应用DPO引物技术同时检测5种蚊媒病毒的多重RT-PCR方法

    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

    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

    摘要：目的 应用双启动寡核苷酸引物（Dual PrimingOligonucleotide，DPO）技术建立同时检测日本脑炎病毒（Japanese Encephalitis Virus,JEV）、黄热病毒（Yellow Fever Virus, YFV）、西尼罗病毒（West Nile Virus,WNV）、圣路易斯脑炎病毒（St. Louis encephalitis virus, SLEV）和登革病毒(Denguevirus，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、377bp、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 × 104 copies/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。

   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

    中图分类号：R183.5 文献标识码：B

    Multiplex RT-PCR method for detecting fivekinds of mosquito-borne

    viruses with DPO system

    XU Huan-zhou*, PING Rui-jin, JI Ru-wu，WANGLin, YANG Peng-fei, ZHANG Li-ping,YUE Qi-an,HU Kong-xin

    *Weifang Medical University, Weifang,Shandong 261053, China

    Abstract:   Objective  To establish a multiplex RT-PCR method for detection of JapaneseEncephalitis Virus(JEV),Yellow Fever Virus(YFV）,West NileVirus(WNV）,St. Louis encephalitis virus(SLEV）and Dengue virus(DV)using a dual priming oligonucleotide system.   Methods   Five pairs of specific DPO primers weredesigned with the Primer Premier 5.0 on the basis of the consensussequences of five viruses and norms of DPO primer designing. Theoptimal reaction conditions of Mg2+ concentration and annealingtemperature were optimized. The specificity and sensitivity of theRT-PCR reaction were checked.   Results   Theestablished multiplex RT-PCR method could amplify 142 bp、293 bp、377bp、630 bp and 521 bp of JEV、YFV、WNV、SLEV and DV with  thesensitivity of  5 000 PPU/ml、4 500 PPU/ml、2.3×105copies/μl、2.6×104 copies/μl and 1.37×104 copies/μl, respectively,and there was non-specific reactivity found in the mosquitosimulation experiment.  Conclusion   The establishedmultiplex RT-PCR reaction using DPO system has good specificity andsensitivity. It could differential diagnosis of JEV, YFV, WNV, SLEVand DV.

    Key words:   Dual primingoligonucleotide (DPO) primers; Multiplex RT-PCR；Japaneseencephalitis virus；Yellow fever virus；West Nile virus；St. Louisencephalitis virus；Dengue virus

    《中国国境卫生检疫杂志》