Kit and method for diagnosis of gastric cancer using analysis of bacteria meta-genome
A kit and technology for gastric cancer, applied in the direction of biochemical equipment and methods, microbial determination/testing, etc.
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Embodiment 1
[0083] Example 1. Analysis of In vivo Absorption, Distribution and Excretion Patterns of Enterobacteria and Bacteria-Derived Extracellular Vesicles
[0084] To assess whether enterobacteria and bacterial-derived extracellular vesicles are systemically absorbed through the gastrointestinal tract, experiments were performed using the following method. More specifically, 50 μg each of fluorescently labeled Pseudomonas intestinal bacteria and extracellular vesicles (EVs) derived from the bacteria were orally administered to the gastrointestinal tract of mice, and at 0 hours, and at Fluorescence was measured after 5 minutes, 3 hours, 6 hours and 12 hours.
[0085] As a result of observing the overall image of the mouse, such as Figure 1A As shown, bacteria were not absorbed systemically at the time of administration, but EVs derived from bacteria were absorbed systemically at 5 minutes after administration, and strong fluorescence was observed in the bladder at 3 hours after admin...
Embodiment 2
[0087] Example 2. Vesicle Isolation and DNA Extraction from Blood
[0088] To isolate vesicles and extract DNA from blood and urine, first add blood and urine to a 10 ml tube, centrifuge at 3,500 x g and 4 °C for 10 min, pellet the suspension, and collect only the supernatant , and place it in a new 10ml tube. The collected supernatant was filtered with a 0.22 μm filter to remove bacteria and impurities, then placed in a central centrifugal filter (50 kD) and centrifuged at 1500 × g and 4° C. for 15 minutes to discard material with a size less than 50 kD, Then concentrate to 10ml. Bacteria and impurities were removed again using a 0.22 μm filter, and then the resulting concentrate was subjected to ultracentrifugation at 150,000 × g and 4° C. for 3 hours to remove the supernatant, and the aggregated precipitate was washed with phosphate buffer ( PBS) to obtain vesicles.
[0089] 100 μl of vesicles isolated from serum according to the method described above were boiled at 100...
Embodiment 3
[0092] Example 3. Bacterial metagenomic analysis using DNA from vesicles extracted from blood
[0093] As shown in Table 2 below, using the same method as in Example 2, using blood samples from 67 patients with gastric cancer and 198 normal individuals, urine samples from 61 patients with gastric cancer and 120 normal individuals, the gender and Age-matched, they were then subjected to PCR using 16S rDNA primers to amplify the DNA and subsequently sequenced (Illumina MiSeq sequencer). Output the results as a Standard Flow Diagram (SFF) file, and convert the SFF file to a sequence file (.fasta) and nucleotide quality score file using GS FLX software (v2.9), then determine credit ratings for reads, And fractions with window (29 bps) average base call accuracy less than 99% (Phred score < 20) were removed. After removal of low-quality parts, only reads with a length of 300 bps or greater (Sickle version 1.33) were used, and for operational taxonomic unit (OTU) analysis clusterin...
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