应用高通量测序技术快速鉴定未分型甲型流感病毒  

Identification of unclassified influenza A virus using high-throughput sequencing technology

作　　者：茅海燕[1] 孙逸[1] 楼秀玉[1] 颜浩 程伟[1] 姚文武 王欣莹[1] 潘军航[1] 张严峻[1] Mao Haiyan;Sun Yi;Lou Xiuyu;Yan Hao;Cheng Wei;Yao Wenwu;Wang Xinying;Pan Junhang;Zhang Yanjun Zhejiang(Provincial Center for Disease Control and Prevention,Hangzhou 310051 , China)

机构地区：[1]浙江省疾病预防控制中心微生物检验所,杭州310051

出　　处：《中华实验和临床病毒学杂志》2018年第3期268-271,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：浙江省医药卫生研究计划（2015KYA050）;浙江省公益技术应用社会发展项目（LGF18H190003）;浙江省自然科学基金青年基金项目（LQ18C010002）

摘　　要：目的利用Ion Torrent半导体二代测序技术开展禽类和外环境中禽流感病毒基因组测序和亚型的快速鉴定，建立快速豁定未分型甲型流感病毒亚型的高通量测序厅法。方法提取9份禽类拭子和外环境标本中的病毒RNA，进行甲型流感病毒通用型、H5N1亚型、H7N9亚型和H9N2业型荧光定量PCR检测；利用PathAmpFluA试剂盒对病毒RNA进行全基因组扩增，利用Next Fast DNA Fragmentation＆Library Prep Set low Ion Torrent试剂盒进行义库制备，并存Ion torrent二代测序仪上完成测序；采用Ion Torrent Suitev3．0进行测序数据提取和质量评估，并且用Flu Atyping v4．0和PathogenAnalyzer两个捕件进行流感序列拼接和数据库比对，最终确定9份禽流感标本的流感病毒业型。结果9份禽类拭子和外环境标本甲型流感病毒核酸阳性但常规监测的H5N1、H7N9和H9N2亚型均为阴性，经二代测序和生物信息学分析共鉴定出7种亚型：H2N3、H5N6、H5N8、H7N1、H7N7、H11N3业型流感病毒阳性标本各1个，H6N6亚型流感病毒阳性标本3个。结论浙江省外环境中禽流感病毒亚型众多，Ion torrent半导体测序技术适用于禽及外环境监测巾发现的末分型甲型流感病毒业型的快速鉴定。Objective To identify the avian influenza virus subtype from the avian and environmental samples using the Ion Torrent new-generation semiconductor sequencing technology and to establish a high-throughput sequencing method to identify unclassified influenza A virus. Methods Virus RNA was extracted from the nine avian swab and environmental samples and real-time RT-PCR was carried out to detect universal fluA, H5N1 , H7N9 and H9N2. The whole genome of influenza A virus was amplified by PathAmpFluA kit. Sequencing library was prepared using Next Fast DNA Fragmentation ＆ Library Prep Set for Ion Torrent kit and high-throughput sequencing was clone by Ion Torrent Personal Genome Machine （PGM）. Data from the PGM was processed and quality evaluated using Ion TorrentSuite v3.0 software. Sequence assembly and influenza database blast were carried out by FluAtyping v4.0 and PathogenAnalyzer bioinformatics software to identify the influenza A virus subtype of these nine samples. Results The results of real-time RT-PCR for universal fluA of these nine samples were positive but the results for HSNI , HTN9 and H9N2 were negative. Seven subtypes of influenza A virus were identified by bigh-throughput sequencing and bioinformatics analysis： six samples were H2N3, H5N6, H5N8, H7N1, H7NT, H11N3 subtype respectively and three samples were H6N6 subtype. Conclusions Avian influenza virus has many subtypes in the environment of Zhejiang province. Ion Torrenl semiconductor sequencing technology is suitable tor fast identification of unclassified influenza virus for avian influenza environment monitoring.

关 键 词：甲型流感病毒 亚型 高通量测序 快速答定 

分 类 号：R446.5[医药卫生—诊断学]