WO2018124742A1 - Procédé de diagnostic d'une maladie prostatique par analyse métagénomique bactérienne - Google Patents

Procédé de diagnostic d'une maladie prostatique par analyse métagénomique bactérienne Download PDF

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WO2018124742A1
WO2018124742A1 PCT/KR2017/015576 KR2017015576W WO2018124742A1 WO 2018124742 A1 WO2018124742 A1 WO 2018124742A1 KR 2017015576 W KR2017015576 W KR 2017015576W WO 2018124742 A1 WO2018124742 A1 WO 2018124742A1
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derived
extracellular vesicles
bacteria
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prostate cancer
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PCT/KR2017/015576
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Korean (ko)
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김윤근
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주식회사 엠디헬스케어
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Priority claimed from KR1020170180014A external-priority patent/KR101942197B1/ko
Application filed by 주식회사 엠디헬스케어 filed Critical 주식회사 엠디헬스케어
Priority to JP2019535312A priority Critical patent/JP7084637B2/ja
Priority to CN201780081402.0A priority patent/CN110418848A/zh
Priority to US16/474,543 priority patent/US11708611B2/en
Priority to EP17886869.1A priority patent/EP3564388B1/fr
Publication of WO2018124742A1 publication Critical patent/WO2018124742A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for diagnosing prostate diseases such as prostate cancer and prostatic hyperplasia through bacterial metagenomic analysis, and more specifically, by performing a bacterial metagenomic analysis using a sample derived from a subject,
  • the present invention relates to a method for diagnosing prostate disease by analyzing the increase or decrease in content.
  • the prostate gland part of the male reproductive system, is an organ that mixes with sperm and creates a fluid that makes semen.
  • Prostatic hyperplasia is a condition in which the urinary flow in the urethra is blocked or decreased due to enlargement of the prostate gland, and the urinary flow in the urethra is blocked or reduced.
  • BPH benign protstate hyperplasia
  • the prostate surrounds the tube (ureter) that carries urine out of the bladder. During puberty, the prostate expands evenly, and as you age, the prostate enlarges, focusing on the side of the urethra of the gland.
  • the cause of benign prostatic hyperplasia is not yet clear, but as with other chronic diseases, several factors are known to work. Since the prostate is a testosterone dependent organ, it requires a constant male hormone to maintain its growth and function.If male hormone is not produced due to castration, the prostate may contract, and genetic factors and family history may also be associated with prostatic hyperplasia. It is known that this is.
  • Prostate carcinoma is a malignant tumor of the prostate gland, most of which is adenocarcinoma (adenocarcinoma of the gland cell) occurring in prostate cells.
  • Risk factors for prostate cancer increase rapidly in old age (50 years and older).
  • Asians have the lowest incidence, genetic predisposition, family history, male hormones, diabetes, obesity, westernized diet (increase in animal fat intake), and infections.
  • blood pressure (serum) prostate-specific antigen (PSA) tests and rectal balance are reported annually for men 50 years of age and older who are generally expected to live longer than 10 years. It is recommended to have a test.
  • Microbiota refers to a microbial community including bacteria, archaea and eukarya that exist in a given settlement.Intestinal microbiota is an important role in human physiology. It is known to have a great effect on human health and disease through interaction with human cells.
  • the symbiotic bacteria secrete nanometer-sized vesicles to exchange information about genes and proteins in other cells.
  • the mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through, so that the symbiotic bacteria cannot pass through the mucosa, but bacterial-derived vesicles are usually less than 100 nanometers in size. It freely speaks to the mucous membrane and is absorbed by our body.
  • Metagenomics also called environmental genomics, can be said to be an analysis of metagenomic data obtained from samples taken from the environment (Korean Patent Publication No. 2011-0073049). Recently, it has become possible to list the bacterial composition of the human microflora by a method based on 16s ribosomal RNA (16s rRNA) sequencing. Next generation sequencing of 16s rDNA sequencing gene of 16s ribosomal RNA is performed. , NGS) platform to analyze.
  • prostate diseases such as prostatic hyperplasia and prostate cancer
  • the present inventors In order to diagnose prostate disease based on the causes of prostatic hyperplasia and prostate cancer, the present inventors extracted a gene from a bacterial-derived extracellular vesicle in a sample-derived urine and performed a metagenome analysis on it. Bacterial-derived extracellular vesicles that can act as causative factors of prostate diseases such as cancer and prostatic hyperplasia have been identified, and thus the present invention has been completed.
  • an object of the present invention is to provide an information providing method for diagnosing prostate cancer through metagenomic analysis of bacterial extracellular vesicles.
