摘要
目的采用成簇规律间隔短回文重复序列相关蛋白Cas13a(CRISPR-Cas13a)联合级联引物交换反应(PER),建立一种基于CRISPR-Cas13a系统的检测信号放大并直接检测新型冠状病毒的方法,即CRISPR/PER。方法筛选新型冠状病毒模板链及针对模板链设计2条特异性CRISPR相关RNA(crRNA),利用Cas13a蛋白的切割活性,对CRISPR反应体系进行条件优化;对PER的配对发卡结构及引物进行筛选,利用筛选后的配对进行PER体系优化并设计特异性探针P2探针和P3探针,利用碱基互补配对使信号探针P3与扩增的发卡结构H27结合,形成反应液,制备胶体金试纸条,并评价CRISPR/PER方法的灵敏度与特异度。结果RNA的最佳配对crRNA为crRNA-1;500 nmol·L^(-1)Cas13a蛋白加入1μL、4 g·L^(-1)的crRNA加入0.5μL、5 U Ribonuclease Inhibitor加入1μL为CRISPR反应体系的最适加入量;H27发卡结构与P27引物的配对效果最好;最适条件为37℃,反应40 min;各组最适加入量分别为4 U Bst DNA聚合酶加入1μL、5μmol·L^(-1)H27发卡结构加入10μL、100 mmol·L^(-1)MgSO_(4)加入1μL、dNTP加入2μL时扩增效率最好。CRISPR/PER方法可在1.5 h左右完成检测,检测新型冠状病毒模板链的最低检测限为100μg·L^(-1);特异性实验表明与3种对照病毒均无交叉反应。结论CRISPR/PER检测方法在新型冠状病毒检测上具有方便快捷,特异性强等优点,可作为核酸检测新方法探究的参考。
Objective To establish a CRISPR/Cas13a-based detection signal amplification method for novel coronaviruses,namely CRISPR/PER,using a spacer-associated short palindromic repeat-associated Cas13a protein(CRISPR-Cas13a)in combination with a cascade primer exchange reaction(PER).Methods Screening of the novel coronavirus template strand and design of two specific CRISPR RNAs(crRNA)for the template strand,optimisation of the CRISPR reaction system using the cleavage activity of Cas13a protein,screening of the paired hairpin structure and primers for the PER reaction,optimisation of the PER reaction system using the screened pairs and design of specific probes P2 probe and the P3 probe were designed,and the signal probe P3 was combined with the amplified hairpin H27 using base complementary pairing to form the reaction solution,and colloidal gold test strips were prepared.Results The best paired crRNA for RNA was crRNA-1,500 nmol·L^(-1)Cas13a protein added to 1μL,4 g·L^(-1)of crRNA added to 0.5μL and 5 U Ribonuclease Inhibitor added to 1μL were the optimal addition amounts for the CRISPR reaction system.The H27 hairpin structure paired best with the P27 primer.the optimum conditions were 37℃and 40 minutes reaction time.The optimum addition amounts of each component were 1μL of 4 U Bst DNA polymerase,10μL of 5μmol·L^(-1)H27 hairpin,1μL of 100 mmol·L^(-1)MgSO_(4)and 2μL of dNTP,respectively.The minimum detection limit of the novel coronavirus template was 100μg·L^(-1),and the specificity test showed no cross-reactivity with the three control viruses.Conclusion The CRISPR/PER assay is convenient,rapid and specific for the detection of novel coronaviruses,and can be used as a reference for the investigation of new nucleic acid detection methods.
作者
王晓军
张怡青
谭娅
王继创
程蕾
王旭东
王云龙
WANG Xiaojun;ZHANG Yiqing;TAN Ya;WANG Jichuang;CHENG Lei;WANG Xudong;WANG Yunlong(Henan Provincial Geriatric Pain Rehabilitation Management Engineering Research Center,Henan Provincial Staff Hospital,Zhengzhou 450001,China;Stomatology Department,Henan Provincial Staff Hospital,Zhengzhou 450001,China;Life and Health Institute,Zhengzhou Technical College,Zhengzhou 450100,China;Quantitative Detection of Biomarkers,Henan Provincial Bioengineering Technology Research Center,Zhengzhou 450100,China;the Second Clinical Medical College of Henan University of Chinese Medicine,Zhengzhou 450000,China)
出处
《河南医学研究》
2025年第5期773-778,共6页
Henan Medical Research
基金
河南省重点研发与推广专项(科技攻关)基金资助项目(222102310164,232102311036)。
关键词
成簇规律间隔短回文重复序列
引物交换反应
核酸检测
新型冠状病毒
扩增
clustered regularly interspaced short palindromic repeats
primer exchange reaction
nucleic acid detection
novel coronavirus
amplification
