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Applied and Environmental Microbiology, March 2003, p. 1383-1390, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1383-1390.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment Culture

Andrew D. Sails,1,{dagger} Andrew J. Fox,2* Frederick J. Bolton,1 David R. A. Wareing,1 and David L. A. Greenway3

Preston Public Health Laboratory, Royal Preston Hospital, Fulwood, Preston, Lancashire PR2 9HG,1 Manchester Public Health Laboratory, Withington Hospital, West Didsbury, Manchester M20 2LR,2 Department of Biological Sciences, University of Central Lancashire, Preston PR1 2HE, United Kingdom3

Received 19 August 2002/ Accepted 11 December 2002

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°C for 24 h, followed by 42°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.

* Corresponding author. Mailing address: Manchester Public Health Laboratory, Withington Hospital, Nell Ln., West Didsbury, Manchester M20 2LR, United Kingdom. Phone: 44 161 291 4631. Fax: 44 161 446 2180. E-mail: ajfox{at}nw.phls.nhs.uk.

{dagger} Present address: National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333.

Applied and Environmental Microbiology, March 2003, p. 1383-1390, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1383-1390.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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