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Applied and Environmental Microbiology, January 2003, p. 290-296, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.290-296.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Burkhard Malorny,1 Jeffrey Hoorfar,2 Cornelia Bunge,1 and Reiner Helmuth1*

Federal Institute for Health Protection of Consumers and Veterinary Medicine, D-12277 Berlin, Germany,1 Danish Veterinary Institute, DK-1790 Copenhagen, Denmark2

Received 24 May 2002/ Accepted 8 October 2002

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.

* Corresponding author. Mailing address: Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin, National Salmonella Reference Laboratory, Diedersdorfer Weg 1, D-12277 Berlin, Germany. Phone: (49 30) 8412 2233. Fax: (49 30) 8412 2953. E-mail: r.helmuth{at}bgvv.de.

Applied and Environmental Microbiology, January 2003, p. 290-296, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.290-296.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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