Quantification of Campylobacter spp. in Chicken Rinse Samples by Using Flotation prior to Real-Time PCR
| Autor: | Börje Norling, Mansel W. Griffiths, Petra F. G. Wolffs, Peter Rådström, Jeffrey Hoorfar |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
DNA
Bacterial Food Handling Colony Count Microbial Buoyant density medicine.disease_cause Polymerase Chain Reaction Applied Microbiology and Biotechnology law.invention Microbiology law Campylobacter Infections Centrifugation Density Gradient Methods medicine Animals Centrifugation Poultry Diseases Polymerase chain reaction Chromatography Ecology biology Campylobacter biology.organism_classification Culture Media Staining Real-time polymerase chain reaction Chickens Quantitative analysis (chemistry) Bacteria Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 71:5759-5764 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.71.10.5759-5764.2005 |
| Popis: | 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 × 10 2 CFU/ml could be detected without culture enrichment and amounts as low as 2.6 × 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°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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|