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
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1.南开大学药学院,天津 300000;2.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,江苏 南京 210009;3.天津国际生物医药联合研究院,天津 300000;4.南京医科大学, 江苏 南京 210029;5.中国科学院生物物理所,北京 100101

作者简介:

安柏霖 男 硕士研究生 研究方向为新型分子诊断技术与病原体基因组分析 E-mail:1315816788@qq.com

通讯作者:

崔仑标 男 教授 研究方向为病原微生物和新发传染病的监测及分子诊断技术研究 E-mail:lbcui@jscdc.cn

中图分类号:

R155

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Rapid detection of four diarrheal bacteria by CRISPR-Cas13a combined with recombinase aided amplification
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1.College of Pharmacy, Nankai University, Tianjin 300000, China;2.NHC Key Laboratory of Enteric Pathogen Microbiology, Jiangsu Provincial Center for Disease Control and Prevention, Jiangsu Nanjing 210009, China;3.Tianjin International Biomedical Joint Research Institute, Tianjin 300000, China;4.Nanjing Medical University, Jiangsu Nanjing 210029, China;5.Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

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    摘要:

    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:H7 4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。

    Abstract:

    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.

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安柏霖,苏璇,郭悦,王祥喜,葛以跃,朱凤才,崔仑标.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[J].中国食品卫生杂志,2023,35(3):381-389.

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  • 收稿日期:2022-10-12
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  • 在线发布日期: 2023-05-24
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