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Method for diagnosing colon tumor via bacterial metagenomic analysis

A technology for colon cancer and bacteria, applied in the field of diagnosing colon tumors, can solve problems such as undiagnosed colon tumors

Pending Publication Date: 2019-10-25
MD HEALTHCARE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at the time of colon tumor onset, there is no information on the identification of colon tumors by isolating bacteria and bacteria-derived vesicles from human-derived material such as feces and by analyzing the metagenome present in bacteria-derived vesicles. A report on the method of diagnosing colon tumors based on pathogenic factors such as colon polyps and colon cancer

Method used

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  • Method for diagnosing colon tumor via bacterial metagenomic analysis
  • Method for diagnosing colon tumor via bacterial metagenomic analysis
  • Method for diagnosing colon tumor via bacterial metagenomic analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1. Analysis of In vivo Absorption, Distribution and Excretion Patterns of Enterobacteria and Bacteria-Derived Extracellular Vesicles

[0124] [1] In order to evaluate whether enterobacteria and extracellular vesicles derived from bacteria are absorbed systemically through the gastrointestinal tract, experiments were performed using the following method. More specifically, 50 μg each of fluorescently labeled intestinal bacteria and extracellular vesicles (EVs) derived from the bacteria were orally administered to the gastrointestinal tract of mice, and were administered at 0 hours, and at 5 minutes, 3 hours, and 6 hours. Fluorescence was measured after 1 hour and 12 hours. As a result of observing the overall image of the mouse, such as Figure 1A As shown, the bacteria were not absorbed systemically at the time of administration, but EVs derived from bacteria were absorbed systemically 5 minutes after administration, and strong fluorescence was observed in the b...

Embodiment 2

[0126] [3] Example 2. Vesicle Isolation and DNA Extraction from Feces and Urine

[0127] [4] In order to isolate extracellular vesicles from feces and urine and extract DNA, feces and urine were first added to a 10ml tube, centrifuged at 3500×g and 4°C for 10 minutes, the suspension was pelleted, and only the supernatant was collected. Supernatant, put the supernatant into 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 x g and 4 °C for 15 min to discard material with a size less than 50 kD , and then concentrated 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 h by using a 90ti type rotor to remove the supernatant and the aggregated precipitate Vesicles were obtained by dissolving with phosphate buffered sal...

Embodiment 3

[0132] Example 3. Metagenomic analysis using DNA extracted from vesicles in feces and urine

[0133] DNA was extracted using the same method as in Example 2, which was then subjected to PCR using the 16S rDNA primers shown in Table 1 to amplify the DNA, followed by sequencing (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 parts with window (20 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 clustering was performed using UCLUST and USEARCH according to sequence similarity. In particular, based on sequence similarity values ​​of 94% for genera, 90% for families, 85% for objects, 80% for...

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Abstract

The present invention relates to a method for predicting the incidence of a colon tumor, such as a colon polyp and colon cancer, and a causative factor thereof through metagenomic analysis of a bacterium-derived vesicle present in a human body-derived substance. More particularly, the present invention relates to a method for diagnosing a causative factor of a colon tumor and the risk of the incidence thereof by sequencing a metagenome present in a bacterium-derived vesicle present in excrement or urine. There are trillions of bacteria in an intestine, and the bacteria secrete vesicles out ofthe cells thereof for the purpose of information exchange. The bacteria are not absorbed into epithelial cells in a colon, but the vesicles secreted therefrom pass through the mucous membrane, are absorbed into the colonic epithelial cells, are distributed throughout the body via blood, and then are excreted through a liver and a kidney. The bacteria-derived vesicles may increase or decrease the occurrence of inflammation and cancer, and the present invention can be useful as a method for diagnosing the risk of the incidence of a colon tumor or a causative factor thereof by sequencing a genetic metagenome of a bacterium-derived vesicle present in a human body-derived substance. Further, an early diagnosis can still be made even after the incidence of a colon polyp or the onset of colon cancer, and the incidence of colon cancer can be lowered, and the treatment effect can be enhanced.

Description

technical field [0001] The present disclosure relates to a method for diagnosing colon tumors by metagenomic analysis of bacteria, and more particularly, by analyzing extracellular vesicles derived from specific bacteria through metagenomic analysis of bacteria using a sample derived from a subject A method for diagnosing colon tumors, such as colon polyps, colon cancer, etc., by increasing or decreasing the content of polyps. Background technique [0002] Colon cancer or colorectal cancer is a malignant tumor that occurs in the appendix, colon, and rectum, and occurs in the mucosa, which is the innermost surface of the large intestine. According to a 2006 announcement, colon cancer is the second most common cancer after gastric cancer in Korea, and its frequency has increased dramatically with the recent westernization of dietary habits, and the mortality rate from colon cancer has increased by approximately 80% in the past decade , the growth rate keeps increasing. Colon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q1/689C12Q2600/158C12Q1/6895C12Q2531/113C12Q1/6806C12Q2600/112
Inventor 金润根
Owner MD HEALTHCARE INC
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