Rapid detection of Campylobacter jejuni in chicken rinse water by melting-peak analysis of amplicons in real-time polymerase chain reaction.
Five DNA extraction protocols for the detection of Campylobacter spp. by polymerase chain reaction (PCR) were compared. A method involving Triton X-100 produced template DNA of sufficient quality to allow the detection of Campylobacter jejuni at levels of 100 CFU/ml in pure culture. Primers were designed on the basis of the cadF gene sequence. With a SYBR Green I real-time PCR assay, these primers amplified only sequences present in C. jejuni to produce a product with a melting temperature of 81.5 degrees C. None of the strains of Campylobacter coli, Campylobacter lari, or Campylobacter fetus tested produced this product during the PCR assay. Other noncampylobacter species tested were shown not to possess the cadF sequence. The real-time PCR combined with a rapid, simple Triton X-100 DNA extraction protocol made it possible to detect < 10 CFU of C. jejuni per ml of chicken rinse within 14 h.
Cheng
Zhihui
Z
Department of Food Science, University of Guelph, 43 McGilvray Street, Guelph, Ontario, Canada N1G 2W1.
Griffiths
Mansel W
MW
D000818:Animals; D001483:Base Sequence; D016123:Campylobacter jejuni / Q000235:genetics / Q000302:isolation & purification*; D002645:Chickens / Q000382:microbiology*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D014871:Water Microbiology
D004269:DNA, Bacterial