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
杨宁 1马君妍 2赵康辰 3吴涛 3朱小娟 3乔乔 3王烁 2虎歌 2吴斌 3崔仑标 3葛以跃3
作者信息
- 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;南京医科大学公共卫生学院
- 2. 南京医科大学公共卫生学院
- 3. 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
- 折叠
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.
Abstract
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 re-porter 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Key words
Multienzyme isothermal rapid amplification/CRISPR/Cas 13a/Quantum dot sensor/Influenza A virus/Rapid detection引用本文复制引用
出版年
2024
NETL
NSTL
万方数据