Easy DNA extraction method and optimisation of PCR-temporal temperature gel electrophoresis to identify the predominant high and low GC-content bacteria from dairy products
2007
Parayre-Breton, Sandrine | Falentin, Hélène | Madec, Marie-Noelle | Sivieri, Katia | Le Dizes, Anne-Sophie | Sohier, Danièle | Lortal, Sylvie
Molecular fingerprinting of bacterial ecosystems has recently increased in food microbiology. The aim of this work was to develop a rapid and easy method to extract DNA from various cheeses, and to optimize the separation of low and high GC-content bacteria by PCR-Temporal Temperature Gel Electrophoresis (PCR-TTGE). Seventy six strains belonging to 50 of the most frequently encountered bacterial species in dairy products were used to construct a database. Specific PCR-TTGE ladders containing 17 species forming a regular scale were created. Amplicons of these species were sequenced and the GCcontent plotted against the migration distance: the correlation coefficients obtained were r2=0.97 and r2=0.99, respectively for high and low GCcontents. The extraction method developed did not use any harmful solvent such as phenol/chloroform. The concentrations of DNA extracted from hard cooked and pressed cheeses, quantified by picogreen molecular probes, were between 0.7 and 6 μg/g for core samples and 8 to 30 μg/g for rind samples. Experimental as well as commercial dairy products were analysed using the developed method and the reproducibility of the profiles was 89%. The method appears to be particularly efficient in the characterization of the ecosystem of cheese rinds.
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