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Food Microbiology

Multicenter Validation of the Analytical Accuracy of Salmonella PCR: towards an International Standard

Burkhard Malorny, Jeffrey Hoorfar, Cornelia Bunge, Reiner Helmuth
Burkhard Malorny
1Federal Institute for Health Protection of Consumers and Veterinary Medicine, D-12277 Berlin, Germany
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Jeffrey Hoorfar
2Danish Veterinary Institute, DK-1790 Copenhagen, Denmark
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Cornelia Bunge
1Federal Institute for Health Protection of Consumers and Veterinary Medicine, D-12277 Berlin, Germany
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Reiner Helmuth
1Federal Institute for Health Protection of Consumers and Veterinary Medicine, D-12277 Berlin, Germany
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  • For correspondence: r.helmuth@bgvv.de
DOI: 10.1128/AEM.69.1.290-296.2003
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ABSTRACT

As part of a major international project for the validation and standardization of PCR for detection of five major food-borne pathogens, four primer sets specific for Salmonella species were evaluated in-house for their analytical accuracy (selectivity and detection limit) in identifying 43 Salmonella spp. and 47 non-Salmonella strains. The most selective primer set was found to be 139-141 (K. Rahn, S. A. De Grandis, R. C. Clarke, S. A. McEwen, J. E. Galán, C. Ginocchio, R. Curtiss III, and C. L. Gyles, Mol. Cell. Probes 6:271-279, 1992), which targets the invA gene. An extended determination of selectivity by using 364 strains showed that the inclusivity was 99.6% and the exclusivity was 100% for the invA primer set. To indicate possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC), which was coamplified with the invA target gene, was constructed. In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139-141 was found to be 100% when a cell suspension containing 104 CFU/ml was used as the template in the PCR (50 CFU per reaction). The primer set was further validated in an international collaborative study that included 16 participating laboratories. Analysis with 28 coded (“blind”) DNA samples revealed an analytical accuracy of 98%. Thus, a simple PCR assay that is specific for Salmonella spp. and amplifies a chromosomal DNA fragment detected by gel electrophoresis was established through extensive validation and is proposed as an international standard. This study addresses the increasing demand of quality assurance laboratories for standard diagnostic methods and presents findings that can facilitate the international comparison and exchange of epidemiological data.

  • Copyright © 2003 American Society for Microbiology
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Multicenter Validation of the Analytical Accuracy of Salmonella PCR: towards an International Standard
Burkhard Malorny, Jeffrey Hoorfar, Cornelia Bunge, Reiner Helmuth
Applied and Environmental Microbiology Jan 2003, 69 (1) 290-296; DOI: 10.1128/AEM.69.1.290-296.2003

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Multicenter Validation of the Analytical Accuracy of Salmonella PCR: towards an International Standard
Burkhard Malorny, Jeffrey Hoorfar, Cornelia Bunge, Reiner Helmuth
Applied and Environmental Microbiology Jan 2003, 69 (1) 290-296; DOI: 10.1128/AEM.69.1.290-296.2003
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KEYWORDS

Polymerase Chain Reaction
Salmonella

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