Biomarker for mental disease
A technique for mental illness, marking
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[0183] Next, the present invention will be described in detail with examples.
[0184] [1] Use strain
[0185] The strains shown in Table 1 stored in the Central Research Institute of Yakult Corporation Headquarters were used. Adjust the initial bacterial count of each strain to make it 1×10 4 around cells.
[0186] Table 1 shows the culture conditions of each strain. The details of the culture conditions A to C are as follows.
[0187] Condition A: In a GAM liquid medium modified with 1% glucose, static culture is carried out at 37°C under anaerobic conditions for 24 to 72 hours.
[0188] Condition B: In an MRS liquid medium, static culture is performed under anaerobic conditions at 37°C for 24 hours to 72 hours.
[0189] Condition C: In the brain heart infusion liquid medium, shaking culture was performed at 37°C under aerobic conditions for 18 hours.
[0190] After measuring the number of bacteria by the DAPI method, these cells are appropriately diluted to a certain number of bacter...
reference example 1
[0194] Atopobacter cluster, Lactobacillus, Lactobacillus brevis, Lactobacillus reuteri subgroup, Lactobacillus sake subgroup, Bifidobacterium, Bacteroides fragilis, Enterococcus, Clostridium sphaeroides, Clostridium tenuis subgroup, Preparation of specific primers for Staphylococcus, Clostridium perfringens and Enterobacteriaceae
[0195] Table 2 shows the primers used in the above-mentioned measurement of the number of intestinal bacteria. In addition, documents describing each primer are also shown in Table 2.
[0196] [Table 2]
[0197]
[0198] A: Matsuki T, Watanabe K, Fujimoto J, et al. Quantitative PCR with16SrRNA-gene-targeted species-specific primers for analysis of human intestinalbifidobacteria.Applied and Environmental Microbiology 2004;70:167-73.
[0199] B: Matsuda K.,Tsuji H.,Asahara T.,Matsumoto K.,Takada T.,and NomotoK.Establishment of an Analytical System for the Human Fecal Microbiota,Basedon Reverse Transcription-Quantitative PCR Targeting of Multicopy rRNAMolecul...
reference example 2
[0207] Preparation of standard curve used in RT-PCR
[0208] Create a standard curve used when quantifying the target intestinal bacteria in the subject. Specifically, in accordance with the following procedure, the number of intestinal bacteria measured by the DAPI counting method was plotted on the horizontal axis, and C T The values are plotted as a standard curve on the vertical axis.
[0209] 1) Add 400 μL of RNAlater (Ambion) to 200 μL of the bacterial solution of each strain prepared with the strain used in [1] above, and let it stand for 5 minutes at room temperature. After that, it was centrifuged at 13,000 g for 5 minutes, and the supernatant was removed by decantation. To the residue after removing the supernatant, add 450 μL of lysis buffer (prepared by mixing 346.5 μL of RLT buffer, 100 μL of TE, and 3.5 μL of β-Mercaptoethanol per sample) and diameter 0.1mm glass beads (TOMY Seiko) 300mg.
[0210] 2) After placing the sample tube in a shaker (Shake Master), shake f...
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