Stool kit

Influenza virus in the lung and small intestine were detected by PCR. The primer sequences to detect the gene encoding the matrix protein within the influenza virus were as follows: 5′-GGACTGCAGCGTAGACGCTT-3′ (forward) and 5′-CATCCTGTTGTATATGAGGCCCAT-3′ (reverse). Intestinal bacterial genomic DNA was extracted from the stool using a stool kit (QIAGEN) according to the manufacturer’s instruction (the optional high-temperature step was performed). The abundance of total and specific intestinal bacterial groups was measured by real-time PCR with corresponding 16S rDNA gene primers (Sangon Biotech; Table S1). The number of E. coli in stool was detected by the 3M Petrifilm E. coli/Coliform Count Plate according to the manufacturer’s instructions.

All fecal samples were thawed, and bacterial DNA was extracted by using the QIAGEN Stool kit (Germany) according to the manufacturer's protocol. Initially, the bead-beating procedure was performed at 5000 rpm for ½ min. DNA concentration was evaluated using NanoPhotometer TM (Germany) [23 (link)].

DNA was extracted from feces and bacterial precipitates using a Qiagen Stool Kit (Hilden, Germany) and a modified cell lysis protocol. Qualitative procedures and the method for detecting bacterial population were performed as described previously38 (link). And the copy number of target DNA was calculated by comparison with serially diluting standards, (10¹ to 10⁸ copies of plasmid DNA containing the respective amplicon for each set of primers) running on the same plate. Bacterial quantity was expressed as log₁₀ bacteria per gram of stool. The specific primers used were as follows: Bacteroides-Prevotella: 5′-GAAGGTCCCCCACATTG-3′ (sense),
5′-CAATCGGAGTTCTTCGTG-3′ (anti-sense);
Bifidobacteria: 5′-GGGTGGTAATGCCGGATG-3′ (sense),
5′-TAAGCCATGGACTTTCACACC-3′ (anti-sense);
Enterobacteriacea: 5′-CATTGACGTTACCCGCAGAAGAAGC-3′ (sense),
5′-CTCTACGAGACTCAAGCTTGC-3′ (anti-sense);
Lactobacillus: 5′-AGCAGTAGGGAATCTTCCA-3′ (sense),
5′-ATTTCACCGCTACACATG-3′ (anti-sense)39 (link).