Optimization of automated immunomagnetic separation for the detection of escherichia coli 0157: H7 by quantitative polymerase chain reaction

Khan, Ayesha (2006) Optimization of automated immunomagnetic separation for the detection of escherichia coli 0157: H7 by quantitative polymerase chain reaction. Masters thesis, University of Central Lancashire.

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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 extraction using a new CST® Nucleic acid extraction kit (Invitrogen Corp.). End point detection was provided by a qPCR assay based on recognition of the rJb gene, and standard plating onto sorbitol MacConkey medium containing cefixime and tellurite.
The use of MyOneTh' Dynabeads® helped to increase the available surface area allowing enhanced capture of target organisms with the new AIMS protocol. This was flirthered by the CST® Nucleic acid extraction kit to provide superior downstream performance in the qPCR assay, which demonstrated a higher degree of sensitivity than culture onto CT-SMAC (Cefixime and Tellurite-Sorbitol MacConkey Agar), after enrichment and AIMS. An evaluation of the three enrichment lengths suggested 6 h to be adequate for sufficient enumeration of target cells, even at low inoculum levels, without producing false positives. This helped to reduce the advised 24 h enrichment (BS EN ISO 16140:2003) down to oh.
The final results demonstrated detection down to approximately 10 CFU from a 25 mg sample when using the improved protocol, whilst the standard (EPEC/VTEC) protocol had a detection limit of 102 CFU.
This study provides an enhanced and reliable sample preparation procedure, which allows detection of Escherichia co/i 0157:1-17 within 10 h, even at significantly low contamination levels. It also reconfirms AIMS as an effective isolation procedure and qPCR as a quicker and more sensitive detection method, in comparison to culture methods. It also introduces the new CST® nucleic acid extraction kit, which provides maximum target concentration for improved detection.

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