基于CRISPR-Cas12a蛋白的副溶血弧菌及tdh基因检测分析

Application of CRISPR-Cas12a system in detection of Vibrio parahaemolyticus and tdh gene

目的将重组酶介导等温核酸扩增技术(RAA)与成簇的规律间隔短回文重复序列(CRISPR)系统相结合,建立一种快速检测并分型副溶血弧菌的检测方法。方法通过纯化CRISPR相关蛋白Cas12a,设计和合成RAA引物、crRNA和单链DNA报告分子,建立副溶血弧菌的荧光和试纸条检测方法。采用煮沸法提取细菌核酸,经RAA扩增后产物加入CRISPR-Cas12a检测系统,测试该方法的灵敏性和特异性。结果本研究检测了110株副溶血弧菌和121株其他肠道致泻性细菌,符合率为99.1%,最低检测菌量为10^(3) CFU/mL,CRISPR-Cas12a最低检测核酸浓度为77.5 pmol/L,灵敏性高于琼脂糖凝胶电泳法。对于50株携带tdh和12株不携带tdh的副溶血弧菌,仅1株tdh+副溶血弧菌未被鉴定出,鉴定成功率为98.4%。结论本研究建立的CRISPR-RAA检测系统可快速、简单地检测副溶血弧菌及tdh基因,且有较强的特异性和灵敏性。

Objective To establish a rapid detection and typing assay for the detection of Vibrio parahaemolyticus by combining recombinase aided amplification(RAA)with clustered regularly interspaced short palindromic repeats(CRISPR)system.Methods By purifying CRISPR associated protein Cas12a,RAA primers,crRNA and single-stranded DNA reporter were designed and synthesized,and fluorescence and lateral flow dipstick methods were established to detect V.parahaemolyticus.The bacterial nucleic acid was extracted by boiling method,and the product amplified by RAA was added to the CRISPR-Cas12a detection system to test the sensitivity and specificity of the assay.Results In this study,110 strains of V.parahaemolyticus and 121 strains of other intestinal diarrheagenic bacteria were detected,and the coincidence rate was 99.1%.The minimum detection limit of bacteria was 10^(3) CFU/mL,and the minimum detection limit of nucleic acid concentration of CRISPR-Cas12a was 77.5 pmol/L.The sensitivity of CRISPR-Cas12a was higher than that of agarose gel electrophoresis.For 50 strains of V.parahaemolyticus with tdh and 12 strains of V.parahaemolyticus without tdh,one strain of V.parahaemolyticus with tdh was not identified,the successful identification rate was 98.4%.Conclusion CRISPR-RAA can be used for the rapid detection of V.parahaemolyticus and tdh gene with high specificity and sensitivity.