A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture.
A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.
Sails
Andrew D
AD
Preston Public Health Laboratory, Royal Preston Hospital, Fulwood, Preston, Lancashire PR2 9HG, UK.
Fox
Andrew J
AJ
Bolton
Frederick J
FJ
Wareing
David R A
DR
Greenway
David L A
DL
D000818:Animals; D016123:Campylobacter jejuni / Q000145:classification / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D002417:Cattle; D002645:Chickens / Q000382:microbiology; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D005516:Food Microbiology; D006801:Humans; D008460:Meat / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012758:Shellfish / Q000382:microbiology
D003470:Culture Media; D004269:DNA, Bacterial