登革2型病毒全长基因组的长链RT-PCR法扩增  

Amplification of complete genome of dengue 2 virus by long RT-PCR

作　　者：贡树基[1] 赵卫[1] 曹虹[1] 张文炳[1] 周浩[1] 陈丽丹[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院微生物学系,广州510515

出　　处：《中国公共卫生》2006年第4期429-430,共2页Chinese Journal of Public Health

基　　金：广州市科技攻关计划重点项目(2004Z2-E0214)

摘　　要：目的采用长链RT-PCR技术扩增登革2型病毒新几内亚株(NGC)全长基因组。方法根据登革2型病毒NGC株基因组全序列设计引物,在上游引物中加入Sp6 RNA聚合酶启动子核心序列,下游引物中加入ClaⅠ内切酶位点。从病毒感染的乳鼠脑中提取RNA,采用长链RT-PCR技术进行扩增。以获得的PCR产物为模板分别扩增3个NGC株特异的基因片段。结果长链RT-PCR法扩增出约11 kb基因片段,经PCR法证实为登革2型病毒NGC株特异序列。结论通过长链RT-PCR技术,获得了登革2型病毒NGC株全长基因组,为进一步构建感染性克隆打下基础。Objective To establish the long reverse transcription PCR for amplification of the complete genome of dengue 2 virus. Methods The primers were designed according to the published nucleotide sequence of DEN2 NC,-C strain. Sp6 was added to 5＇ terminal and restriction endonuclease site for Cla Ⅰwas added to 3＇ terminal. After virus RNA was extracted from the brains of the infected new - born mice, the complete genome of DEN2 NGC strains was amplified by the long RT- PCR. Three fragments of specific lengths were re- amplified from PCR products for identification. Results The llkb full - length genome of dengue 2 virus was amplified successfully by the long RT - PCR. Conclusion The complete genome of dengue 2 virus was successful to be amplified using the long RT - PCR, and it will be advantageous for further constructing infectious clone.

关 键 词：登革2型病毒 全长CDNA 长链RT—PCR 

分 类 号：R373.33[医药卫生—病原生物学]