The reactions were carried out in a Veriti 96-well selleck inhibitor thermal cycler (Applied Biosystems, California, USA) as follows: 95°C for 3 min; 30 cycles of 30 s at 95°C, 30 s at the annealing temperature (Tm, Additional file 2: Primers and their annealing temperatures (Tm)), and 90 s at 72°C; 10 min at 72°C, and cooling to 12°C. PCR products were verified by gel (1.2%) electrophoresis and observed by UV selleck compound fluorescence. DNA sizing Size determination of SSR amplification products with motif lengths of 66 bp, 90 bp and 480 bp was performed by 2% agarose gel electrophoresis. Sizing of the other seven SSR loci was performed by capillary electrophoresis on an ABI 3130 genetic analyzer, using fluorophore-labeled primers. The amplification
products were loaded into the genetic analyzer together with 9 μl formamide and 0.5 μl GeneScan 500 LIZ size standard (Applied Biosystems). The results were analyzed
with GeneMapper 4.0 software (Applied Biosystems). DNA sequencing PCR amplification products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). Purified DNA (20–50 ng) was sequenced on both strands using Selleck Trametinib a BigDye terminator v1.1 cycle sequencing kit (Applied Biosystems) and loaded into the ABI 3130 genetic analyzer. Results were analyzed with SeqScape 2.5 software (Applied Biosystems) and DNA sequencing analysis 5.2 software (Applied Biosystems). GenBank numbers of nucleotide sequences for genes LJ_0017, LJ_0648 and LJ_1632: JN012103 – JN 012141, JN 012142 – JN 012180 Axenfeld syndrome and JN 012181 – JN 012219 respectively. Data and statistical analyses tRFLP: The relative abundance of each tRFLP peak was calculated as the peak area divided by the total area summed over all peaks in
a sample. A statistical analysis was performed for each of the four main tRFLP peaks (74 bp, 181 bp, 189 bp and 566 bp) separately. M-ANOVA (JMP 8.0) was performed based on the relative abundance of each tested peak in each sample to compare its presence among the 50 tested samples under three parameters (geographical location, taxonomy and food classification). The software R was used to present the relative abundances of the tRFLP patterns, split into eight levels. Sequence comparison: The obtained 16 S rDNA sequences were compared to all available sequences using the NCBI BLAST algorithm for species identification. The analysis of the sequence variation data was performed on the combined sequences of the three conserved hypothetical genes for each of the 46 strains. One strain (LJ_56) did not give any amplification product and was therefore excluded from the MLST analysis. Multiple sequence alignments were performed using CLUSTALW software . The alignment files were converted to MEGA format and used to evaluate genetic relationships among the strains by the unweighted pair group method with arithmetic mean (UPGMA) (MEGA 4.0 ). Allele analysis: A nonparametric analysis of allelic variation was used for all 47 L.