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Quantification of Campylobacter spp. in chicken rinse samples by using flotation prior to real-time PCR

Wolffs, Petra LU ; Norling, B; Hoorfar, J; Griffiths, M and Rådström, Peter LU (2005) In Applied and Environmental Microbiology 71(10). p.5759-5764
Abstract
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... (More)
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 10(2) CFU/ml could be detected without culture enrichment and amounts as low as 2.6 X 10(3) 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 degrees 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 degrees C; 11% +/- 4% and 10% +/- 2%, respectively, at 20 degrees C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Applied and Environmental Microbiology
volume
71
issue
10
pages
5759 - 5764
publisher
American Society for Microbiology
external identifiers
  • wos:000232504000012
  • pmid:16204485
  • scopus:26844444533
ISSN
0099-2240
DOI
10.1128/AEM.71.10.5759-5764.2005
language
English
LU publication?
yes
id
799d96f4-e433-4740-9d64-bdfc2612d422 (old id 151208)
date added to LUP
2007-06-28 14:47:32
date last changed
2017-01-01 05:01:13
@article{799d96f4-e433-4740-9d64-bdfc2612d422,
  abstract     = {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 10(2) CFU/ml could be detected without culture enrichment and amounts as low as 2.6 X 10(3) 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 degrees 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 degrees C; 11% +/- 4% and 10% +/- 2%, respectively, at 20 degrees C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method.},
  author       = {Wolffs, Petra and Norling, B and Hoorfar, J and Griffiths, M and Rådström, Peter},
  issn         = {0099-2240},
  language     = {eng},
  number       = {10},
  pages        = {5759--5764},
  publisher    = {American Society for Microbiology},
  series       = {Applied and Environmental Microbiology},
  title        = {Quantification of Campylobacter spp. in chicken rinse samples by using flotation prior to real-time PCR},
  url          = {http://dx.doi.org/10.1128/AEM.71.10.5759-5764.2005},
  volume       = {71},
  year         = {2005},
}