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

• Published in: Journal of microbiological methods Vol. 69; no. 3; pp. 431 - 441
• Main Authors: Parayre, Sandrine, Falentin, Hélène, Madec, Marie-Noëlle, Sivieri, Katia, Le Dizes, Anne-Sophie, Sohier, Danièle, Lortal, Sylvie
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.06.2007; Elsevier Science; Elsevier
• Subjects: Bacteria; Bacteria - chemistry; Bacteria - classification; Bacteria - genetics; Bacteria - isolation & purification; Base Composition; Biological and medical sciences; Biotechnology - methods; Cheese - microbiology; Colony Count, Microbial; Dairy products; Dairy Products - microbiology; DNA, Bacterial - analysis; DNA, Bacterial - isolation & purification; Electrophoresis, Agar Gel - methods; Food industries; Fundamental and applied biological sciences. Psychology; GC-content; Identification; Life Sciences; Microbiology and Parasitology; Milk and cheese industries. Ice creams; Polymerase Chain Reaction - methods; Reproducibility of Results; Temperature; Time Factors; Yogurt - microbiology; Bacteria; Dairy products; Identification; GC-content; Polymerase chain reaction; GC rich sequence; Gel electrophoresis; Dairy industry; Identification test; Extraction; bacterium; PCR-TTGE; bacteria; dairy product; adn; electrophoresis; lactic acid bacteria; identification; dna; produit laitier; bactérie; température; électrophorèse; dairy products; pcr; bactérie lactique
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2007.02.011

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 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.