禽流感RT-RAA侧向流试纸条可视化检测方法的建立  

Establishment of visual detection method for avian influenza RT-RAA lateral flow dipstick

作　　者：王文静 李馨月 张鹏[1] 刘静茹 姚姗姗 王春光 张铁[1] WANG Wen-jing;LI Xin-yue;ZHANG Peng;LIU Jing-ru;YAO Shan-shan;WANG Chun-guang;ZHANG Tie(College of Veterinary Medicine,Hebei Agricultural University,Baoding,Hebei 07100)

机构地区：[1]河北农业大学动物医学院,河北保定071000

出　　处：《中国兽医学报》2020年第11期2159-2163,共5页Chinese Journal of Veterinary Science

基　　金：河北省农业高质量发展关键共性技术攻关专项资助项目(20326621D);石家庄市科学技术研究与发展计划资助项目(201500412A);家畜疫病病原生物学国家重点实验室开放基金资助项目(BYSWX2020KFKT02)。

摘　　要：禽流感(AI)是威胁我国养禽业健康发展的重要疫病,为建立该病高效、便捷的检测方法进行本项研究。本试验根据禽流感病毒(AIV)保守基因(NP)序列设计引物和探针,利用反转录重组聚合酶介导恒温扩增(RT-RAA)技术对NP基因进行快速扩增并形成双标产物,扩增产物通过侧向流试纸条(LFD)进行快速检测,通过对反应时间、温度和引物浓度等条件的优化,建立了1种能够检测禽流感病毒的RT-RAA-LFD检测方法。结果显示,该方法37℃条件下,反应24 min,即可实现NP目的基因片段的有效扩增,且扩增产物可通过侧向流试纸条肉眼观察;特异性良好,与传染性支气管炎病毒(IBV)、传染性喉气管炎病毒(AILV)、新城疫病毒(NDV)无核酸交叉反应;敏感性达到102 copies/μL,是常规PCR的100倍,临床检测符合率为94.8%。该方法具有特异性强、敏感性高、便捷快速等优点,为田间检测禽流感病毒提供一种可行方法。Avian influenza(AI)is an important epidemic that threatens the healthy development of China’s poultry industry.This study was conducted to establish an efficient and convenient detection method for the disease.In this test,primers and probes were designed based on the avian influenza virus(AIV)conserved gene(NP)sequence.The reverse transcription recombinase Add amplification(RT-RAA)technology was used to rapidly amplify the NP gene and form a double standard.Products,amplified products are quickly detected by a lateral flow dipstick(LFD).By optimizing conditions such as reaction time,temperature,and primer concentration,an RT-RAA-LFD detection method for detecting avian influenza virus was established.The results show that the method can effectively amplify the NP target gene fragment at 37℃for 24 minutes,and the amplified product can be visually observed through a lateral flow test strip;the specificity is good,and infectious bronchitis virus(IBV),infectious laryngotracheitis virus(AILV),and Newcastle disease virus(NDV)have no nucleic acid cross-reactivity;the sensitivity reaches 102copies/μL,which is 100 times more than that of conventional PCR,and the clinical detection compliance rate is 94.8%.This method has the advantages of strong specificity,high sensitivity,convenience and fastness,etc.,and provides a feasible method for field detection of avian influenza virus.

关 键 词：禽流感 重组酶介导等温核酸扩增技术 侧向流试纸条 NP基因 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]