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Applied and Environmental Microbiology, June 2004, p. 3588-3592, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3588-3592.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters

M. H. Josefsen, N. R. Jacobsen, and J. Hoorfar*

Danish Institute for Food and Veterinary Research, DK-1790 Copenhagen V, Denmark

Received 10 December 2003/ Accepted 2 March 2004

As part of a large international project for standardization of PCR (Food-PCR; www.pcr.dk), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 102 to 107 CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of 1 x 103 and 2 x 103 CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 x 101 to 1 x 107 (R2 = 1.00) for the RotorGene and 103 to 107 (R2 = 0.99) for the ABI-PRISM. In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively. The results showed a correlation (R2) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment. Signal from the internal amplification control was detected in all culture-negative samples (VIC Ct: 23.1 to 28.1). The method will be taken further and validated in an international collaborative trial with regard to standardization.

* Corresponding author. Mailing address: Danish Institute for Food and Veterinary Research, 27 Bülowsvej, DK-1790 Copenhagen V, Denmark. Phone: (45) 72346251. Fax: (45) 72346360. E-mail: jho{at}dfvf.dk.

Applied and Environmental Microbiology, June 2004, p. 3588-3592, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3588-3592.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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