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Appl. Environ. Microbiol. doi:10.1128/AEM.02823-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella in meat

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

^* To whom correspondence should be addressed. Email: jho{at}dfvf.dk.

	Abstract

We developed a 12-hour Salmonella detection method, based on 8 hours of pre-enrichment followed by automated DNA extraction, and 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 pre-enrichment media, and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of 1) analysing larger volumes (1 to 5 ml) from pre-enriched samples, and introducing wash steps prior to DNA-extraction, 2) regulating the amount of paramagnetic particles (60 to 90 µl) in the DNA-extraction, 3) eluting the DNA in reduced volumes (100 to 25 µl), and 4) increasing the PCR template volume (5 to 20 µl) were investigated.

After 8 hours of pre-enrichment, buffered peptone water (BPW) yielded the highest number of Salmonella. Analysing 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 resulted in approx. 2 Ct-values improvement.

The improved 12-hour PCR method was successfully compared to a reference culture method on 100 minced meat and poultry samples, with a relative accuracy of 99 %, a relative sensitivity of 98 % and a relative specificity of 100 %.