- DNA extraction DNA was extracted

from culture broths obtained after the enrichment step (from non-diluted to 10-6 dilution). One ml of each homogenized content from each dilution was transferred in a microcentrifuge tube and centrifuged at 12,000 × g for 2 min using a bench-top centrifuge. The pellets were transferred into 1 ml of sterile molecular grade water. The DNA was extracted using the Wizard Genomic DNA purification kit (Promega, Madisson, WI, USA) with addition of lysozyme (10 mg/ml, Eurogentec, Seraing, Belgium), as recommended for Gram-positive bacteria. DNA samples https://www.selleckchem.com/products/KU-60019.html were analyzed pure or 10 fold-diluted in case of PCR inhibition. Molecular protocols for bifidobacteria detection PCR-RFLP protocol based on the 16S rDNA gene (PCR-RFLP) The PCR method for the detection of the Bifidobacterium genus consisted of primers targeting the 16SrDNA gene followed by a digestion using 2 restriction enzymes for species detection. A 1050 bp amplicon of the 16S rDNA gene was generated using primers: 16S up: 5′-AAT AGC TCC TGG AAA CGG GT-3′ and 16S down: 5′-CGT AAG GGG CAT GAT GAT CT-3′ (Eurogentec, Seraing, Belgium; Genbank PUID: updown16S EOY_1) as previously described [23]. The digestion of the PCR products for species detection was performed using two enzymes: AluI and TaqI (Roche;

Basel, Switzerland) as described previously BAY 63-2521 ic50 [23]. Following the digestion, the products were analyzed by gel electrophoresis using 2.5% agarose gel. The profiles were analyzed using the Kodak 1D software (Thermolabsystems, Brussels, Belgium). Real-time PCR protocol based on the hsp60 gene A first step consisted in PCR targeting the hsp60 gene for detection of positive samples for bifidobacteria. Next, real-time PCR was applied to positive samples for species identification. The PCR procedure for detection of the Bifidobacterium genus

was described in a previous study [15]. The following primers were used: B11 up: 5′-GTS CAY GAR GGY CTS AAG AA-3′ and B12 down: 5′-CCR TCC TGG CCR ACC TTG T-3′ Atorvastatin (Sigma Genosys, UK; Genbank PUID: hsp60updown EOY_2), to obtain a 217 bp amplicon of the hsp60 gene. An internal DNA control was included in each reaction. The products were analyzed by gel electrophoresis using 1.5% agarose gels. Species detection was carried out by real-time PCR using TaqMan technology. The degenerated primers specific to the Bifidobacterium genus were the same than those utilized for the PCR on the hsp60 gene. One probe was chosen from hsp60 sequences of B. pseudolongum after hsp60 gene sequencing of 40 bifidobacteria strains: 3 B. adolescentis, 3 B. pseudocatenulatum, 2 B. breve, 2. B. longum, 2 B. bifidum, 14 B. pseudolongum and 10 B. thermophilum (data not shown). The bifidobacteria sequences were aligned using the program LY294002 chemical structure clustalw from the European Bioinformatics Institute (http://​www.​ebi.​ac.​uk/​clustalw/​). The alignments revealed specific sequences for B. pseudolongum.