Total DNAs were obtained as described by De Los Reyes-Gavilàn et al. . The concentration selleck screening library and purity of DNA was assessed by a NanoDrop® ND-1000 Spectrophotometer
(Thermo Fisher Scientific Inc.). A primer pair (Invitrogen Life Technologies, Milan, Italy), LpigF/LpigR (5′-TACGGGAGGCAGCAGTAG-3′ and 5′-CATGGTGTGACGGGCGGT-3′) , corresponding to the position 369-386, and 1424-1441, respectively, of the 16S rRNA gene sequence of L. mucosae, (accession number AF126738) was used to amplify the 16S rRNA gene fragment of presumptive lactic acid bacteria. Fifty microliters of each PCR mixture contained 200 μM of each dNTP, 1 μM of both forward and reverse primer, 2 mM MgCl2, 2 U of Taq DNA polymerase (Invitrogen Life Technologies)
in the supplied buffer, and approximately 50 ng of DNA. PCR amplification Defactinib research buy was carried out using the GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, USA). PCR products were separated by electrophoresis on 1.5% (wt/vol) agarose gel (Gibco BRL, France) stained with ethidium bromide (0.5 mg/ml). The amplicons were eluted from gel and purified by the GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences, Milan, Italy). DNA sequencing reactions were carried out by MWG Biotech AG (Ebersberg, Germany) using both, forward and reverse, primers. Taxonomic identification of strains was performed by comparing the sequences of each isolate with those reported in the Basic BLAST database http://www.ncbi.nlm.nih.gov. Primers casei/para were used to discriminate between the species L. casei, L. paracasei and L. rhamnosus . Primers pheS-21-F/pheS-23-R were used to identify Enterococcus species . Primers designed on recA gene were also used to discriminate between the species L. plantarum, L. pentosus and L. paraplantarum. Part of the recA gene was amplified using the degenerate
primer pair (MWG Biotech AG, Ebersberg, Germany) recALb1F 5′-CRRTBATGCGBATGGGYG-3′/recALb1R Pembrolizumab datasheet 5′-CGRCCYTGWCCAATSCGRTC-3′ derived from the homologous regions of the recA gene sequences of L. plantarum (accession no. AJ621668). PCR reactions and separation, and purification and sequencing of amplicons were carried out as described for 16S rRNA gene. Genotypic characterization by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis Genomic DNA from each isolates was extracted as described above. Three oligonucleotides, P4 5′-CCGCAGCGTT-3′, P7 5′-AGCAGCGTGG-3′ and M13 5′-GAGGGTGGCGGTTCT-3′ [55, 56], with arbitrarily chosen sequences, were used for isolates biotyping. Reaction mixture and PCR conditions for primers P4 and P7, and primer M13 were according to De Angelis et al. [55, 56]. PCR products (15 μl) were separated by electrophoresis at 100 V for 200 min on 1.