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Method for diagnosing breast cancer via microbial metagenomic analysis

A breast cancer and group technology, applied in the field of breast cancer diagnosis, can solve unreported problems

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

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Problems solved by technology

[0007] However, it has never been reported in the pathogenesis of breast cancer that the pathogenicity of breast cancer has been identified by metagenomic analysis of microorganism-derived vesicles isolated from human-derived substances (such as blood, urine, etc.) factors, and ways to predict breast cancer

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  • Method for diagnosing breast cancer via microbial metagenomic analysis
  • Method for diagnosing breast cancer via microbial metagenomic analysis
  • Method for diagnosing breast cancer via microbial metagenomic analysis

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0072] Example 1. Analysis of In vivo Absorption, Distribution and Excretion Patterns of Enterobacteria and Bacteria-Derived Extracellular Vesicles

[0073] 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 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 5 minutes after administration, EVs derived from bacteria were absorbed systemically, and 3 hours after administration, a strong fluorescence was obs...

Embodiment 2

[0075] Example 2. Vesicle Isolation and DNA Extraction from Blood and Urine

[0076]To isolate extracellular vesicles from blood or urine and extract DNA, blood and urine were first added to a 10 ml tube, centrifuged at 3500 x g and 4°C for 10 minutes, the suspension was pelleted, and only the supernatant was collected, Place the supernatant 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 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 saline (PBS).

[0077] 100 μl o...

Embodiment 3

[0080] Example 3. Metagenomic analysis using DNA extracted from blood and urine

[0081] As shown in Table 2 below, DNA was extracted using the same method as in Example 2, and then subjected to PCR using the 16SrDNA 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 a sequence similarity of 94% for genera, 90% for families, 85% for orders, 80% for...

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Abstract

The present invention relates to a method for diagnosing breast cancer via bacterial and archaeal metagenomic analysis and, more particularly, to a method for diagnosing breast cancer by performing bacterial and archaeal metagenomic analysis by using a subject-derived sample, and analyzing an increase or decrease in the content of a specific bacterium or archaeon-derived extracellular vesicle. Anextracellular vesicle secreted from a bacterium or an archaeon present in the environment can be absorbed into the body and directly affect the occurrence of cancer, and breast cancer is difficult todiagnose early on before any symptom appears, which makes efficient treatment difficult. As such, through the bacterial and archaeal metagenomic analysis using a human body-derived sample according tothe present invention, the risk of the onset of breast cancer can be predicted in advance, enabling early diagnosis and prediction of a breast cancer risk group and delay of the time of the onset orprevention of the onset with proper care, and early diagnosis is still possible even after the onset, which can lower the incidence rate of breast cancer and enhance the treatment effect.

Description

technical field [0001] The present disclosure relates to methods for diagnosing breast cancer by metagenomic analysis of microorganisms, and more particularly, to metagenomic analysis of microorganisms such as bacteria, archaea, etc. A method for the diagnosis of breast cancer by the increase or decrease in the content of extracellular vesicles of specific bacteria and archaea. Background technique [0002] Breast cancer collectively refers to all malignant tumors that occur in the breast. Breast cancer is a fatal disease that continues to grow in abnormal breast tissue or spread to other organs. Although breast cancer is the most studied of all cancers, it is only known with certainty that it occurs due to two factors: environmental factors and genetic factors, and there is currently no established theory about the cause of breast cancer. However, according to some research results, it is agreed that several factors are highly correlated, especially estrogen, which is a f...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/689C12Q1/6851
CPCC12Q1/6895C12Q1/6886C12Q1/689C12Q2600/158C12Q2531/113C12Q1/6806C12Q2600/16
Inventor 金润根
Owner MD HEALTHCARE INC
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