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Applied and Environmental Microbiology, May 2007, p. 3040-3048, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02823-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Optimization of a 12-Hour TaqMan PCR-Based Method for Detection of Salmonella Bacteria in Meat{dagger} ,{triangledown}

M. H. Josefsen,1 M. Krause,1 F. Hansen,2 and J. Hoorfar1*

National Food Institute, Bülowsvej 27, DK-1790 Copenhagen,1 Danish Meat Research Institute, Maglegårdsvej 2, DK-4000 Roskilde, Denmark2

Received 5 December 2006/ Accepted 27 February 2007

We developed a 12-h Salmonella detection method, based on 8 h of preenrichment, followed by automated DNA extraction and a sensitive real-time PCR. The method was optimized to obtain the highest possible yield of cells and DNA. The growth of different Salmonella strains in various preenrichment media and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of (i) analyzing larger volumes (1 to 5 ml) from preenriched samples and introducing wash steps prior to DNA extraction, (ii) regulating the amount of paramagnetic particles (increasing it from 60 to 90 µl) in the DNA extraction, (iii) eluting the DNA in reduced volumes (25 or 50 µl rather than 100 µl), and (iv) increasing the PCR template volume (from 5 to 20 µl) were investigated. After 8 h of preenrichment, buffered peptone water yielded the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 µl were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 µl) had no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 µl improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples, with a relative accuracy of 99%, a relative sensitivity of 98%, and a relative specificity of 100%.

* Corresponding author. Mailing address: National Food Institute, Bülowsvej 27, DK-1790 Copenhagen, Denmark. Phone: 45-72346251. Fax: 45-72346001. E-mail: jho{at}dfvf.dk

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{triangledown} Published ahead of print on 9 March 2007.

Applied and Environmental Microbiology, May 2007, p. 3040-3048, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02823-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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