不同提取及扩增方法检测大肠杆菌DNA的性能比较  

Comparison of the performance of different extraction and amplification methods for detecting Escherichia coli

作　　者：余小妹 钱纯亘 聂立波 汤建新 吴力强 黄钊 易辉 DNA YU Xiaomei;QIAN Chungen;NIE Libo;TANG Jianxin;WU Liqiang;HUANG Zhao;YI Hui(Hunan Key Laboratory of Biomedical Nanomaterials and Devices,College of Life Science and Chemistry,Hunan University of Technology,Zhuzhou 412007,China;Shenzhen YHLO Biotech Co.,Ltd.,Shenzhen 518116,China;Zhuzhou People’s Hospital,Zhuzhou 412008,China)

机构地区：[1]湖南工业大学生命科学与化学学院,生物医用纳米材料与器件湖南省重点实验室,湖南株洲412007 [2]深圳市亚辉龙生物科技股份有限公司,广东深圳518116 [3]株洲市人民医院,湖南株洲412008

出　　处：《湘潭大学学报（自然科学版）》2024年第2期110-122,共13页Journal of Xiangtan University(Natural Science Edition)

基　　金：湖南省重点研发计划(2022SK2009);湖南省自然科学基金(2022JJ50099);湖南省教育厅优秀青年项目(23B0561)。

摘　　要：大肠杆菌O157∶H7是一种危害性极大的食源性致病菌,为实现大肠杆菌O157∶H7的快速检测,分别采用碱加热裂解法、TritonX-100加热裂解法、溶菌酶加十二烷基硫酸钠(SDS)裂解法、SDS加蛋白酶K裂解法及热裂解法5种方法提取大肠杆菌O157∶H7基因组DNA,考察了环介导等温扩增(LAMP)和实时荧光定量聚合酶链式反应(qPCR)扩增法对所提取DNA的检测效果,并建立最佳的核酸快速提取样品预处理方法.结果表明,以碱加热裂解预处理提取的大肠杆菌O157∶H7基因组DNA为检测样本时,LAMP扩增及qPCR扩增法的检测性能最好,检出限达到10^(1)CFU/mL,与常规商品化试剂盒提取所得菌液基因组DNA的检测性能一致.所建立的碱加热裂解样品预处理方法耗时短、成本低,无须进行复杂的核酸提取,操作简单,可用于大肠杆菌O157∶H7的快速检测.Escherichia coli O157∶H7 is a highly hazardous pathogenic foodborne bacterium.In order to achieve rapid detection of Escherichia coli O157∶H7,five methods including alkaline thermal lysis,TritonX-100 thermal lysis,lysozyme with SDS lysis,SDS with proteinase K lysis,and thermal lysis were used to extract genomic DNA of Escherichia coli O157∶H7.The detection effects of loop-mediated isothermal amplification(LAMP)and real-time fluorescence quantitative polymerase chain reaction(qPCR)amplification methods on the extracted DNA were investigated to establish the optimal nucleic acid rapid extraction sample pretreatment method.The results showed that using the extracted Escherichia coli O157∶H7 genomic DNA obtained through alkaline thermal lysis pretreatment as the testing sample,both LAMP and qPCR amplification methods showed the best detection performance,with a detection limit of 10^(1) CFU/mL,consistent with the detection performance of genomic DNA obtained from conventional commercial kits.The established alkaline thermal lysis sample pretreatment method is time-efficient,cost-effective,without complex nucleic acid extraction,and simple to operate,which is suitable for rapid detection of Escherichia coli O157∶H7.

关 键 词：大肠杆菌O157∶H7 LAMP qPCR 碱加热裂解法 核酸提取 

分 类 号：Q93-332[生物学—微生物学]