|本期目录/Table of Contents|

[1]王大刚,王雅杰,潘美晨,等.甲型流感病毒快速基因分型POCT 方法的建立[J].传染病信息,2019,04:307-311.
 WANG Da-gang,WANG Ya-jie*,PAN Mei-chen,et al.Establishment of POCT method for rapid genotyping of influenza A virus[J].Infectious Disease Information,2019,04:307-311.
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甲型流感病毒快速基因分型POCT 方法的建立(PDF)

《传染病信息》[ISSN:1007-8134/CN:11-3886/R]

期数:
2019年04期
页码:
307-311
栏目:
论著
出版日期:
2019-09-12

文章信息/Info

Title:
Establishment of POCT method for rapid genotyping of influenza A virus
文章编号:
 1007-8134(2019)04-0307-05
作者:
王大刚王雅杰潘美晨焦炳欣郭 杰王 爽郭晶晶徐 飞吴吟妮
100015,首都医科大学附属北京地坛医院检验科(王大刚、王雅杰、潘美晨、焦炳欣、郭杰、王爽、郭晶晶、徐飞);100070,北京英诺特生物技术有限公司(吴吟妮)
Author(s):
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
*Corresponding author, E-mail: wangyajie@ccmu.edu.cn
关键词:
甲型流感HyBeacon 探针熔解曲线ParaDNA基因分型
Keywords:
 influenza A HyBeacon probe melting curve ParaDNA genotype
分类号:
R511.7
DOI:
10.3969/j.issn.1007-8134.2019.04.005
文献标识码:
A
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。 
Abstract:
 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.    

参考文献/References


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备注/Memo

备注/Memo:

[ 通信作者] 王雅杰,E-mail: wangyajie@ccmu.edu.cn
更新日期/Last Update: 2019-09-15