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Applied and Environmental Microbiology, October 2005, p. 5759-5764, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5759-5764.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Quantification of Campylobacter spp. in Chicken Rinse Samples by Using Flotation prior to Real-Time PCR

Department of Applied Microbiology, Lund Institute of Technology, Lund University, SE-221 00 Lund, Sweden,¹ Canadian Research Institute for Food Safety, Guelph University, Guelph, Ontario N1G2W1, Canada,² Quintessence Research AB, SE-746 93 Bålsta, Sweden,³ Danish Institute for Food and Veterinary Research, DK-1790 Copenhagen, Denmark⁴

Received 18 January 2005/ Accepted 29 April 2005

Real-time PCR is fast, sensitive, specific, and can deliver quantitative data; however, two disadvantages are that this technology is sensitive to inhibition by food and that it does not distinguish between DNA originating from viable, viable nonculturable (VNC), and dead cells. For this reason, real-time PCR has been combined with a novel discontinuous buoyant density gradient method, called flotation, in order to allow detection of only viable and VNC cells of thermotolerant campylobacters in chicken rinse samples. Studying the buoyant densities of different Campylobacter spp. showed that densities changed at different time points during growth; however, all varied between 1.065 and 1.109 g/ml. These data were then used to develop a flotation assay. Results showed that after flotation and real-time PCR, cell concentrations as low as 8.6 x 10² CFU/ml could be detected without culture enrichment and amounts as low as 2.6 x 10³ CFU/ml could be quantified. Furthermore, subjecting viable cells and dead cells to flotation showed that viable cells were recovered after flotation treatment but that dead cells and/or their DNA was not detected. Also, when samples containing VNC cells mixed with dead cells were treated with flotation after storage at 4 or 20°C for 21 days, a similar percentage resembling the VNC cell fraction was detected using real-time PCR and 5-cyano-2,3-ditolyl tetrazolium chloride-4',6'-diamidino-2-phenylindole staining (20% ± 9% and 23% ± 4%, respectively, at 4°C; 11% ± 4% and 10% ± 2%, respectively, at 20°C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method.

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