检索国际基因序列数据库初步验证其特异性,随后对镁离子、dNTP及引物浓度,PCR反应条件进行优化,建立稳定、特异的套式RTPCR快速检测黄热病毒方法,并以同属于黄病毒科的登革病毒1～4型、流行性乙型脑炎病毒为对照,验证其特异性。结果外引物扩增可获得404bp左右的特异性扩增片断,阴性对照毒株未见设计大小的扩增片断,但可见非特异性片断;内引物扩增可获得349bp左右片断,阴性对照无扩增条带。结论套式RTPCR可快速、灵敏、特异的检测黄热病毒。Objective To established quick, special and sensitive method for the detection of Yellow Fever virus.Methods Based on the genomes sequence analysis of Yellow Fever virus, two pair of primers were designed .The specificity of the primers was tested by searching the GenBank DNA sequence database.Then the optimal reaction conditions of the RT-PCR were established.The specificity of the RT-PCR were tested by use the homologous virus, dengue virus type 1~4 and Japanese encephalitis virus.Results A positive segment about 404bp could be seen after the RT-PCR reaction using the outer primers .A positive segment about 348bp could be seen after the RT-PCR reaction using the nested primers.There WAS no positive segments in the RT-PCR productions of virus, dengue virus type 1~4 and Japanese encephalitis virus .Conclusion The method of nested ploymerase chain reaction could be used for the early detection of Yellow Fever virus.

关 键 词：套式逆转录聚合酶链反应 快速检测 黄热病毒 套式RT-PCR 逆转录聚合酶联反应 流行性乙型脑炎病毒 病毒方法 引物扩增 阴性对照 套式引物 序列设计 基因序列 反应条件 登革病毒 黄病毒科 非特异性 片断 标准株 数据库 镁离子 

分 类 号：R446.5[医药卫生—诊断学]