重组酶介导的等温扩增技术联合CRISPR-Cas13a快速检测4种腹泻病原菌

Rapid detection of four diarrheal bacteria by CRISPR-Cas13a combined with recombinase aided amplification

目的重组酶介导的等温扩增技术(RAA)联合规律间隔性成簇短回文重复序列相关Cas13a蛋白(CRISPR-Cas13a),建立对沙门菌、志贺菌、霍乱弧菌、肠出血性大肠杆菌O157:H74种腹泻病原菌的快速分子检测方法,即RAA-Cas13a。方法设计4种腹泻病原菌的RAA特异性引物和CRISPR RNA(crRNA),利用RAA技术扩增样本核酸,并进行CRISPR-Cas13a恒温荧光检测,以实时荧光定量聚合酶链式反应(RT-qPCR)为对照方法,评价RAA-Cas13a优化方法的灵敏度与特异性。结果RAA-Cas13a方法可在35 min内完成检测。检测志贺菌、霍乱弧菌、肠出血性大肠杆菌O157:H7的最低检测限为10 copies/μL,沙门菌最低检测限为1 copy/μL;特异性实验表明每一种病原菌与其余10种对照细菌均无交叉反应。应用建立的方法检测200份实际样本和40份人工污染样本,结果表明同RT-qPCR检测结果一致性高(分别为Kappa=0.927和Kappa=1.000)。结论RAA-Cas13a检测方法具有灵敏度高,特异性强等优点,能用于4种腹泻病原菌的快速检测。

Objective To establish a rapid,sensitive and specific detection method for 4 diarrheal bacteria(Salmonella,Shigella,Vibrio cholera and Escherichia coli O157:H7)by the Clustered regularly interspaced short palindromic repeats associated protein 13a(CRISPR-Cas13a)combined with recombinant enzyme-mediated isothermal amplification(RAA),called RAA-Cas13a.Methods In this study,the specific primer for RAA and CRISPR RNA(crRNA)of 4 different diarrheal bacteria were designed.The sample nucleic acids were amplified by RAA,and the amplification products were then detected with CRISPR-Cas13a.Compared with real-time quantitative polymerase chain reaction(RT-qPCR),the sensitivity and specificity of the RAA-Cas13a method were evaluated.Results The established RAA-Cas13a detection method for Shigella,Vibrio cholera and Escherichia coli O157:H7 had the detection limit of 10 copies/μL,the detection limit for Salmonella was 1 copy/μL,and each bacteria did not have cross-reaction with the other ten bacteria.Meantime,the detection of the RT-qPCR and RAA-Cas13a were highly consistent in 200 suspected samples and 40 artificial simulation samples(Kappa=0.927 and 1.000,respectively).Conclusion The established RAA-Cas13a detection method has the advantages of high sensitivity and strong specificity.It can quickly detect and screen diarrheal diseases caused by 4 pathogenic bacteria.