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.
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 GC-content plotted against the migration distance: the correlation coefficients obtained were r(2)=0.97 and r(2)=0.99, respectively for high and low GC-contents. 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 microg/g for core samples and 8 to 30 microg/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.
Parayre
Sandrine
S
Institut National de la Recherche Agronomique, UMR1253, Science et Technologie du Lait et de l'Oeuf, Agrocampus Rennes, 65 rue de Saint Brieuc, F-35042 Rennes Cedex, France.
Falentin
Hélène
H
Madec
Marie-Noëlle
MN
Sivieri
Katia
K
Le Dizes
Anne-Sophie
AS
Sohier
Danièle
D
Lortal
Sylvie
S
D001419:Bacteria / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001482:Base Composition; D001709:Biotechnology / Q000379:methods; D002611:Cheese / Q000382:microbiology; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D003611:Dairy Products / Q000382:microbiology*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors; D015014:Yogurt / Q000382:microbiology
D004269:DNA, Bacterial