新型冠状病毒RT-LAMP检测方法的建立  

Establishment of RT-LAMP method for detection of Severe Acute Respiratory Syndrome Coronavirus 2

作　　者：周杨[1] 万强[1] 蔡芷荷 卢勉飞[1] 杨绪伟[1] 吴清平[2] 李健顺 ZHOU Yang;WAN Qiang;CAI Zhi-he;LU Mian-fei;YANG Xu-wei;WU Qing-ping;LI Jian-shun(Guangdong Huankai Microbial Science and Technology Co.,Ltd.,Guangzhou,Guangdong 510663,China)

机构地区：[1]广东环凯微生物科技有限公司,广东广州510663 [2]广东省微生物研究所 [3]广东环凯生物科技有限公司

出　　处：《现代预防医学》2021年第1期128-134,共7页Modern Preventive Medicine

基　　金：国家重点研发计划(2018YFC1604201);肇庆市引进西江创新团队项目(肇人才领2018-8);广东省自然科学基金(2017A030310090)。

摘　　要：目的建立基于逆转录环介导恒温扩增技术(reverse transcription loop-mediated isothermal amplification,RT-LAMP)的新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)快速检测方法。方法针对SARS-CoV-2的ORF1ab和N基因序列保守区,设计引物,优化RT-LAMP检测体系,评价其检测特异性和灵敏度。结果成功建立了检测SARS-CoV-2的可视化RT-LAMP(Visual RT-LAMP,V-RT-LAMP)和实时荧光RT-LAMP(Real-time fluorescence RT-LAMP,RF-RT-LAMP)方法,经检测人工合成靶位基因RNA样品和各种实际核酸样品,其结果均与实际结果相符;V-RT-LAMP和RF-RT-LAMP方法的最低检出限均可达到10拷贝数,与实时荧光逆转录聚合酶链反应(quantatitive Reverse Transcription Polymerase Chain Reaction,qRT-PCR)方法相一致。结论 V-RT-LAMP和RF-RT-LAMP方法其检测特异性好、灵敏度高,适用于SARS-CoV-2的快速检测。Objective To establish a rapid assay for detection of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)based on reverse transcription loop-mediated isothermal amplification(RT-LAMP).Methods The LAMP primers were respectively designed according to the conserved sequence regions of ORF1ab and N genes of SARS-CoV-2.The RT-LAMP reaction condition was optimized,and specificity and sensitivity of the assay were evaluated as well.Results Visual RT-LAMP(V-RT-LAMP)and real-time fluorescence RT-LAMP(RF-RT-LAMP)assays were successfully established for detection of SARS-COV-2.All artificial synthesized SARS-COV-2 target gene RNA samples and various actual nucleic acid samples were tested,and the results were all consistent with the actual results.The limit of detection(LoD)of the two LAMP assays could reach 10 copies/testing,which was consistent with the counterpart result gained by quantitative reverse transcription polymerase chain reaction(qRT-PCR)assay.Conclusion TheV-RT-LAMP and RF-RT-LAMP assays represent high specificity and sensitivity,which enable them apply for rapid detection of SARS-COV-2.

关 键 词：新型冠状病毒 逆转录环介导恒温扩增技术 可视化 实时荧光 

分 类 号：R563.1[医药卫生—呼吸系统] R115[医药卫生—内科学]