基于RPA/CRISPR-Cas12a技术的单核细胞增生李斯特菌快速检测方法建立

Development of a Rapid Detection Method for Listeria monocytogenes Based on Recombinase Polymerase Amplification Combined with CRISPR-Cas12a Technology

建立基于RPA/CRISPR-Cas12a技术单核细胞增生李斯特菌的快速检测方法。选取单增李斯特菌溶菌素毒力基因hly(GeneID:987033),设计重组酶聚合酶扩增(recombinase polymerase amplification,RPA)引物结合CRISPR-Cas12a蛋白研制两步法单增李斯特菌快速检测试剂盒,并对其灵敏度、特异性以及反应速率进行分析。结果表明:该法检测速率快,30 min内可检出单核细胞增生李斯特菌,同时具有较高的灵敏度,20μL体系可检测到最低0.0015 ng靶标核酸。将该试剂盒进一步用于检测人工模拟污染食品三文鱼肉片,检测限可达10 CFU/mL。本方法检测30份实际样本,结果均与荧光定量聚合酶链式反应法阳性检出率一致。RPA/CRISPR-Cas12a技术是快速检测食源性单增李斯特菌的可行方法。

Objective:To develop a rapid detection method for Listeria monocytogenes based on clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 12a(Cas12a)combined with recombinase polymerase amplification(RPA).Methods:The virulence gene hly(GeneID:987033)of L.monocytogenes was selected to design RPA primers,and a two-step rapid detection kit for L.monocytogenes was developed using RPA combined with CRISPR-Cas12a.The sensitivity,specificity,and reaction rate of the kit were analyzed.Results:This method was rapid and sensitive;it could detect L.monocytogenes within 30 minutes,and the minimum level of the target nucleic acid of 0.0015 ng was detected using 20μL of the system.Furthermore,when this kit was applied to detect artificially contaminated salmon fillets,the limit of detection was 10 CFU/mL,and the results for 30 actual samples were consistent with the positive detection rate obtained by fluorescence quantitative PCR.Conclusion:RPA combined with CRISPR-Cas12a is a feasible method for rapid detection of foodborne L.monocytogenes.