Optimization of automated immunomagnetic separation for the detection of escherichia coli 0157: H7 by quantitative polymerase chain reaction
type: CreativeWork
description: Escherichia coil 0157:1-17 is a virulent human pathogen responsible for severe bloody diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome. Carriage of the organism through the food chain can result in contamination of raw meat, dairy products, crops and produce during production. Numerous outbreaks of the diseases have made it essential to produce detection methods that are rapid, sensitive and specific, especially as the number of Escherichia coil 0157:1-17 in a sample can be low and obscured by the presence of other organisms. Molecular techniques such as quantitative polymerase chain reaction (qPCR) assays have been developed, which are specific, quick and reliable. The major drawback of the technique is prior DNA extraction, which can be time consuming and result in partial loss of target material, followed by small sample input into the qPCR assay. The objective of this study was to develop a sensitive, quick and technically simple sample concentration and DNA extraction protocol which would allow maximum yield for detection of Escherichia coii 0157 by qPCR assays. An improved automated immunomagnetic separation (AIMS) protocol was designed for the detection of Escherichia coil 0157:1-17 with specific, antibody coated, MyOneTm Dynabeads®. The new protocol was found to increase capture by approximately 60 % in comparison to the standard AIMS protocol for Escherichia coil. The improved protocol was used to test spiked food samples according to the BS EN ISO 16140:2003 'Microbiology of food and animal feeding stuffs - Protocol for the validation of alternate methods'. Briefly, four different food types were homogenised and spiked with Escherichia coil 0157:1-17 Vt negative (NCTC 12900) at a level between 10 and 104 CFU (colony forming units). The spiked samples were then enriched in TSB (Tryptic soy broth) for 4, 6 and 24 h at 37°C. On completion of the enrichment step, the samples were subjected to the improved AIMS protocol, followed by DNA extractio…
abstract: Escherichia coil 0157:1-17 is a virulent human pathogen responsible for severe bloody diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome. Carriage of the organism through the food chain can result in contamination of raw meat, dairy products, crops and produce during production. Numerous outbreaks of the diseases have made it essential to produce detection methods that are rapid, sensitive and specific, especially as the number of Escherichia coil 0157:1-17 in a sample can be low and obscured by the presence of other organisms. Molecular techniques such as quantitative polymerase chain reaction (qPCR) assays have been developed, which are specific, quick and reliable. The major drawback of the technique is prior DNA extraction, which can be time consuming and result in partial loss of target material, followed by small sample input into the qPCR assay. The objective of this study was to develop a sensitive, quick and technically simple sample concentration and DNA extraction protocol which would allow maximum yield for detection of Escherichia coii 0157 by qPCR assays. An improved automated immunomagnetic separation (AIMS) protocol was designed for the detection of Escherichia coil 0157:1-17 with specific, antibody coated, MyOneTm Dynabeads®. The new protocol was found to increase capture by approximately 60 % in comparison to the standard AIMS protocol for Escherichia coil. The improved protocol was used to test spiked food samples according to the BS EN ISO 16140:2003 'Microbiology of food and animal feeding stuffs - Protocol for the validation of alternate methods'. Briefly, four different food types were homogenised and spiked with Escherichia coil 0157:1-17 Vt negative (NCTC 12900) at a level between 10 and 104 CFU (colony forming units). The spiked samples were then enriched in TSB (Tryptic soy broth) for 4, 6 and 24 h at 37°C. On completion of the enrichment step, the samples were subjected to the improved AIMS protocol, followed by DNA extractio…
author: Khan, Ayesha
language: en
keywords: C990 - Biological sciences not elsewhere classified
date published: 2006-06
date modified: 2026-07-24T01:36:00Z
provider: University of Central Lancashire
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Central Lancashire