应用DPO引物技术同时检测5种蚊媒病毒的多重RT-PCR方法  

Multiplex RT-PCR method for detecting five kinds of mosquito-borne viruses with DPO system

作　　者：徐焕洲[1,2] 平芮巾[2] 季汝武[3] 王琳[2] 杨鹏飞[2] 张丽萍[2] 岳启安[1] 胡孔新[2] 

机构地区：[1]潍坊医学院,山东潍坊261053 [2]中国检验检疫科学研究院 [3]安徽出入境检验检疫局

出　　处：《中国国境卫生检疫杂志》2012年第2期73-77,共5页Chinese Journal of Frontier Health and Quarantine

基　　金：国家科技重大专项课题(2011ZX10004-001);国家质检总局科研基金项目(2008IK210)

摘　　要：目的应用双启动寡核苷酸引物(Dual Priming Oligonucleotide,DPO)技术建立同时检测日本脑炎病毒(JapaneseEncephalitisVirus,JEV)、黄热病毒(YellowFeverVirus,YFV)、西尼罗病毒(WestNileVirus,WNV)、圣路易斯脑炎病毒(St.Louis encephalitis virus,SLEV)和登革病毒(Dengue virus,DV)5种蚊媒病毒的多重RT-PCR方法。方法利用Primer Premier5.0软件设计5种病毒的特异性DPO引物,对引物浓度、Mg2+浓度和退火温度进行优化,测定多重RT-PCR反应的特异性和敏感性。结果应用DPO引物技术建立的多重RT-PCR方法可特异性扩增JEV、YFV、WNV、SLEV和DV的142 bp、293 bp、377 bp、630 bp和521 bp基因片段,灵敏度分别为5 000 PFU/ml、4 500 PFU/ml、2.3×105copies/μl、2.6×104copies/μl和1.37×104copies/μl,蚊虫模拟添加实验未发生非特异反应。结论应用DPO引物技术建立的针对5种蚊媒病毒的多重RT-PCR检测方法有良好的敏感性和特异性,可以同时对JEV、YFV、WNV、SLEV和DV进行检测。Objective To establish a multiplex RT-PCR method for detection of Japanese Encephalitis Virus（JEV）,Yellow Fever Virus（YFV）,West Nile Virus（WNV）,St.Louis encephalitis virus（SLEV）and Dengue virus（DV） using a dual priming oligonucleotide system.Methods Five pairs of specific DPO primers were designed with the Primer Premier 5.0 on the basis of the consensus sequences of five viruses and norms of DPO primer designing.The optimal reaction conditions of Mg2 ＋ concentration and annealing temperature were optimized.The specificity and sensitivity of the RT-PCR reaction were checked.Results The established multiplex RT-PCR method could amplify 142bp、293bp、377 bp、630 bp and 521 bp of JEV、YFV、WNV、SLEV and DV with the sensitivity of 5 000 PPU/ml、4 500 PPU/ml、2.3×105 copies/μl、2.6×104 copies/μl and 1.37×104 copies/μl,respectively,and there was non-specific reactivity found in the mosquito simulation experiment.Conclusion The established multiplex RT-PCR reaction using DPO system has good specificity and sensitivity.It could differential diagnosis of JEV,YFV,WNV,SLEV and DV.

关 键 词：DPO引物 多重RT-PCR 日本脑炎病毒 黄热病毒 西尼罗病毒 圣路易斯脑炎病毒 登革病毒 

分 类 号：R183.5[医药卫生—流行病学]