A Robotic DNA Purification Protocol and Real-Time PCR for the Detection of Campylobacter jejuni in Foods
Abstract:Primers designed to amplify a Campylobacter jejuni cadF gene sequence were used in an SYBR Green I real-time PCR assay as an alternative to conventional bacteriological methods for the rapid detection of C. jejuni in foods. Twenty-five grams of chicken skin (breast and thigh) was contaminated by adding approximately 1, 10, or 50 CFU of C. jejuni ATCC 35560. Twenty-five grams of pork and 25-ml aliquots of milk were also inoculated with 1 and 10 CFU of the pathogen. The samples were incubated in Bolton broth for different periods at 37 and 42°C under microaerophilic conditions. Using a commercial robotic DNA purification system, DNA was extracted and purified from 1-ml aliquots of the enrichment cultures before and after centrifugation of the 250-ml enrichment broth at 15,900 × g for 10 min at 4°C. The DNA was used as the template in a real-time PCR assay. C. jejuni was detected after 12 h of enrichment from samples inoculated with about 50 CFU/25-g sample. After centrifugation, an enrichment step of 8 h was sufficient to allow detection of pathogen in samples inoculated with 10 CFU/25 g. However, 24 h of enrichment was necessary to detect pathogen in samples inoculated with approximately 10 CFU/25 g. The real-time PCR protocol developed in this study significantly reduced the detection time of C. jejuni in foods.
Document Type: Research Article
Affiliations: 1: Department of Food and Drug Technology, University of Londrina, Londrina, Parana 86.060-090, Brazil 2: Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 3: Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1; Canadian Research Institute for Food Safety, 43 McGilvray Street, Guelph, Ontario, Canada N1G 2W1
Publication date: October 1, 2005
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