基于荧光探针重组酶介导等温扩增技术快速检测食源性单增李斯特菌方法的建立

Establishment of a Rapid Detection Method for Foodborne Listeria monocytogenes Using Fluorescence Probe Recombinant Enzyme Mediated Isothermal Amplification Technology

目的:建立一种基于荧光探针重组酶介导等温核酸扩增(Recombinant Enzyme Mediated Isothermal Nucleic Acid Amplification,RAA)技术的快速检测食源性单增李斯特菌的方法。方法:针对单增李斯特菌hlyA基因的保守序列设计特异性引物和探针,通过构建含hlyA靶基因片段的重组质粒和不同浓度单增李斯特菌核酸,评价其灵敏度;通过检测大肠杆菌、沙门氏菌、金黄色葡萄球菌、副溶血性弧菌等菌液核酸,评价其特异性。结果:所建立的荧光RAA法可在39℃、20 min内完成样本核酸的检测。采用所建立的方法对质粒和核酸进行检测,质粒最低检出限为1 copy/μL,菌液核酸最低检测限为103 CFU·mL^(-1),且与其他菌株核酸无交叉反应。结论:成功建立了一种基于荧光RAA法的单增李斯特菌核酸检测方法,该方法具有特异性强和快速的特点,可用于疑似污染单增李斯特菌食品的快速检测。

Objective:To establish a rapid detection method for foodborne Listeria monocytogenes based onfluorescence probe recombinant enzyme mediated isothermal nucleic acid amplification(RAA)technology.Method:Specific primers and probes were designed for the conserved sequence of the hlyA gene of Listeria monocytogenes.The sensitivity was evaluated by constructing recombinant plasmids containing hlyA target gene fragments and different concentrations of Listeria monocytogenes nucleic acid;evaluate the specificity of E.coli,Salmonella,Staphylococcus aureus,Vibrio parahaemolyticus and other bacterial fluids by detecting their nucleic acid levels.Result:Thefluorescence RAA method established can complete the detection of sample nucleic acid within 39℃and 20 minutes.The established method is used to detect plasmids and nucleic acids,with a minimum detection limit of 31 copy/μL.The minimum detection limit for bacterial nucleic acid is 10 CFU·mL^(-1),and there is no cross reaction with other strains.Conclusion:Afluorescence RAA based nucleic acid detection method for Listeria monocytogenes has been successfully established.This method has strong specificity and is fast,and can be used for rapid detection of food suspected to be contaminated with Listeria monocytogenes.