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Method for diagnosing cholangiocarcinoma via bacterial metagenomic analysis

A technique for cholangiocarcinoma, bacteria, used in the field of diagnosing cholangiocarcinoma

Active Publication Date: 2020-09-04
MD HEALTHCARE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, regarding the occurrence of cholangiocarcinoma, there are no reports on a method for identifying causative factors of cholangiocarcinoma from human-derived substances such as blood by analyzing the metagenome present in vesicles derived from bacteria and diagnosing cholangiocarcinoma

Method used

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  • Method for diagnosing cholangiocarcinoma via bacterial metagenomic analysis
  • Method for diagnosing cholangiocarcinoma via bacterial metagenomic analysis
  • Method for diagnosing cholangiocarcinoma via bacterial metagenomic analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1. Analysis of In vivo Absorption, Distribution and Excretion Patterns of Enterobacteria and Bacteria-Derived Vesicles

[0074] To assess whether enterobacteria and vesicles derived from bacteria are absorbed systemically through the mucosa, the following method was used to perform experiments. 50 μg each of fluorescently labeled enterobacteria and extracellular vesicles (EVs) derived from the enterobacteria were administered to the gastrointestinal tract of mice, and at 0 hours, and at 5 minutes, 3 hours, 6 hours and 12 hours Fluorescence was then measured. As a result of observing the overall image of the mouse, such as Figure 1A As shown, bacteria were not taken up systemically at the time of administration, whereas 5 minutes after administration, EVs derived from bacteria were taken up systemically, and 3 hours after administration, strong fluorescence was observed in the bladder, from which it was confirmed that EVs are excreted via the urinary system a...

Embodiment 2

[0076] Example 2. Vesicle Isolation and DNA Extraction from Blood

[0077] To isolate vesicles from blood and extract DNA, blood was first added to a 10 ml tube, centrifuged at 3,500 x g for 10 minutes at 4°C, the suspension was pelleted, and only the supernatant was collected, which was placed in 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 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 h by using a 90ti type rotor to remove the supernatant and the aggregated precipitate Vesicles were obtained by dissolving with phosphate buffered saline (PBS).

[0078] 100 μl of extracellular vesicles isolated from blood according...

Embodiment 3

[0081] Example 3. Metagenomic analysis using DNA extracted from blood

[0082] DNA was extracted using the same method as that used 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 removing 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 a sequence similarity of 94% for genera, 90% for families, 85% for orders, 80% for classes, and 75% fo...

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Abstract

The present invention relates to a method for diagnosing cholangiocarcinoma via a bacterial metagenomic analysis, and more specifically, to a method for diagnosing cholangiocarcinoma by performing a bacterial metagenomic analysis using a sample derived from a subject so as to analyze the content increase / decrease of extracellular vesicles derived from specific bacteria. The extracellular vesicles,secreted from bacteria present in the environment, may directly affect the incidence of cancer by being absorbed in the human body, and early diagnosis of cholangiocarcinoma before symptoms appear isdifficult, and thus the efficient treatment thereof is difficult. Thus, by preemptively predicting the risk for the onset of cholangiocarcinoma via the metagenomic analysis, according to the presentinvention, of extracellular vesicles derived from bacteria using a sample derived from a human body, cholangiocarcinoma risk groups may be diagnosed and predicted at an early stage so as to delay thetime of onset or prevent the onset of cholangiocarcinoma through proper care, and early diagnosis may be possible even after the onset, and thus the incidence rate of cholangiocarcinoma may be lowered, and a therapeutic effect therefor may be increased.

Description

technical field [0001] The present invention relates to a method for diagnosing cholangiocarcinoma by bacterial metagenomic analysis, and more particularly, to analyzing the content of extracellular vesicles derived from a specific bacterium by performing bacterial metagenomic analysis using a sample derived from a subject method for diagnosing cholangiocarcinoma and so on by the increase or decrease of Background technique [0002] Cholangiocarcinoma or biliary tract cancer is a malignant tumor that occurs in the intrahepatic or extrahepatic bile ducts, and is used as a concept including gallbladder cancer. Histologically, most types of cholangiocarcinoma are adenocarcinomas. To date, the cause of cholangiocarcinoma is unknown. However, various biliary tract diseases are known to be associated with the development of cholangiocarcinoma, and genetic and environmental factors are expected to be involved in the development of cholangiocarcinoma. Risk factors include primary...

Claims

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Application Information

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