The PCR was carried out under the following conditions: 1 cycle of 95°C for 7 min, 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min and 1 cycle of 72°C for 7 min. 500 ng of DNA of PCR product from each sample were used to perform the subsequent TTGE experiments. TTGE buy ABT-888 Analysis of PCR amplicons We used the DCode Universal mutation detection system (Bio-Rad, Paris, France) for the sequence-specific separation https://www.selleckchem.com/products/thz1.html of PCR products. Electrophoresis was performed as previously described [17]. TTGE runs were conducted in triplicate and gel photographed with DigiDoc-It system (UVP, Cambridge, UK). Species-specific PCR We choose to detect those particular species whose presence seems

to be involved in celiac disease [7, 9]. 16S rDNA gene-targeted primers were utilized to detect them. The primers used were ECO-1 5′-gacctcggtttagttcacaga-3′, ECO-2 5′-cacacgctgacgctgacca-3′ for Escherichia coli (585 bp); BV-1 5′-gcatcatgagtccgcatgttc-3′, BV-2 5′-tccatacccgactttattcctt-3′ for

Bacteroides vulgatus (287 bp); g-Ccoc-F 5′-aaatgacggtacctgactaa-3′, g-Ccoc-R 5′-ctttgagtttcattcttgcgaa-3′ www.selleckchem.com/products/MGCD0103(Mocetinostat).html for Clostridium coccoides group (438-441 bp), g-Bifid-F 5′-ctcctggaaacgggtgg-3′, g-bifid-R 5′-ggtgttcttcccgatatctaca-3′ for Bifidobacterium spp (549-563 bp). The PCR were performed as previously described [18]. Data Analysis Agglomerative Hierarchical Classification (AHC.) Dendrogram generated with XLStat 7.5 (Addinsoft, NY, USA) on binary matrix of TTGE variables was evaluated by one-tailed chi-squared test. Data were automatically mean centred and unit variance (UV) scaled. A P value equal or less 0.05 was considered statistically significant. Dice similarity index (S D , mean % ± SD) was calculated within the

respective HC and CD groups to assess inter-individual similarity by the formula S D = (2n AB )/(nA + nB), where n A is the total 17-DMAG (Alvespimycin) HCl number of bands in pattern A, n B is the total number of bands in pattern B and n AB is the number of bands common to pattern A and B. Ecological features. Doc-It LS software (UVP, Cambridge, UK) was used for TTGE bands densitometry peak height quantification, and the correspondent data were analyzed for the microbial biodiversity by Shannon-Wiener index with SigmaPlot 9.0 software. Intra-group variance value (V value) was also calculated. V value defines the variance of data points in each cohort, representing the data dispersion, and indicating the homogeneity/heterogeneity between individuals within a population. In addition, the range-weighted richness (Rr), reflecting the carrying capacity of the duodenal system, was calculated by the formula Rr = N2 XTg, where N is the total number of bands in the TTGE profile and Tg the temperature gradient comprised between the first and the last band of the same pattern [19]. Principal Component Analysis (PCA). Linearly-dependent TTGE variables were ortogonalized in new factorial axes (F1,F2…Fn) through PCA by XLStat 7.5 (Addinsoft).