MIRA-CRISPR/Cas13a结合磁珠量子点传感器快速检测甲型流感病毒方法的建立  

Establishment of a method for rapid detection of influenza A virus with multienzyme isothermal rapid amplification-CRISPR/Cas13a in combination with magnetic bead-quantum dot sensors

作　　者：杨宁 马君妍 赵康辰[1] 吴涛[1] 朱小娟[1] 乔乔 王烁 虎歌 吴斌[1] 崔仑标[1] 葛以跃[1] YANG Ning;MA Jun-yan;ZHAO Kang-chen;WU Tao;ZHU Xiao-juan;QIAO Qiao;Wang Shuo;HU Ge;WU Bin;CUI Lun-biao;GE Yi-yue(National Health Commission Key Laboratory of Enteric Pathogenic Microbiology,Jiangsu Provincial Medical Key Laboratory of Pathogenic Microbiology in Emerging Major Infectious Diseases,Jiangsu Provincial Center for Disease Control and Prevention,Jiangsu Nanjing 210009,China;不详)

机构地区：[1]国家卫生健康委员会肠道病原微生物重点实验室、江苏省新发突发重大传染病病原微生物重点实验室、江苏省疾病预防控制中心,江苏南京210009 [2]南京医科大学公共卫生学院

出　　处：《江苏预防医学》2024年第4期403-407,共5页Jiangsu Journal of Preventive Medicine

基　　金：国家重点研发计划(2023YFC2605100,2023YFC2605104);江苏省自然科学基金(BK20231374,BK20221413,BK20211373);江苏省重点研发计划(BE2022804)。

摘　　要：目的将多酶恒温快速扩增(MIRA)、成簇的规则间隔短回文重复序列及其相关蛋白Cas13a(CRISPR/Cas13a)与研发的磁珠量子点(MBQD)传感器相结合,建立一种甲型流感病毒(IAV)快速检测方法。方法针对IAV的M基因设计并筛选MIRA扩增引物、CRISPR RNA(crRNA)、荧光报告探针及连接探针,对反应条件进行优化,建立MIRA-CRISPR/Cas13a-MBQD检测体系;评价该体系的检测灵敏度与特异性;比较建立的方法与实时荧光RT-PCR(RT-qPCR)法的一致性。结果建立的检测体系可以在60 min内完成检测,对IAV的检测灵敏度达1 copy RNA分子/反应;特异性试验表明其与新型冠状病毒(SARS-CoV-2)、乙型流感病毒(IBV)、呼吸道合胞病毒(RSV)、人偏肺病毒(HMPV)、副流感病毒(PIV)、腺病毒(ADV)、肺炎支原体(MP)均无交叉反应;临床样本检测结果显示,MIRA-CRISPR/Cas13a-MBQD与RT-qPCR法具有极高的检测一致性(Kappa=1.00)。结论建立的MIRA-CRISPR/Cas13a-MBQD检测体系可快速检测IAV,灵敏性、特异性较好。Objective To establish a method for rapid detection of influenza A virus(IAV)by combining multienzyme isothermal rapid amplification(MIRA),clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 13(Cas13)and magnetic bead-quantum dot(MBQD)sensors.Methods MIRA amplification primers,CRISPR RNA(crRNA),fluorescent reporter probes and connected probes were designed and screened targeting the M gene of IAV,and the reaction conditions were optimized to establish a MIRA-CRISPR/Cas13a-MBQD system.The detection sensitivity and specificity of the system were evaluated.In addition,the agreement between this system and real-time quantitative reverse-transcription PCR(RT-qPCR)assay was examined for detection of clinical samples.Results TheMIRA-CRISPR/Cas13a-MBQD system detected IAV within 60 min.The sensitivity of the system for detection of IAV was 1 copy RNA per reaction,and no cross-reaction was found with SARS-CoV-2,influenza virus B(IBV),respiratory syncytial virus(RSV),human metapneumovirus(HMPV),parainfluenza virus(PIV1),and three(HPIV3),adenovirus(ADV)or Mycoplasma pneumonia(MP).In addition,there a high agreement between the MIRA-CRISPR/Cas13a-MBQD system and RT-qPCR assay for detection of clinical samples(Kappa=1.00).Conclusions The established MIRA-CRISPR/Cas13a-MBQD system is rapid,sensitive and specific for detection of IAV.

关 键 词：多酶恒温快速扩增 CRISPR/Cas13a 量子点传感器 甲型流感病毒 快速检测 

分 类 号：R373.1[医药卫生—病原生物学]