甲型流感病毒快速基因分型POCT方法的建立  

Establishment of POCT method for rapid genotyping of influenza A virus

作　　者：王大刚 王雅杰 潘美晨 焦炳欣[1] 郭杰[1] 王爽 郭晶晶[1] 徐飞[1] 吴吟妮 WANG Da-gang;WANG Ya-jie;PAN Mei-chen;JIAO Bing-xin;GUO Jie;WANG Shuang;GUO Jing-jing;XU Fei;WU Yin-ni(Department of Clinical Laboratory,Beijing Ditan Hospital,Capital Medical University,100015,China)

机构地区：[1]首都医科大学附属北京地坛医院检验科,100015 [2]北京英诺特生物技术有限公司,100070

出　　处：《传染病信息》2019年第4期307-311,共5页Infectious Disease Information

摘　　要：目的建立一种无需核酸提取,直接检测鼻咽拭子中甲型流感病毒H1HA pdm09、H3HA亚型和季节性流感病毒H1HA non-pdm09的POCT快速分型方法。方法针对3种流感病毒的血凝素(hemagglutinin,HA)基因序列保守区设计高度特异性的引物和HyBeacon探针,同时以人类RNaseP基因作为内参。优化多重PCR反应条件,建立用熔解曲线分析甲型流感病毒基因分型的方法,并将其应用到ParaDNA核酸POCT检测仪,对50例甲型流感病毒抗原初筛阳性且经过实时荧光定量PCR检测的临床鼻咽拭子标本进行检测。结果该方法可在1.5 h内完成对3种甲型流感病毒亚型HA基因的特异性扩增和分型,与其他7种呼吸道病原微生物无交叉反应,其核酸检测下限为50 copies/μl。对临床50例甲型流感病毒抗原阳性的鼻咽拭子标本进行直接检测,检出H1HA pdm09阳性标本48例,H3HA阳性标本2例,未检出季节性流感H1HA nonpdm09阳性标本。结论基于HyBeacon探针和ParaDNA核酸POCT检测仪所建立的甲型流感病毒快速分型方法能同时检测H1HA pdm09、H3HA和季节性流感病毒H1HA non-pdm09。该方法无需核酸提取,操作简便,检测时间短,适于对甲型流感病毒抗原初筛阳性的鼻咽拭子开展现场基因分型检测,可为流感的诊治和防控提供及时快速的实验室依据。Objective To establish a rapid POCT genotyping method for direct detection of influenza A virus H1HA pdm09,H3HA subtypes and seasonal influenza virus H1HA non-pdm09 in nasopharyngeal swabs without nucleic acid extraction.Methods Highly specific primers and HyBeacon probes were designed for the conserved region of hemagglutinin(HA)gene sequence of the 3 influenza viruses.Human RNaseP gene was used as internal reference.The multiplex PCR formulation was optimized and a melting curve analysis method for influenza A virus genotypes was established.Then the multiplex PCR assay was applied to ParaDNA nucleic acid POCT instrument to detect 50 clinical nasopharyngeal swabs with influenza A virus antigen positive by primary screening.All of these specimens were detected by real-time fluorescence quantitative PCR.Results The specific amplification and typing of HA gene of the 3 influenza A viruses could be completed within 1.5 h by this method.There were no cross-reactions with other 7 respiratory pathogenic microbes.The lower limit of nucleic acid detection was 50 copies/μl.A total of 50 clinical nasopharyngeal swabs with influenza A virus antigen positive results were directly detected.There were 48 samples positive for H1HA pdm09,2 samples positive for H3HA and 0 sample positive for seasonal influenza H1HA non-pdm09.Conclusions The rapid genotyping method for influenza A virus based on HyBeacon probe and ParaDNA nucleic acid POCT instrument can simultaneously discriminate H1HA pdm09,H3HA viruses and seasonal influenza virus H1HA non-pdm09.This method doesn’t require nucleic acid extraction,has convenient operations and short detection period,so it is suitable for on-site genotyping of nasopharyngeal swabs with influenza A antigen positive by primary screening,which is expected to offer timely and rapid laboratory basis of diagnosis,treatment,prevention and control for influenza A.

关 键 词：甲型流感 HyBeacon探针 熔解曲线 ParaDNA 基因分型 

分 类 号：R511.7[医药卫生—内科学]