甲型肝炎病毒、帕斯拉属和罗卡属戊型肝炎病毒三重荧光定量RT-PCR检测方法的建立和应用  

Establishment and application of triple qRT-PCR for detection of hepatitis A virus,genus Paslahepevirus and genus Rocahepevirus

作　　者：毕敬琛 尹文娇[2] 杨磊[1,3] Bi Jingchen;Yin Wenjiao;Yang Lei(School of Public Health,Hebei Medical University,Shijiazhuang 050017,China;NHC Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Hebei Key Laboratory of Environment and Human Health,Shijiazhuang 050017,China)

机构地区：[1]河北医科大学公共卫生学院,石家庄050017 [2]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委医学病毒和病毒病重点实验室,北京102206 [3]河北省环境与人群健康重点实验室,石家庄050017

出　　处：《中华实验和临床病毒学杂志》2025年第1期115-121,共7页Chinese Journal of Experimental and Clinical Virology

基　　金：国家科技重大专项(2018ZX10201002-009-002)。

摘　　要：目的为弥补目前临床检验方法对肝炎患者感染来自啮齿动物的罗卡属(genus Rocahepevirus)戊型肝炎病毒(HEV-C)的漏检情况,以及与甲型肝炎病毒(HAV)感染进行快速鉴别,本研究建立一种可同时检测HAV、帕斯拉属(genus Paslahepevirus)戊型肝炎病毒(HEV-A)和HEV-C的三重实时荧光定量RT-PCR(qRT-PCR)检测方法。方法选取3对扩增引物和3条TaqMan探针,对反应体系及扩增温度进行优化,构建三重qRT-PCR方法;利用质粒建立标准曲线,并评价该方法的灵敏度;利用其它标本评价特异度;利用HAV、HEV-A和HEV-C的核酸进行稳定性评价;对疑似甲型或戊型肝炎病例标本进行检测,并与HAV(或HEV)IgM抗体和巢式RT-PCR(nRT-PCR)检测结果比较。结果三重qRT-PCR的三条标准曲线的相关系数R 2均大于0.99,斜率均接近-3.3;对HAV、HEV-A和HEV-C质粒的最低检出限分别为8拷贝/μl、5拷贝/μl和8拷贝/μl;与乙型肝炎病毒、丙型肝炎病毒等无交叉反应;批内和批间试验变异系数均小于5%。HAV IgM抗体检测法、HAV nRT-PCR和三重qRT-PCR对疑似甲型肝炎病例血清的检测阳性率分别为95.1%(58/61)、83.6%(51/61)和83.6%(51/61),其中HAV IgM抗体检测法与HAV nRT-PCR(或三重qRT-PCR)的检测一致率为85.2%(52/61),差异具有统计学意义(χ^(2)=4.00,P=0.039)。HEV IgM抗体检测法、HEV nRT-PCR和三重qRT-PCR对疑似戊型肝炎病例血清的检测阳性率分别为89.7%(61/68)、69.1%(47/68)和72.1%(49/68),其中HEV IgM抗体检测法与三重qRT-PCR的检测一致率为79.4%(54/68),差异具有统计学意义(χ^(2)=8.64,P=0.002);HEV nRT-PCR和三重qRT-PCR对疑似戊型肝炎病例粪便的检测阳性率分别为80.0%(20/25)、76.0%(19/25),检测一致率为96%(24/25),差异无统计学意义(χ^(2)=0,P>0.05)。结论本研究建立了一种可同时快速检测甲型肝炎病毒、帕斯拉属或罗卡属戊型肝炎病毒的三重实时荧光定量RT-PCR检测方法,其灵敏度、特异度和稳定性高,适用于新�ObjectiveIn order to make up for the missed detection of the genus Rocahepevirus(HEV-C)infection in the current clinical test method and to quickly differentiate the infection from the hepatitis A virus(HAV)infection,a triple real-time fluorescence quantitative RT-PCR(qRT-PCR)detection method was established to simultaneously detect HAV,genus Paslahepevirus(HEV-A),and HEV-C.MethodsThree pairs of amplification primers and three TaqMan probes were selected to optimize the reaction system and amplification temperature to construct a triple qRT-PCR method;standard curves were established by plasmid and the sensitivity of the method was evaluated;specificity was evaluated by other viruses;stability was evaluated by nucleic acid of HAV,HEV-A and HEV-C;samples from suspected hepatitis A or E cases were detected and compared with HAV(or HEV)IgM antibody and nested RT-PCR(nRT-PCR)detection result.ResultsThe correlation coefficient R 2 of three standard curves of triple qRT-PCR were all greater than 0.99,and the slopes were close to-3.3.The minimum detection limits of HAV,HEV-A and HEV-C plasmids were 8 copies/μl,5 copies/μl and 8 copies/μl,respectively.There was no cross reaction with hepatitis B virus and hepatitis C virus,etc.The coefficients of variation of intra-and inter-batch tests were less than 5%.The positive rates of HAV IgM antibody test,HAV nRT-PCR and triple qRT-PCR were 95.1%(58/61),83.6%(51/61)and 83.6%(51/61)for serum from suspected hepatitis A cases,respectively.The coincidence rate of HAV IgM antibody test and HAV nRT-PCR(or triple qRT-PCR)was 85.2%(52/61),the difference was statistically significant(χ^(2)=4.00,P=0.039).The positive rates of HEV IgM antibody test,HEV nRT-PCR and triple qRT-PCR were 89.7%(61/68),69.1%(47/68)and 72.1%(49/68)for serum from suspected hepatitis E cases,respectively.The coincidence rate of HEV IgM antibody test and triple qRT-PCR was 79.4%(54/68),difference was statistically significant(χ^(2)=8.64,P=0.002).The positive rates of HEV nRT-PCR and triple qRT-PCR for stool sam

关 键 词：甲型肝炎病毒 帕斯拉属戊型肝炎病毒 罗卡属戊型肝炎病毒 三重实时荧光定量RT-PCR 

分 类 号：R51[医药卫生—内科学]