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Applied and Environmental Microbiology, September 2003, p. 5664-5669, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5664-5669.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Assay Development and Analytical Validation

P. S. Lübeck,1 P. Wolffs,2 S. L. W. On,1 P. Ahrens,1 P. Rådström,2 and J. Hoorfar1*

Danish Veterinary Institute, DK-1790 Copenhagen, Denmark,1 Applied Microbiology, Lund University, 22100 Lund, Sweden2

Received 29 January 2003/ Accepted 23 June 2003

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.

* Corresponding author. Mailing address: Danish Veterinary Institute, Bülowsvej 27, DK-1790 Copenhagen V, Denmark. Phone: 45-35 300 251. Fax: 45-35 300 120. E-mail: jho{at}vetinst.dk.

Applied and Environmental Microbiology, September 2003, p. 5664-5669, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5664-5669.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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