A robotic DNA purification protocol and real-time PCR for the detection of Campylobacter jejuni in foods.
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 degrees 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 x g for 10 min at 4 degrees 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 1 CFU/25 g. The real-time PCR protocol developed in this study significantly reduced the detection time of C. jejuni in foods.
Oliveira
Tereza C R M
TC
Department of Food and Drug Technology, University of Londrina, Londrina, Parana 86.060-090, Brazil. terezaoliveira@yahoo.com
Barbut
Shai
S
Griffiths
Mansel W
MW
D000818:Animals; D016123:Campylobacter jejuni / Q000302:isolation & purification*; D002645:Chickens / Q000382:microbiology; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis*; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D006801:Humans; D008460:Meat / Q000382:microbiology*; D008892:Milk / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D013552:Swine / Q000382:microbiology; D013696:Temperature
D004269:DNA, Bacterial