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Applied and Environmental Microbiology, June 2006, p. 3896-3900, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02112-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

Petra F. G Wolffs,^1,2* Kari Glencross,¹ Romain Thibaudeau,¹ and Mansel W. Griffiths¹

Canadian Research Institute for Food Safety, 43 McGilvray St., Guelph, Ontario, Canada N1G 2W1,¹ Department of Medical Microbiology, University Hospital Maastricht, Maastricht, The Netherlands²

Received 7 September 2005/ Accepted 17 March 2006

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 µm to remove large particles and of 0.22 µm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 x 10² CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.

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* Corresponding author. Mailing address: Department of Medical Microbiology, University Hospital Maastricht, P. Debyelaan 25, 6202 AZ Maastricht, The Netherlands. Phone: 31 43-3876642. Fax: 31 43-3876643. E-mail: pwolf{at}lmib.azm.nl.

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Applied and Environmental Microbiology, June 2006, p. 3896-3900, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02112-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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