一步法MGB荧光定量RT-PCR检测埃博拉病毒扎伊尔亚型和苏丹亚型方法的建立  

Establishment of one step MGB real-time RT-PCR for detection of Ebola virus Zaire and Sudan subtypes

作　　者：刘阳[1,2] 史子学[1] 王水明[3] 刘学辉[3] 马玉堃[2] 马志永[1] 

机构地区：[1]中国农业科学院上海兽医研究所兽医公共卫生研究室,上海200241 [2]黑龙江大学生命科学学院,哈尔滨150080 [3]南京出入境检验检疫局,南京211136

出　　处：《中国人兽共患病学报》2012年第4期343-346,350,共5页Chinese Journal of Zoonoses

基　　金：江苏出入境检验检疫局科研项目(2011KJ03)

摘　　要：目的为了建立一种快速准确的检测埃博拉病毒(EBOV)亚型的方法。方法本研究根据GenBank中公布的EBOV NP基因序列,设计引物和MGB探针,通过优化反应条件,建立了一种检测EBOV扎伊尔亚型和苏丹亚型的一步法MGB荧光定量RT-PCR方法。结果以体外转录的EBOV RNA为模板进行试验,该方法检测的灵敏度可以达到1.0×103个拷贝/μL,检测范围达到8个数量级为103～1010。建立的方法对马尔堡病毒(MARV)、登革病毒(DENV)、新疆出血热病毒(XHFV)、乙型脑炎病毒(JEV)、流感病毒(H1N1和H3N2)和猪繁殖和呼吸综合征病毒(PRRSV)E基因组RNA无非特异性扩增。结论本文将荧光定量RT-PCR技术用于埃博拉病毒的定量检测中,并且建立了EBOV扎伊尔亚型和苏丹亚型荧光定量RT-PCR检测方法,具有一定的创新性。该方法的建立对加快我国EBOV诊断技术的研究具有重要推动作用。In order to establish a rapid and accurate method for detection of Ebola virus（EBOV） subtypes,the primers and MGB probe used in MGB real-time RT-PCR were designed according to the EBOV NP gene sequence published in GenBank.One step MGB real-time RT-PCR that detects Zaire and Sudan subtypes of EBOV was established and optimized.The EBOV RNA that was transcribed in vitro was used as template.The sensitivity of this method was found to reach 1.0×103 copies/μL and the detection range was 103-1010.This method did not react with RNA samples from Marburg virus（MARV）,Dengue virus（DENV）,Xinjiang hemorrhagic fever virus（XHFV）,Japanese encephalitis virus（JEV）,Influenza virus（H1N1 and H3N2） and Porcine reproductive and respiratory syndrome virus E genomic RNA.It has some innovation that the study quantitatively detected EBOV with real-time RT-PCR and established One step MGB real-time RT-PCR for detection of Ebola virus Zaire and Sudan subtypes.The method would be useful for detection and inspection of EBOV in China.

关 键 词：埃博拉病毒 荧光定量RT-PCR MGB探针 检测 

分 类 号：S855.3[农业科学—临床兽医学]