基于RAA-CRISPR/Cas13a的禽流感病毒检测方法的建立  

Detection of avian influenza virus by RAA-CRISPR/Cas13a

作　　者：乐翔云 冯之航 樊彦莉 张强 蔡一村 熊炜 王翔[2] 董庆利[2] 李健 薛俊欣 王艳 LE Xiangyun;FENG Zhihang;FAN Yanli;ZHANG Qiang;CAI Yicun;XIONG Wei;WANG Xiang;DONG Qingli;LI Jian;XUE Junxin;WANG Yan(Technical Center for Animal&Plant and Food Inspection and Quarantine,Shanghai Customs,Shanghai 200135,China;School of Health Science and Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China)

机构地区：[1]上海海关动植物与食品检验检疫技术中心,上海200135 [2]上海理工大学健康科学与工程学院,上海200093

出　　处：《中国兽医学报》2024年第10期2153-2158,2171,共7页Chinese Journal of Veterinary Science

基　　金：上海市“科技创新行动计划”技术标准资助项目(20DZ2200100);上海市“科技创新行动计划”农业科技领域资助项目(22N31900200)。

摘　　要：对NCBI文库公布的120条不同亚型禽流感病毒的M基因序列进行分析,筛选得到一处高保守片段并进行重组酶介导等温核酸扩增(recombinase-aided amplification,RAA)引物和crRNA的设计。待测样品先进行RAA核酸扩增,再将扩增产物转移至有规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/Cas13a检测体系中,通过监测该体系中荧光值的变化情况进行结果判定。使用梯度稀释(10^(6)~10^(0)拷贝/μL)的标准阳性质粒以及其他7种禽病病毒验证方法的灵敏度和特异性,并利用本试验所建方法和国标荧光RT-PCR方法对50份临床样品(21份阳性样品、29份阴性样品)进行检测以评价方法的检测性能。结果显示,本试验建立的检测方法灵敏度为10^(2)拷贝/μL,较普通RAA方法灵敏度提高了2个数量级;能特异性检出AIV,不与NDV、IBV、ILTV、IBDV、ALV、AMPV、AAV-1发生交叉反应;与国标荧光RT-PCR方法相比,该方法检测的特异性、准确性和一致性分别为100%、95.24%和98.00%。结果表明,本试验建立了检测AIV通用型的RAA-CRISPR/Cas13a快速诊断技术,其在37℃条件下60 min内即可完成检测,具有快速、灵敏、特异等优势,为AIV的现场快速检测提供了手段。An innovative on-site real-time avian influenza virus(AIV)detection method was established by integratingrecombinase-aided amplification(RAA)with the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)system.After analyzing 120 sequences of the M gene of avian influenza viruses of different subtypes publicly available on NCBI,the RAA primers and crRNA were designed based on the identified highly conserved segment and used for RAA nucleic acid amplification.After the amplified products were transferred to a CRISPR/Cas13a detection system,the fluorescence values were monitored throughout the reaction process to indicate the results.The sensitivity and specificity of the RAA-CRISPR/Cas13a method were validated using gradient dilutions(10^(6)~10^(0) copies/μL)of positive plasmids and seven other avian viruses.Fifty clinical samples were tested using this method and compared with the national standard fluorescence RT-PCR method.The results indicated that the detection limit for RAA-CRISPR/Cas13a method was 10^(2) copies/μL,a two-fold improvement over the standard RAA.Specificity assay showed the established method only detected AIV with no cross-reactivity with other seven avian viruses.Compared to the national standard fluorescence RT-PCR method,this method exhibited 100%specificity,95.24%accuracy,and 98.00%consistency in detection of clinical samples.In conclusion,a universal and rapid RAA-CRISPR/Cas13afor detection of AIV was established with the capacity of achieving detection within 60minutes at 37℃,which provides a rapid,sensitive,and specific on-site detection method for AIV.

关 键 词：禽流感病毒 RAA CRISPR/Cas13a 

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