H7亚型和N9亚型禽流感病毒RT-LAMP可视化检测方法的建立

doi:10.11843/j.issn.0366-6964.2015.07.014

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (7): 1176-1183.doi: 10.11843/j.issn.0366-6964.2015.07.014

H7亚型和N9亚型禽流感病毒RT-LAMP可视化检测方法的建立

罗思思，谢芝勋^*，谢丽基，邓显文，谢志勤，黄娇玲，曾婷婷

（广西壮族自治区兽医研究所广西畜禽疫苗新技术重点实验室，南宁 530001）

收稿日期:2014-10-28 出版日期:2015-07-23 发布日期:2015-07-20
通讯作者: 谢芝勋，研究员，E-mail: xiezhixun@126.com
作者简介:罗思思（1985-），女，广西平南人，助理研究员，主要从事禽流感病原学和防控技术的研究
基金资助:
广西特聘专家专项 ( 2011B020)；广西科技攻关重大专项 (14121003-4-2；1222003-2-4)

Visual Detection of H7 and N9 Subtypes Avian Influenza Viruses by Reverse Transcription Loop-mediated Isothermal Amplification Assay

LUO Si-si，XIE Zhi-xun^*，XIE Li-ji，DENG Xian-wen，XIE Zhi-qin，HUANG Jiao-ling，ZENG Ting-ting

(Guangxi Key Laboratory of Animal Vaccines and New Technology，Guangxi Veterinary Research Institute，Nanning 530001，China)

Received:2014-10-28 Online:2015-07-23 Published:2015-07-20

摘要/Abstract

摘要：

应用反转录环介导等温扩增技术（RT-LAMP），建立一种H7亚型和N9亚型禽流感病毒（AIV）可视化快速检测方法。根据GenBank中H7亚型和N9亚型AIV HA和NA基因序列，在其保守区域设计筛选出H7亚型和N9亚型AIV的特异性LAMP引物各一套，优化反应条件，建立能检测H7亚型和N9亚型AIV RT-LAMP方法，并进行特异性和敏感性检验。结果显示，该法用实时浊度仪63 ℃反应1 h，能特异性地检测H7亚型和N9亚型AIV，而对其他15个H亚型和8个N亚型的AIV和禽类呼吸道病原体均无扩增，最低能检出10 拷贝•μL^-1目的基因。所建立的H7亚型和N9亚型RT-LAMP方法特异性强、敏感性高，结果可视化，操作简便、快速，为H7、N9和H7N9亚型AIV的有效防控提供技术支撑，具有较好的应用前景。

关键词: H7亚型禽流感病毒, N9亚型禽流感病毒, 反转录环介导等温扩增, 可视化检测

Abstract:

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of H7 and N9 subtype avian influenza viruses (AIV).According to the conservative sequences of hemagglutinin (HA) and neuraminidase (NA) genes of H7 and N9 subtype AIV in GenBank，two sets of primers were designed，then the reaction conditions were optimized.The specificity and sensitivity of the RT-LAMP assay were evaluated.RT-LAMP amplification was completed in 1 h by incubating all of the reagents in a single tube by real-time turbidimeter at 63 ℃.The specific test demonstrated that the RT-LAMP assay was specific for detecting H7 and N9 subtypes of AIV，and no cross reaction with other subtypes of AIV and avian respiratory pathogens.The detection limit of the RT-LAMP assay was 10 copies using vitro transcription RNA of target genes.The newly developed RT-LAMP assay is specific，sensitive，simple，rapid and can identify H7，N9 and H7N9 subtype AIV visually，and it will be a very useful screening assay for prevention and control these diseases.

Key words: H7 subtype avian influenza viruses, N9 subtype avian influenza viruses, reverse transcription loop-mediated isothermal amplification, visual detection

中图分类号:

S852.659.5

罗思思，谢芝勋，谢丽基，邓显文，谢志勤，黄娇玲，曾婷婷. H7亚型和N9亚型禽流感病毒RT-LAMP可视化检测方法的建立[J]. 畜牧兽医学报, 2015, 46(7): 1176-1183.

LUO Si-si，XIE Zhi-xun，XIE Li-ji，DENG Xian-wen，XIE Zhi-qin，HUANG Jiao-ling，ZENG Ting-ting. Visual Detection of H7 and N9 Subtypes Avian Influenza Viruses by Reverse Transcription Loop-mediated Isothermal Amplification Assay[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(7): 1176-1183.

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H7亚型和N9亚型禽流感病毒RT-LAMP可视化检测方法的建立

Visual Detection of H7 and N9 Subtypes Avian Influenza Viruses by Reverse Transcription Loop-mediated Isothermal Amplification Assay

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