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Poster communications

Culture-Independent Analysis of Bacterial Ecosystems in Milk and Cheese Samples Using Denaturing High Performance Liquid Chromatography

Abstract : Denaturing high performance liquid chromatography (DHPLC) technique is an ion-pair reverse-phase chromatography. It associates the principles of HPLC with methods of DNA fragments separation in denaturing conditions (i.e. TTGE/DGGE). It uses specific cartridges that elute double stranded DNA molecules according to their melting temperature (Tm) and size. In non-denaturing conditions, it allows the detection of nucleotide polymorphisms. DHPLC has been commonly used since the 1990s in medical research to detect DNA mutations and polymorphisms. More recently, DHPLC protocols were adapted for the separation of bacterial PCR-amplified fragments. We tested the feasibility of applying DHPLC (WAVE system; Transgenomic Ltd) to the field of microbial ecology of dairy products for: i) identification and separation of pure strains in mixed cultures (lactic acid bacteria, staphylococci), ii) analysis of bacterial biodiversity in dairy products, iii) diagnosis of mastitis (by analysis of milk samples), and iv) following the dynamics of bacterial populations from milk to ripened cheese. We then compared the potential of DHPLC with TTGE/DGGE methods (Ogier et al. 2004). Results showed different advantages of the DHPLC compared to TTGE/DGGE methods. For example, we observed a better separation of the staphylococci species. Moreover, DHPLC offers a greater detection sensitivity using fluorescence, and an automatic collection of DNA fragments for further sequencing or other uses. We conclude that the DHPLC technique is a promising novel tool that complements the current repertory of analytic methods at the molecular level, and is valuable for studies the complex ecosystem of dairy products. Keywords: Bacterial ecosystem, dairy products, 16S rDNA, DHPLC
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Poster communications
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https://hal.archives-ouvertes.fr/hal-03164496
Contributor : Marina Gregoire <>
Submitted on : Wednesday, March 10, 2021 - 6:59:54 AM
Last modification on : Tuesday, March 16, 2021 - 3:21:41 AM

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Marina Aigle, Jean-Claude Ogier, Agnes Delacroix-Buchet. Culture-Independent Analysis of Bacterial Ecosystems in Milk and Cheese Samples Using Denaturing High Performance Liquid Chromatography. 5th IDF Symposium on Cheese Ripening, Mar 2008, BERN, Switzerland. ⟨hal-03164496⟩

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