ena-STUDY-LNU-30-04-2015-16:13:49:716-19
Gut microbiome pattern in infants developing IgE-associated  eczema in the first 2 years of life and in controls
Early intestinal colonization may impact the risk of developing eczema. We analyzed the gut microbiome using barcoded 16S rRNA 454 pyrosequencing in collected stool samples at 1 week, 1 month and 12 months of age in 10 infants that developed IgE-associated eczema and in 10 infants that did not develop any manifestation of allergic disease or IgE-sensitization. Children were clinically assessed by a pediatric allergist and they were skin-pricktested to verify or exclude allergic sensitization. DNA from collected stool samples was extracted with Ultra clean fecal DNA isolation (MoBio, Naxo ltd, California, USA) from 100 mg of each stool sample according to the manufacturer’s instructions. Final elution was made with 2x50 µl elution buffer (solution S5).  For each sample, three 50 µl PCR mixes were prepared containing 1X PCR buffer, 200 µM dNTP PurePeak DNA polymerase (Pierce Nucleic Acid Technologies, Milwaukee, USA), 50 mM of each primer, 0.5 U Phusion F-530L enzyme (Finnzyme, Massachusetts, USA) and 1 µL template DNA for the fecal samples. Primer pairs used amplify the hypervariable 16 rRNA regions V3-V4 were 341f (5'CCTACGGGNGGCWGCAG) with adaptor B and 805r, (5' GACTACHVGGGTATCTA ATCC) with adaptor A, and a sample specific sequence tag of 7 nucleotides as a development of previous study conducted by Andersson et al 2008. A PCR negative template control was also made for each primer pair. The PCR conditions used were 95°C for 5 minutes, followed by 25 cycles of 95°C for 40 sec, 58°C for 40 sec, and 72°C for 1 min, followed by a final extension of 72°C for 7 min for the fecal samples. The samples and the negative template control were then run on an agarose gel (1% wv in TBE buffer) for quality control. The three PCR reactions were pooled and for the fecal samples 45 µl of each pooled PCR reaction was purified using Agencourt AMPure beads (Beckman Coulter, California, USA) according to manufacturer’s instructions with final elution in 15 µl 1X TE buffer. The purified PCR products were diluted to a concentration of approximately 3 ng/µl before pooling them together for the multiplexed pyrosequencing. All concentrations were determined using the Qubit system (Invitrogen, California, USA). The DNA pools were finally sequenced on the 454-FLX GS-100 using Titanium kit (Roche 454 Life Sciences, Branford, CT, USA) .
Early intestinal colonization may impact the risk of developing eczema. We analyzed the gut microbiome using barcoded 16S rRNA 454 pyrosequencing in collected stool samples at 1 week, 1 month and 12 months of age in 10 infants that developed IgE-associated eczema and in 10 infants that did not develop any manifestation of allergic disease or IgE-sensitization. Children were clinically assessed by a pediatric allergist and they were skin-pricktested to verify or exclude allergic sensitization. DNA from collected stool samples was extracted with Ultra clean fecal DNA isolation (MoBio, Naxo ltd, California, USA) from 100 mg of each stool sample according to the manufacturer’s instructions. Final elution was made with 2x50 µl elution buffer (solution S5).  For each sample, three 50 µl PCR mixes were prepared containing 1X PCR buffer, 200 µM dNTP PurePeak DNA polymerase (Pierce Nucleic Acid Technologies, Milwaukee, USA), 50 mM of each primer, 0.5 U Phusion F-530L enzyme (Finnzyme, Massachusetts, USA) and 1 µL template DNA for the fecal samples. Primer pairs used amplify the hypervariable 16 rRNA regions V3-V4 were 341f (5'CCTACGGGNGGCWGCAG) with adaptor B and 805r, (5' GACTACHVGGGTATCTA ATCC) with adaptor A, and a sample specific sequence tag of 7 nucleotides as a development of previous study conducted by Andersson et al 2008. A PCR negative template control was also made for each primer pair. The PCR conditions used were 95°C for 5 minutes, followed by 25 cycles of 95°C for 40 sec, 58°C for 40 sec, and 72°C for 1 min, followed by a final extension of 72°C for 7 min for the fecal samples. The samples and the negative template control were then run on an agarose gel (1% wv in TBE buffer) for quality control. The three PCR reactions were pooled and for the fecal samples 45 µl of each pooled PCR reaction was purified using Agencourt AMPure beads (Beckman Coulter, California, USA) according to manufacturer’s instructions with final elution in 15 µl 1X TE buffer. The purified PCR products were diluted to a concentration of approximately 3 ng/µl before pooling them together for the multiplexed pyrosequencing. All concentrations were determined using the Qubit system (Invitrogen, California, USA). The DNA pools were finally sequenced on the 454-FLX GS-100 using Titanium kit (Roche 454 Life Sciences, Branford, CT, USA) .