Quantitative Detection of Strains in Feces Using Strain-Specific Primers.

We adopted a bioinformatics-based technique to identify strain-specific markers, which were then used to quantify the abundances of three distinct sup. strains in fecal samples of humans and mice. A pangenome analysis of 205 sup. genomes revealed the accumulation of considerable strain-specific genes within this species; specifically, 28.7% of the total identified genes were strain-specific. We identified 32, 14, and 49 genes specific to sup. RG4-1, sup. M1-20-R01-3, and sup. FGSZY6M4, respectively. After performing an in silico validation of these strain-specific markers using a nucleotide BLAST against both the sup. genome database and an NR/NT database, RG4-1_01874 (1331 bp), M1-20-R01-3_00324 (1745 bp), and FGSZY6M4_01477 (1691 bp) were chosen as target genes for strain-specific quantification. The specificities of the qPCR primers were validated against 47 non-target microorganisms and fecal baseline microbiota to ensure that they produced no PCR amplification products. The performance of the qPCR primer-based analysis was further assessed using fecal samples. After oral administration, the target strains appeared to efficiently colonize both the human and mouse guts, with average population levels of >10 CFU/g feces. The bioinformatics pipeline proposed here can be applied to the quantification of various bacterial species.