基于RT-RAA的禽流感H5亚型核酸CRISPR-Cas13a检测方法的建立  

Establishment of Nucleic Acid Detection Methods for Avian Influenza H5 Subtype Based on CRISPR-Cas13a and RT-RAA

作　　者：杨芷翊 王新凯 史玉婷 付思源 张钰炘 曹琛福 贾伟新[1] YANG Zhiyi;WANG Xinkai;SHI Yuting;FU Siyuan;ZHANG Yuxin;CAO Chenfu;JIA Weixin(National Avian Influenza Para-Reference Laboratory(Guangzhou),Guangdong Engineering Laboratory for Medicament of Zoonosis Prevention and Control,National Local Joint Engineering Laboratory of Zoonosis Prevention and Control Agents,Key Laboratory of Zoonoses of Ministry of Agriculture and Rural Affairs,Guangdong Key Laboratory for Prevention and Control of Zoonotic Diseases,College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center,Shenzhen 518045,China)

机构地区：[1]华南农业大学兽医学院国家禽流感专业实验室(广州)/广东省人兽共患病防控制剂工程实验室/人兽共患病防控制剂国家地方联合工程实验室/农业农村部人畜共患病重点实验室/广东省动物源性人兽共患病防控重点实验室,广州510642 [2]深圳海关动植物检验检疫技术中心,深圳518045

出　　处：《畜牧兽医学报》2023年第9期3803-3811,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：广东省科技计划项目(2021B1212030015);国家现代农业产业技术体系(CARS-41)。

摘　　要：采用逆转录-重组酶介导等温核酸扩增技术(RT-RAA)和成簇的规律间隔的短回文重复序列(CRISPR)检测系统,建立一种快速、高灵敏度和高特异性的禽流感病毒(AIV)H5亚型核酸检测新方法。通过分析342条禽流感病毒H5亚型的HA基因序列,选取保守性区域设计具有强特异性的CRISPR RNA(crRNA)序列以及RT-RAA引物,并对检测体系中的主要成分LwaCas13a蛋白、crRNA、TaqMan探针、T7 RNA Polymerase、NTP Buffer Mix含量进行优化,建立了基于RT-RAA的AIV H5亚型核酸CRISPR-Cas13a检测方法。取105~1 copies·μL^(-1)含有目的基因的质粒标准品和其他亚型禽流感病毒以及其他禽病病毒分别验证和评价该方法的灵敏性和特异性。结果表明,本文设计的RT-RAA引物与crRNA特异且灵敏,其最佳反应体系中主要成分用量分别为LwaCas13a蛋白(60μg·mL^(-1))2.0μL、crRNA(100μmol·L^(-1))3.2μL、TaqMan探针(50μmol·L^(-1))1.28μL、T7 RNA Polymerase 0.25μL、NTP Buffer Mix 2.0μL。该方法检测灵敏度可达1 copy·μL^(-1),且与AIV H3、H6、H7、H9、H10亚型以及NDV、IBV、IBDV、DTMUV无交叉反应。对56份已知的临床样品进行检测,检测结果与荧光定量RT-PCR符合率为98.2%。综上,本研究通过RT-RAA等温扩增技术结合CRISPR-Cas13a基因编辑技术建立了针对AIV H5亚型的快速检测平台,该方法从样本的核酸抽提到获得检测结果的时间控制在1 h 20 min以内,为AIV H5亚型高灵敏度和高特异性的临床快速检测提供了新的技术手段,具有良好的应用前景。A new rapid,highly sensitive and highly specific nucleic acid detection method for avian influenza virus(AIV)H5 subtype was established by using reverse transcription-recombinase aided amplification(RT-RAA)and clustered regularly interspaced short palindromic repeats(CRISPR).By analyzing the HA gene sequences of 342 avian influenza virus H5 subtype genes,the conserved regions were selected to design highly specific CRISPR RNA(crRNA)sequences and RT-RAA primers.The contents of main components in the detection system were optimized,including LwaCas13a protein,crRNA,TaqMan probe,T7 RNA Polymerase and NTP Buffer Mix.Thus,the RT-RAA based detection method of AIV H5 subtype nucleic acid CRISPR-Cas13a was established.The sensitivity and specificity of the method were evaluated by taking 105~1 copies·μL^(-1)of standard plasmid containing the target gene and other subtypes of avian influenza viruses and other avian disease viruses.The results showed that the RT-RAA primer and crRNA designed in this paper were specific and sensitive.The main components in the optimal reaction system were LwaCas13a protein(60μg·mL^(-1))2.0μL,crRNA(100μmol·L^(-1))3.2μL,TaqMan probe(50μmol·L^(-1))1.28μL,T7 RNA Polymerase 0.25μL and NTP Buffer Mix 2.0μL,respectively.The detection sensitivity of this method was up to 1 copy·μL^(-1),and there was no cross reaction with AIV H3,H6,H7,H9,H10 subtypes,neither with NDV,IBV,IBDV or DTMUV.The method established in this study and fluorescence quantitative RT-PCR were used to detect 56 clinical samples,and the coincidence rate of the two methods was 98.2%.In conclusion,a rapid detection method for AIV H5 subtype was established by using RT-RAA isothermal amplification technology combined with CRISPR-Cas13a gene editing technology in this study.The results were obtained within 1 h and 20 min.It provides a new technique for the rapid clinical detection of AIV H5 subtype with high sensitivity and high specificity,and has a good application prospect.

关 键 词：H5亚型禽流感病毒 CRISPR 等温扩增 核酸检测 

分 类 号：S852.65[农业科学—基础兽医学]