Application of magnetic carriers for whole DNA isolation and detection of probiotic Lactobacillus gasseri K7 strain in complex samples

Application of magnetic carriers for whole DNA isolation and detection of probiotic Lactobacillus gasseri K7 strain in complex samples

Abstrakt

The importance of lactic acid bacteria (LAB) for dairy industry is well known. Increasing interest has been focused on some LAB strains that are proposed to be probiotic. Lactobacillus gasseri K7 is a probiotic strain, producer of gassericins K7 A and B1, which was tested in vivo in mice. Falsely negative results can occur in PCR identification of target cells due to the presence of extracellular PCR inhibitors in tested samples - mouse faeces. The problem can be solved by solid phase systems adsorbing genomic DNA. Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl dimethylmethacrylate) (P(HEMA-co-GMA) were used for the isolation of whole DNA from mouse faeces. These microspheres reversibly bind DNA in the presence of high concentrations of poly(ethylene glycol) (PEG 6000) and sodium chloride2. The release of adsorbed DNA from the microsphereses was optimalised using a low-ionic-strength TE buffer of different pH and temperature. The presence of target DNA and quality of isolated DNA were checked by PCR amplification with bacteriocin-specific primers1. It was shown that DNA eluted from microspheres can be quantified in real-time PCR. The sensitivity of PCR was evaluated.

The importance of lactic acid bacteria (LAB) for dairy industry is well known. Increasing interest has been focused on some LAB strains that are proposed to be probiotic. Lactobacillus gasseri K7 is a probiotic strain, producer of gassericins K7 A and B1, which was tested in vivo in mice. Falsely negative results can occur in PCR identification of target cells due to the presence of extracellular PCR inhibitors in tested samples - mouse faeces. The problem can be solved by solid phase systems adsorbing genomic DNA. Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl dimethylmethacrylate) (P(HEMA-co-GMA) were used for the isolation of whole DNA from mouse faeces. These microspheres reversibly bind DNA in the presence of high concentrations of poly(ethylene glycol) (PEG 6000) and sodium chloride2. The release of adsorbed DNA from the microsphereses was optimalised using a low-ionic-strength TE buffer of different pH and temperature. The presence of target DNA and quality of isolated DNA were checked by PCR amplification with bacteriocin-specific primers1. It was shown that DNA eluted from microspheres can be quantified in real-time PCR. The sensitivity of PCR was evaluated.

Lactobacillus gasseri K7, real time PCR, magnetic particles

Lactobacillus gasseri K7, real time PCR, magnetic particles

TRACHTOVÁ, S.; OBERMAJER, T.; SPANOVA, A.; MATIJASIC, B.B.; ROGELJ,I.; HORAK,D.; RITTICH, B.

2010

28.10.2009

OOO "Gastro"

Sankt-Petersburg

Abstracts of XXXII International Congress of the Society for Microbial Ecology and Diease

A27

A28

1

http://hdl.handle.net/