PCR screening of Lactobacillus species in which the detection rate significantly differed between UC patients and healthy controls

The primers used in the investigation are listed in Table 1. BLAST searches were performed to determine the specificity of the primers. For DNA extracts from each fecal sample, PCR amplification reactions (in a total volume of 20 μL) were performed on a PCR thermal cycler (PC-808; Astec, Fukuoka, Japan). Each reaction was performed in a 20 μL reaction volume containing 2 μL of template DNA, 2 μL of 25 mM MgCl₂, 0.5 μL of 10 mM dNTPs, 5 μL of 10×PCR buffer, 10 pmol of each primer, and 1.0 U of Taq DNA polymerase (Promega, Madison, WI, USA). PCR was performed using the following parameters: an initial denaturation at 95°C for 3 min, followed by 36 cycles of 94°C for 30 s; 56°C for 40 s; 72°C for 40 s; and a final elongation period at 72°C for 5 min. The amplifications were confirmed by standard electrophoresis of the PCR products (5 μL) using 2% agarose gels (Sigma–Aldrich, St. Louis, MO, USA) prepared in 0.5× TBE buffer (45 mM Tris base, 45 mM boric acid, and 1 mM EDTA [pH 8.0]) and visualized by silver staining. A Lactobacillus species was considered to be detectable in the fecal samples of participants when primed PCR products were obtained. Thus, we screened out the Lactobacillus species that had significantly different detection rates between the patients and controls.