Scholarlink

<p>Diarrheagenic <i>Escherichia coli</i> (DEC) are strains of <i>Escherichia coli</i> (<i>E. coli</i>) that can induce diarrhea symptoms in the host, as well as cause disease through contaminated food. To accurately and efficiently identify the five DEC types, this study retrieved the corresponding gene sequences from NCBI based on the virulence genes specified in Chinese national standards: invE, stx1, stx2, sth, stp, lt, aggR, astA, pic, bfpB, and escV. Probes and primers were designed for the conserved regions of these genes, and the amplification system, temperature, and other parameters were optimized. A TaqMan<sup>®</sup>single-plex real-time PCR assay was established for simultaneous detection of Enteropathogenic <i>E. coli</i>, Enteroinvasive <i>E. coli</i>, Enterotoxigenic <i>E. coli</i>, Enterohemorrhagic <i>E. coli</i>, and Enteroaggregative <i>E. coli</i>. The results demonstrated that the minimum detection limit for bacterial genomic DNA was 1.60 × 10<sup>1</sup>&#xa0;copies/μL (except for <i>stx2</i> which was 1.60 × 10<sup>2</sup>&#xa0;copies/μL). The within-group variation rate ranged from 0.12 to 0.88%, while the between-group variation rate ranged from 0.67 to 1.62%. Moreover, R<sup>2</sup> values for the standard curve generated by this method were between 0.999 and 1 with an amplification efficiency ranging from 98.4 to 100%. We evaluated a total of 122 clinical specimens using both conventional PCR and the qPCR method developed in this study. The findings indicated that the qPCR method exhibited high accuracy. Therefore, this study successfully developed a Taqman real-time fluorescence quantitative PCR assay with high specificity, sensitivity, repeatability, and amplification efficiency as well as significant regression effect. This assay can be utilized for clinical detection of DEC.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

A TaqMan qPCR for precise detection and quantification of diarrheagenic Escherichia coli

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年5月14日

查看全文
  • Bingyu Xue,
  • Huanrong Zhang,
  • Xia Yan,
  • Xiaoyan Su,
  • Yuling Zhou,
  • Junjin Xie,
  • Sheng Li,
  • Yang Zhou,
  • Lin Li

摘要

Diarrheagenic Escherichia coli (DEC) are strains of Escherichia coli (E. coli) that can induce diarrhea symptoms in the host, as well as cause disease through contaminated food. To accurately and efficiently identify the five DEC types, this study retrieved the corresponding gene sequences from NCBI based on the virulence genes specified in Chinese national standards: invE, stx1, stx2, sth, stp, lt, aggR, astA, pic, bfpB, and escV. Probes and primers were designed for the conserved regions of these genes, and the amplification system, temperature, and other parameters were optimized. A TaqMan®single-plex real-time PCR assay was established for simultaneous detection of Enteropathogenic E. coli, Enteroinvasive E. coli, Enterotoxigenic E. coli, Enterohemorrhagic E. coli, and Enteroaggregative E. coli. The results demonstrated that the minimum detection limit for bacterial genomic DNA was 1.60 × 101 copies/μL (except for stx2 which was 1.60 × 102 copies/μL). The within-group variation rate ranged from 0.12 to 0.88%, while the between-group variation rate ranged from 0.67 to 1.62%. Moreover, R2 values for the standard curve generated by this method were between 0.999 and 1 with an amplification efficiency ranging from 98.4 to 100%. We evaluated a total of 122 clinical specimens using both conventional PCR and the qPCR method developed in this study. The findings indicated that the qPCR method exhibited high accuracy. Therefore, this study successfully developed a Taqman real-time fluorescence quantitative PCR assay with high specificity, sensitivity, repeatability, and amplification efficiency as well as significant regression effect. This assay can be utilized for clinical detection of DEC.