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 . The digestion of the PCR products for species detection was performed using two enzymes: AluI and TaqI (Roche; Basel, Switzerland) as described previously . 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 . 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' (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 ClustalW from the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw/). The alignments revealed specific sequences for B. pseudolongum. From these sequences, the probe was designed using the primers and probes design guidelines provided by Applied Biosystems (Applied Biosystems, Foster city, USA). To check for specificity, the selected probes were compared to all available hsp60 gene sequences using the BLAST database search program (http://www.ncbi.nlm.nih.gov/BLAST/). The B. pseudolongum probe was VIC- CTCCGACGCGATCGT-DQ (Applied Biosystems, Foster city, USA; Genbank PUID: TaqManPseudolongum EOY_3).
Amplification reaction mixtures contained between 10 to 50 ng of DNA, 12.5 ml of qPCR tm Mastermix (Eurogentec, Seraing, Belgium), 960 nM of each primer, 50 to 150 nM of fluorogenic probe, and 5 mM MgCl2 in a total volume of 25 μl. In each microwell plate, one well was used as non-template control, which contained all the reagents except the DNA sample. The amplification, 50°C for 2 min, 95°C for 10 min, and then 40 cycles of two-temperature PCR (95°C for 30 s and 60°C for 90 s) and detection was carried out on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster city, USA). The PCR results for the samples were expressed as delta Rn (relative sensitivity) fluorescence signal. A sample was considered as positive when the relative fluorescence value was higher than 500.
The degenerated pair of primers specific to the Bifidobacterium genus was tested for its specificity in a previous study . To check specificity of the probe, a real-time PCR was performed on 55 strains belonging to 13 different Bifidobacterium species (Table 1). The limit of detection was of minimum 10 ng of DNA/reaction.