基于LAMP-CRISPR-Cas12b-侧层析技术检测A、B型流感病毒的方法建立  

Development of influenza A and B viruses detection method based on LAMP-CRISPR-Cas12b-lateral chromatography

作　　者：黄佳 齐适雨 黄顺武 刘慧[2] 孙颖 武伟华[2] 王昕[2] 邹旋 房师松 Huang jia;Qi Shiyu;Huang Shunwu;Liu Hu;Sun Ying;Wu Weihua;Wang Xin;Zou Xuan;Fang Shisong(School of Public Health,Guangdong Pharmaceutical University,Guangzhou 510006,China;Department of Microbiology Laboratory,Shengzhen Center for Disease Control and Prevention,Shengzhen 518055,China)

机构地区：[1]广东药科大学公共卫生学院,广州510006 [2]深圳市疾病预防控制中心微生物检验科,518055 [3]南华大学公共卫生学院,衡阳421001

出　　处：《国际病毒学杂志》2024年第2期128-133,共6页International Journal of Virology

基　　金：深圳市科技研发资金项目(KCXFZ20211020172545006);深圳市科技研发资金(JCYJ20200109142418595)。

摘　　要：目的为在疫情现场和基层医疗卫生机构实现对A、B型流感病毒的快速检测,而建立一种快速流感病毒诊断方法。方法将等温扩增技术与CRISPR-Cas12b系统联用,结合侧层析技术,建立基于LAMP-CRISPR-Cas12b-侧层析技术检测A、B型流感病毒的方法。设计A、B型流感病毒的特异性LAMP引物组及crRNA。采用梯度稀释法验证该方法的灵敏度;利用其他4种呼吸道病毒验证该方法的特异性;应用该方法对52例ILI监测样本进行检测,验证该方法的实用性。结果成功建立了LAMP-CRISPR-Cas12b-侧层析检测A、B型流感病毒的方法,最低检测极限为10 copies/μL;该方法特异性强,与其他4种呼吸道病毒无交叉反应;52例ILI监测样本结果与qRT-PCR一致,一致性好(kappa=1)。结论建立了一种适用于疫情现场和基层医疗卫生单位对流感病毒快速检测的方法。Objective To develop a diagnostic method for the rapid detection of influenza A and B viruses in epidemiological investigation site and primary healthcare institutions.Methods A method based on LAMP-CRISPR-Cas12b-lateral chromatography was established,which combined isothermal amplification technology with CRISPR-Cas12b system with Lateral Flow Immunoassay technology to detect influenza A and B viruses.Specific LAMP primer sets and crRNAs for influenza A and B viruses were designed.The sensitivity of the method was verified by using the gradient dilution method while the specificity was verified by using four other respiratory viruses.The method was applied for analysis of 52 ILI surveillance samples to verify practicality.Results A method for the detection of influenza A and B viruses by LAMP-CRISPR-Cas12b-side Lateral Flow Immunoassay was successfully established,with a lower detection limit of 10 copies/μL.The method was highly specific and had no cross-reactivity with the other 4 respiratory viruses.The results of 52 ILI surveillance samples were in good agreement and consistency(kappa=1)with qRT-PCR.Conclusions A method suitable for rapid detection of influenza viruses at the epidemiological investigation site and in primary healthcare institute was established.

关 键 词：流感病毒 RT-LAMP CRISPR-Cas 侧层析技术 

分 类 号：R373.1[医药卫生—病原生物学]