湘潭大学学报(自然科学版)2024,Vol.46Issue(2) :110-122.DOI:10.13715/j.issn.2096-644X.20231106.0002

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 DNA

余小妹 钱纯亘 聂立波 汤建新 吴力强 黄钊 易辉
湘潭大学学报(自然科学版)2024,Vol.46Issue(2) :110-122.DOI:10.13715/j.issn.2096-644X.20231106.0002

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 DNA

余小妹 1钱纯亘 2聂立波 3汤建新 3吴力强 2黄钊 3易辉4
扫码查看

作者信息

  • 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;深圳市亚辉龙生物科技股份有限公司,广东深圳 518116
  • 2. 深圳市亚辉龙生物科技股份有限公司,广东深圳 518116
  • 3. 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
  • 4. 株洲市人民医院,湖南株洲 412008
  • 折叠

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101 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.

Abstract

Escherichia coli O157∶H7 is a highly hazardous pathogenic foodborne bacterium.In order to a-chieve rapid detection of Escherichia coli O157∶H7,five methods including alkaline thermal lysis,Tri-tonX-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)amplifica-tion 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 ge-nomic 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 101 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 com-plex 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

Key words

Escherichia coli O157∶H7/LAMP/qPCR/alkaline thermal lysis/nucleic acid extraction

引用本文复制引用

基金项目

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)

出版年

2024
湘潭大学学报(自然科学版)
湘潭大学

湘潭大学学报(自然科学版)

CSTPCD
影响因子:0.403
ISSN:2096-644X
参考文献量25
段落导航相关论文