bAsthma cGrass Pollen Sensitization dAllergic Atopic Dermatitis

bAsthma. cGrass Pollen Sensitization. dAllergic Atopic Dermatitis. eOral Allergy Idasanutlin datasheet Syndrome. fCow’s Milk Allergy. Allergometric tests Skin prick tests were performed following established guidelines [26]. The following allergens were tested: cow’s milk, egg, soy bean, wheat, peanut, codfish, grass pollen, Dermatophagoides pteronyssinus Dermatophagoides farinae, and cat dander. Other allergens were tested on the basis of the child’s history. Data of the skin prick tests

were used to determine the presence of atopic sensitization in the definition of allergic or non-allergic atopic dermatitis. The determination of total serum IgE was performed by ELISA test; the values were assumed as normal or increased in comparison with the ones from children of the same age group [27]. The determination of specific IgE was performed by UNICAP 1000 (Phadia) in all patients for the following allergens: cow’s milk, egg, soy bean, wheat, peanut, BAY 63-2521 cost codfish, Bermuda grass, timothy grass, D. pteronyssinus D. farinae, and cat dander. Other allergens were tested on the basis of the child’s history. DNA extraction and polymerase chain reaction (PCR) Total DNA from faecal material was ARS-1620 extracted

using QIAamp DNA Stool Mini Kit (Qiagen) according to the modified protocol reported by Candela et al.[24]. Final DNA concentration was determined using NanoDrop ND-1000 (NanoDrop Technologies). PCR amplifications were performed with Biometra Thermal Cycler T Gradient (Biometra). The 16 S rRNA gene was amplified using universal forward primer 27 F and reverse primer r1492, following the protocol described by Candela et al.[24]. PCR products were purified by using the Wizard

SV gel and PCR clean-up System kit (Promega), eluted in 20 μl of sterile water and quantified with the DNA 7500 LabChip Assay kit and BioAnalyzer 2100 (Agilent Technologies). All the oligonucleotide Acesulfame Potassium primers used for PCR reactions and probe pairs employed for the array construction were synthesized by Thermo Electron. HTF-microbi.Array analysis The HTF-Microbi.Array utilized in this study is based on the Ligase Detection Reaction-Universal Array (LDR-UA) approach [28] and enables specific detection and quantification of the 16 S rRNA from 31 phylogenetically related groups of the human intestinal microbiota (Additional file 1). The original HTF-Microbi.array [24] was updated to include a probe for the detection of A. muciniphila. The new probe was designed and validated as reported by Candela et al.[24] (Additional file 2). Sequences of the entire probe set of the HTF-Microbi.Array are reported in Additional file 3.

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