Document Detail

Quantification of Campylobacter spp. in chicken rinse samples by using flotation prior to real-time PCR.
MedLine Citation:
PMID:  16204485     Owner:  NLM     Status:  MEDLINE    
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.
Petra Wolffs; Börje Norling; Jeffrey Hoorfar; Mansel Griffiths; Peter Rådström
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Publication Detail:
Type:  Evaluation Studies; Journal Article    
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  71     ISSN:  0099-2240     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2005 Oct 
Date Detail:
Created Date:  2005-10-05     Completed Date:  2005-12-02     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5759-64     Citation Subset:  IM    
Canadian Research Institute for Food Safety, 43 McGilvray St., Guelph, ON N1G2W1, Canada.
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MeSH Terms
Campylobacter / classification,  genetics,  isolation & purification*
Campylobacter Infections / microbiology*,  veterinary*
Centrifugation, Density Gradient / instrumentation,  methods*
Chickens / microbiology*
Colony Count, Microbial
Culture Media
DNA, Bacterial / analysis
Food Handling / methods*
Polymerase Chain Reaction
Poultry Diseases / microbiology*
Reg. No./Substance:
0/Culture Media; 0/DNA, Bacterial

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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