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).