重组酶介导的等温扩增技术结合CRISPR-Cas13a蛋白检测8种境外输入性病毒  

Detection of eight imported viruses using recombinase aided isothermal amplification combined with CRISPR-Cas13a

作　　者：郭悦 安柏霖 刘丹丹 罗君红 赵康辰[3] 朱小娟[3] 葛以跃[3] 吴宏斌 崔仑标[1,3] Guo Yue;An Bailin;Liu Dandan;Luo Junhong;Zhao Kangchen;Zhu Xiaojuan;Ge Yiyue;Wu Hongbin;Cui Lunbiao(Nanjing Medical University,Nanjing 210029,China;College of Pharmacy,Nankai University,Tianjin International Biomedical Joint Research Institute,Tianjin 300071,China;NHC Key Laboratory of Enteric Pathogen Microbiology,Jiangsu Provincial Center for Disease Control and Prevention,Nanjing 210009,China;Taizhou Medical City Medical Detection,Ltd,Taizhou 225300,China)

机构地区：[1]南京医科大学,南京210029 [2]南开大学药学院天津国际生物医药联合研究院,天津300071 [3]江苏省疾病预防控制中心国家卫生健康委员会肠道病原微生物重点实验室,南京210009 [4]泰州医药城医学检验有限公司,泰州225300

出　　处：《中华实验和临床病毒学杂志》2022年第3期245-251,共7页Chinese Journal of Experimental and Clinical Virology

基　　金：江苏省社会发展项目(BE2019761,BE2020682)。

摘　　要：目的基于重组酶介导的扩增技术(recombinase aided amplification,RAA)和CRISPR(clustered regularly interspaced short palindromic repeats)-Cas13a(CRISPR-associated protein 13a)检测,建立一种快速、灵敏且特异的重要境外输入性病毒检测方法。方法以流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)、黄热病毒(Yellow fever virus,YEV)、西尼罗病毒(West Nile virus,WNV)、中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)、埃博拉病毒(Ebola virus,EBOV)、登革病毒(Dengue virus,DENV)、裂谷热病毒(Rift Valley fever virus,RVFV)、寨卡病毒(Zika virus,ZIKV)为检测对象,设计特异性RAA扩增引物和CRISPR RNA(crRNA),建立RAA扩增结合CRISPR-Cas13a蛋白检测反应,评价方法的灵敏度和特异性,使用登革热疑似临床样本进行检测,并与荧光RT-PCR技术进行比较,同时检测其余7种病毒临床模拟样本。结果RAA扩增结合CRISPR-Cas13a蛋白检测方法能在39℃条件下,40~52 min内检测出8种输入性传染病病原体,灵敏度达到1~10拷贝/μl,并且这8种病原体之间无交叉反应,临床样本均可100%检测出。结论建立的RAA扩增结合CRISPR-Cas13a蛋白检测方法能够灵敏、特异且快速地检测出8种境外输入性传染病病原体。Objective To establish a rapid,sensitive and specific detection method for important imported viruses based on the recombinase aided amplification(RAA)method and clustered regularly interspaced short palindromic repeats-associated protein 13a(CRISPR-Cas13a)system.Methods In this study,we selected Japanese encephalitis virus(JEV),Yellow fever virus(YEV),West Nile virus(WNV),Middle East respiratory syndrome coronavirus(MERS-CoV)、Ebola virus(EBOV),Dengue virus(DENV),Rift Valley fever virus(RVFV),Zika virus(ZIKV)as subjects,and designed specific RAA primers and CRISPR RNA(crRNA).The sensitivity and specificity of the method were evaluated.We detected suspected clinical samples of dengue fever and compared with the fluorescent reverse transcriptase-polymerase chain reaction(RT-PCR)technology.Clinical simulation samples of the remaining seven viruses were also detected.Results The RAA method combined with CRISPR-Cas13a can detect eight pathogens within 40-52 min at 39℃.The sensitivity was 1-10 copies/μl.There was no cross-reaction among eight viruses and all clinical samples could be detected by this method.Conclusions The established RAA combined with CRISPR-Cas13a detection method can sensitively,specifically and quickly detect eight imported infectious disease pathogens.

关 键 词：输入性传染病 病毒 重组酶介导的扩增技术 CRISPR-Cas13a 

分 类 号：R446[医药卫生—诊断学]