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Journal of Clinical Microbiology, September 2000, p. 3429-3435, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Automated 5' Nuclease PCR Assay for Identification of Salmonella enterica

J. Hoorfar,1,* P. Ahrens,1 and P. Rådström2

Danish Veterinary Laboratory, Copenhagen, Denmark,1 and Department of Applied Microbiology, Lund University, Lund, Sweden2

Received 21 March 2000/Returned for modification 1 June 2000/Accepted 26 June 2000

A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [Delta Rn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (Delta Rn, <= 0.5). All 100 rough Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20°C. Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for identification of Salmonella, particularly in large reference laboratories.

* Corresponding author. Mailing address: Department of Microbiology, Danish Veterinary Laboratory, 27 Bülowsvej, DK-1790 Copenhagen V, Denmark. Phone: 45-35300251. Fax: 45-35300120. E-mail: jho{at}svs.dk.

Journal of Clinical Microbiology, September 2000, p. 3429-3435, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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