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Applied and Environmental Microbiology, February 2004, p. 790-797, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.790-797.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification

Knut Rudi,1* Hilde Kristin Høidal,2 Tone Katla,3 Birgit Klungseth Johansen,2 John Nordal,3 and Kjetill S. Jakobsen2,4

MATFORSK, Norwegian Food Research Institute, 1430 Ås,1 Genpoint AS, 0884 Oslo,2 Prior Norge AS, 0483 Oslo,3 Division of Molecular Biology, University of Oslo, 0315 Oslo, Norway4

Received 21 August 2003/ Accepted 11 November 2003

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log10 CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5'-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log10 were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log10 CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.

* Corresponding author. Mailing address: MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway. Phone: 47 64 97 01 00. Fax: 47 64 97 03 33. E-mail: knut.rudi{at}matforsk.no.

Applied and Environmental Microbiology, February 2004, p. 790-797, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.790-797.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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