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A reporter bacteriophage - beta-galactosidase assay for detection of generic Escherichia coli from beef carcasses.

机译：记者噬菌体-β-半乳糖苷酶测定法，用于检测牛肉car体中的一般大肠杆菌。

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摘要

Recent outbreaks of foodborne illness during the past decade have highlighted the need for fundamental changes in meat inspection programs. The Hazard Analysis Critical Control Point (HACCP) program is a method for evaluating and controlling the safety and quality of all processed food. Central to the HACCP plan is the requirement for rapid and reliable microbiological detection methods. This thesis describes the development and characterization of a reporter bacteriophage - beta-galactosidase assay for detection and enumeration of generic Escherichia coli from beef carcasses. Generic E. coli testing is often employed as an indicator of fecal contamination. Two bacteriophages capable of growing on many serotypes of E. coli were characterized according to their morphological, host range, and genetic properties. The host range genes of one of these bacteriophages, bacteriophage AR1, were cloned into a plasmid and this host range plasmid was used to create several T4 bacteriophages that had an expanded host range by growing the T4 bacteriophages on cells harboring the host range plasmid and allowing for homologous recombination between the plasmid and the T4 bacteriophages. The expanded host range T4 bacteriophages were further genetically modified to carry the E. coli beta-galactosidase (lacZ) gene. These reporter bacteriophages were used in the reporter - bacteriophage, beta-galactosidase assay. The sensitivity of the assay for E. coli in pure suspensions was determined. The specificity of the assay was determined with E. coli isolates from beef and with other bacterial genera. The reporter bacteriophage beta-galactosidase assay was capable of detecting 5 x 102 CFU/ml of E. coli without an enrichment step if a chemiluminescent substrate for beta-galactosidase was employed. However, the assay could only detect 5 of 19 E. coli isolates from beef carcasses. None of the non E. coli bacteria tested were detected by the assay. A most probable number method (MPN) with three parallels, based on the use of the lacZ reporter bacteriophages is described. The lacZ reporter bacteriophages were used to differentiate between positive and negative samples in the MPN. The MPN method is sensitive, and enables the enumeration of 10' CFU/ml of E. coli cells in broth culture within 8 hours.
机译：在过去的十年中，最近爆发的食源性疾病突显了肉类检查计划的根本变化。危害分析关键控制点（HACCP）程序是一种评估和控制所有加工食品的安全性和质量的方法。快速，可靠的微生物检测方法要求是HACCP计划的核心。本文描述了一种报道噬菌体-β-半乳糖苷酶检测试剂的开发和表征，该检测试剂用于检测和计数牛肉car体中的普通大肠杆菌。常规大肠杆菌检测通常被用作粪便污染的指标。根据它们的形态，宿主范围和遗传特性，对能够在多种血清型大肠杆菌上生长的两种噬菌体进行了表征。将这些噬菌体之一的噬菌体AR1的宿主范围基因克隆到质粒中，并使用该宿主范围质粒通过在容纳宿主范围质粒的细胞上培养T4噬菌体并允许其生长，从而创建了多个具有扩大宿主范围的T4噬菌体用于质粒和T4噬菌体之间的同源重组。进一步扩大了宿主范围的T4噬菌体的基因，使其携带大肠杆菌β-半乳糖苷酶（lacZ）基因。这些报道细菌噬菌体用于报道分子-噬菌体，β-半乳糖苷酶测定。确定了该测定对纯悬液中大肠杆菌的敏感性。用牛肉和其他细菌属的大肠杆菌分离物测定测定的特异性。如果采用了β-半乳糖苷酶的化学发光底物，则报告噬菌体的β-半乳糖苷酶测定法无需富集步骤即可检测5 x 102 CFU / ml的大肠杆菌。但是，该检测方法只能检测出19个来自牛尸体的大肠杆菌分离物中的5个。该测定法未检测到所测试的非大肠杆菌细菌。描述了基于lacZ报告基因噬菌体的最可能的编号方法（MPN），具有三个平行项。 lacZ报告基因噬菌体用于区分MPN中的阳性和阴性样品。 MPN方法灵敏，能够在8小时内计数10'CFU / ml肉汤培养中的大肠杆菌细胞。

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  • 作者

    Goodridge, Lawrence David.;

  • 作者单位

    University of Guelph (Canada).;

  • 授予单位 University of Guelph (Canada).;
  • 学科 Biology Microbiology.
  • 学位 Ph.D.
  • 年度 2002
  • 页码 196 p.
  • 总页数 196
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 微生物学;
  • 关键词

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