Sample Collection and DNA Extraction
Meconium was collected from newborns who had not been weaned within 2 hours after birth, and was labeled control, EB, and C-EB groups. While wearing sterile gloves, the central part of fresh feces was probed and sampled with a sterile cotton swab the size of a soybean (3-5 g), placed into a 2-mL sterile storage tube(Stool collection tube,Zhejiang Gongdong Medical Equipment Co.,Ltd,Zhe jiang ,China), and immediately place into an -80°C freezer for storage.
DNA was extracted from fecal samples according to the instructions of a Fecal Bacteria Genomic DNA Extraction Kit (centrifugal column type,Beijing Tiangen Inc, Beijing, China), then stored in a freezer at -20°C.
All samples of bacterial DNA were tested for quality. The 16S V3-V4 region of the sample was amplified by PCR. The 16S rRNA v3-v4 specific primers (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′ and 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′) were selected for PCR amplification. Then, the sample library was mixed quantitatively and the library was sequenced by Illumina MiSeq system (MiSeq,Illumina, San Diego, CA, USA).