Comparative, Collaborative, and On-Site Validation of a TaqMan PCR Method as a Tool for Certified Production of Fresh, Campylobacter -Free Chickens
- 1 August 2006
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 72 (8), 5463-5468
- https://doi.org/10.1128/aem.00291-06
Abstract
Certified Campylobacter -free poultry products have been produced in Denmark since 2002, the first example of fresh (unprocessed and nonfrozen) chickens labeled “ Campylobacter free.” This success occurred partly through use of a 4-hour gel-based PCR testing scheme on fecal swabs. In this study, a faster, real-time PCR approach was validated in comparative and collaborative trials, based on recommendations from the Nordic system for validation of alternative microbiological methods (NordVal). The comparative real-time PCR trial was performed in comparison to two reference culture protocols on naturally contaminated samples (99 shoe covers, 101 cloacal swabs, 102 neck skins from abattoirs, and 100 retail neck skins). Culturing included enrichment in both Bolton and Preston broths followed by isolation on Preston agar and mCCDA. In one or both culture protocols, 169 samples were identified as positive. The comparative trial resulted in relative accuracy, sensitivity, and specificity of 98%, 95%, and 97%, respectively. The collaborative trial included nine laboratories testing neck skin, cloacal swab, and shoe cover samples, spiked with low, medium, and high concentrations of Campylobacter jejuni . Valid results were obtained from six of the participating laboratories. Accuracy for high levels was 100% for neck skin and cloacal swab samples. For low levels, accuracy was 100% and 92% for neck skin and cloacal swab samples, respectively; however, detection in shoe cover samples failed. A second collaborative trial, with an optimized DNA extraction procedure, gave 100% accuracy results for all three spiking levels. Finally, on-site validation at the abattoir on a flock basis was performed on 400 samples. Real-time PCR correctly identified 10 of 20 flocks as positive; thus, the method fulfilled the NordVal validation criteria and has since been implemented at a major abattoir.Keywords
This publication has 15 references indexed in Scilit:
- Detection of Campylobacter spp. in ChickenFecal Samples by Real-TimePCRJournal of Clinical Microbiology, 2004
- A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic CampylobacterMolecular and Cellular Probes, 2004
- Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samplesJournal of Microbiological Methods, 2004
- Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant CampylobactersApplied and Environmental Microbiology, 2004
- Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA PurificationApplied and Environmental Microbiology, 2004
- Application of real-time PCR for quantitative detection ofCampylobacter jejuniin poultry, milk and environmental waterFEMS Immunology & Medical Microbiology, 2003
- Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Validation in a Multicenter Collaborative TrialApplied and Environmental Microbiology, 2003
- Toward an International Standard for PCR-BasedDetection of Food-Borne Thermotolerant Campylobacters: AssayDevelopment and AnalyticalValidationApplied and Environmental Microbiology, 2003
- Neurogenetics: Methods and Protocols, Methods in Molecular Biology Vol. 217, edited by Nicholas T. PotterAmerican Journal of Medical Genetics Part A, 2003
- A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment CultureApplied and Environmental Microbiology, 2003