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Title: Detection of Campylobacter jejuni and Campylobacter coli in high risk foods and environmental waters using the polymerase chain reaction
Author: Sails, Andrew David
ISNI:       0000 0001 3545 3892
Awarding Body: University of Central Lancashire
Current Institution: University of Central Lancashire
Date of Award: 2000
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Nucleic acid amplification methods (PCR) were investigated for the detection of Campylobacter species in foods and environmental waters. A novel species-specific PCR was adapted into a high-throughput, colorimetric end-stage detection format (PCR ELISA). The sensitivity of detection of the PCR ELISA assay was equivalent to one genome copy and was demonstrated to be 100-fold more sensitive than a conventional gel electrophoresis-based PCR method. The novel species-specific PCR assay was adapted into a quantitative real-time format (TaqMan) for accurate quantification of the numbers of C. jejuni cells present in food samples. Quantification was demonstrated over six orders of magnitude between 1.2 x 101 to 1.2 x 107 genome equivalents per reaction, with a limit of detection of 10 genome equivalents. The PCR ELISA and real-time PCR assays were validated for the specific and sensitive detection of C. jejuni and C. coil in naturally contaminated food and environmental water samples. Methods for the extraction of DNA from foods and enrichment cultures were investigated and an optimised method (PrepMan) determined. The PCR ELISA and real-time PCR assays significantly reduced the time taken for the detection of C. jejuni and C. co/i in foods and waters to seven hours and two and a half hours respectively. Previous studies have indicated that detection of mENA by reverse transcriptase PCR (RT-PCR) may reflect viability in the detected organism. RT-PCR assays were m developed for the detection of thee C. jejuni mRNA targets, DNase treatment of extracted RNA was optimised and appropriate controls for RT-PCR in bacteria established. The assays were applied to the detection of heat-killed and chlorine-killed cell suspensions with diflèrentiation between viable and heat-killed cells being demonstrated. However, problems with target-dependent variations in the halilives of the mRNA targets were identified which have major implications for the use of RT-PCR to establish the viability of contaminating pathogens in processed food samples. The PCR ELISA and real-time PCR assays are high-throughput methods for the sensitive and specific detection of C. jejuni and C. coli in foods, and are an important model for other foodborne pathogens.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: C910 - Applied biological sciences