Method for diagnosing parkinson's disease through bacterial metagenome analysis

A technology for Parkinson's disease and bacteria, applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as movement disorders and uncomfortable movements

Pending Publication Date: 2019-12-06
MD HEALTHCARE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, dyskinesias can occur, making movement uncomfortable

Method used

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  • Method for diagnosing parkinson's disease through bacterial metagenome analysis
  • Method for diagnosing parkinson's disease through bacterial metagenome analysis
  • Method for diagnosing parkinson's disease through bacterial metagenome analysis

Examples

Experimental program
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Embodiment 1

[0055] Example 1. Analysis of In vivo Absorption, Distribution and Excretion Patterns of Enterobacteria and Bacteria-Derived Extracellular Vesicles

[0056] 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 whole-body images of mice, as shown in Fig. 1A, 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, at Strong fluorescence was observed in th...

Embodiment 2

[0058] Example 2. Vesicle Isolation and DNA Extraction from Urine

[0059] To isolate extracellular vesicles from urine and extract DNA, urine was first added to a 10 ml tube, centrifuged at 3500 x g for 10 minutes at 4°C, the suspension was pelleted, and only the supernatant was collected, which was Place 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).

[0060] 100 μl of extracellular vesicles isolated fr...

Embodiment 3

[0063] Example 3. Metagenomic analysis using DNA extracted from urine

[0064]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 classes, a...

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Abstract

The present invention relates to a method for diagnosing Parkinson's disease through bacterial metagenome analysis and, more specifically, to a method for performing bacterial metagenome analysis by using a sample derived from a subject so as to analyze increases and decreases in the amount of extracellular vesicles derived from specific bacteria, thereby diagnosing Parkinson's disease. The extracellular vesicles secreted from microbes, such as bacteria and archaea, present in the environment are absorbed into the body so as to be distributed to the brain, thereby directly influencing inflammatory responses and brain functions, and since early diagnosis of Parkinson's disease, which is characterized by inflammation, before symptoms occur is difficult, effective treatment has been difficult. Through the bacteria-derived extracellular vesicle metagenome analysis using a human-derived sample, according to the present invention, the risk of onset of Parkinson's disease can be predicted inadvance such that Parkinson's disease risk groups are diagnosed and predicted in an early stage, thereby enabling the time of onset of the disease to be delayed or the onset of the disease to be prevented through appropriate management, and early diagnosis is enabled even after the onset of the disease, thereby enabling the incidence of Parkinson's disease to be lowered and therapeutic effects tobe increased.

Description

technical field [0001] The present disclosure relates to a method for diagnosing Parkinson's disease by metagenomic analysis of bacteria, and more particularly, by analyzing cells derived from specific bacteria through metagenomic analysis of bacteria using a sample derived from a subject. A method for diagnosing Parkinson's disease by increasing or decreasing the content of exovesicles. Background technique [0002] Parkinson's disease is a progressive neurodegenerative disorder characterized by parkinsonian symptoms such as slow motion, resting tremor, muscle stiffness, abnormal gait, and bent posture. The disease occurs due to reduced stimulation of the motor cortex due to imperfect production and action of dopamine (mainly in the substantia nigra). Severe cognitive impairment and mild language impairment also occur, and they are chronic and progressive. Parkinson's disease can even occur with Japanese encephalitis, cerebral syphilis, carbon dioxide poisoning, manganism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895
CPCC12Q1/689C12Q1/6883C12Q2600/158C12Q1/6895C12Q2531/113
Inventor 金润根
Owner MD HEALTHCARE INC
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