  • an object of the present invention is to provide a method for providing information for diagnosing prostate cancer in patients with enlarged prostate through metagenomic analysis of bacterial extracellular vesicles.
  • an object of the present invention is to provide an information providing method for diagnosing prostatic hyperplasia through metagenomic analysis of bacterial extracellular vesicles.
  • the present invention provides a method for providing information for diagnosing prostate disease, comprising the following steps.
  • the present invention also provides a prostate disease diagnostic method comprising the following steps.
  • the present invention also provides a method for predicting the risk of developing a prostate disease, comprising the following steps.
  • At least one phylum bacteria-derived extracellular vesicle selected from the group consisting of Tenericutes, Euryarchaeota, Verrucomicrobia, Gemmatimonadetes, Acidobacteria, and Planctomycetes compared to the sample derived from the normal person in the step (c)
  • a phylum bacteria-derived extracellular vesicle selected from the group consisting of Tenericutes, Euryarchaeota, Verrucomicrobia, Gemmatimonadetes, Acidobacteria, and Planctomycetes compared to the sample derived from the normal person in the step (c)
  • Stramenopiles Alteromonadales, RF39, Rickettsiales, Neisseriales, Methanobacteriales, Verrucomicrobiales, Myxococcales, Acidimicrobiales, Chthoniobacterales, iii1-15, Acidobacteriales, Ellin3ales, and Pedospha It may be to diagnose prostate cancer by comparing the increase or decrease in the content of one or more order bacteria-derived extracellular vesicles selected from the group consisting of.
  • Peptococcaceae In another embodiment of the present invention, Peptococcaceae, Exiguobacteraceae, Actinomycetaceae, Cellulomonadaceae, mitochondria, Fusobacteriaceae, S24-7, Porphyromonadaceae, Flavobacteriaceae, Moraxellaceae, Neisseriaceae, Methanobacteriabiaceae, Verrucomicrobiaceae Prostate cancer may be diagnosed by comparing the increase or decrease in the content of one or more family bacteria-derived extracellular vesicles selected from the group consisting of Weeksellaceae, Streptomycetaceae, Helicobacteraceae, Chthoniobacteraceae, and Koribacteraceae.
  • prostate cancer may be diagnosed by comparing the increase or decrease in the content of Verrucomicrobia phylum bacteria-derived extracellular vesicles compared to the sample from the prostatic hyperplasia in step (c).
  • the content of at least one class bacteria-derived extracellular vesicles selected from the group consisting of Verrucomicrobiae, Acidimicrobiia, Saprospirae, and Pedosphaerae as compared to the prostatic hypertrophy-derived sample in step (c) It may be to diagnose prostate cancer by comparing the increase and decrease.
  • At least one order bacterial-derived extracellular vesicle selected from the group consisting of Verrucomicrobiales, Acidimicrobiales, Saprospirales, Pedosphaerales, and Ellin329 compared to the sample from the prostatic hyperplasia patient in step (c) By comparing the increase and decrease in the amount of prostate cancer may be to diagnose.
  • the amount of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of Ruminococcus, Akkermansia, and Flexispira is increased compared to the sample from the prostatic hyperplasia in step (c). In comparison, it may be to diagnose prostate cancer.
  • prostate hyperplasia by comparing the increase or decrease of the content of one or more phylum bacteria-derived extracellular vesicles selected from the group consisting of Euryarchaeota, and Acidobacteria in comparison to the sample derived from normal in step (c) It may be to diagnose.
  • compared to the normal sample derived from the step (c) compared to increase or decrease the content of one or more class bacteria-derived extracellular vesicles selected from the group consisting of Methanobacteria, Acidobacteria, and Acidobacteriia It may be to diagnose an enlarged prostate.
  • comparing the increase and decrease of the content may be to diagnose prostatic hyperplasia.
  • At least one family bacteria selected from the group consisting of Exiguobacteraceae, Flavobacteriaceae, Actinomycetaceae, Moraxellaceae, Ruminococcaceae, Rikenellaceae, Methanobacteriaceae, and Koribacteraceae compared to a sample derived from a normal person in step (c). By comparing the increase or decrease in the content of the derived extracellular vesicles may be to diagnose prostatic hyperplasia.
  • Rhizobium, Proteus, Acinetobacter, SMB53, Halomonas, Ruminococcus, Faecalibacterium, Klebsiella, Roseburia, Leuconostoc, Bilophila, Chromohalobacter, and Methanobrevibacter By comparing the increase or decrease in the content of one or more genus bacteria-derived extracellular vesicles selected may be to diagnose prostatic hyperplasia.
  • the sample may be urine.
  • Extracellular vesicles secreted from the bacteria present in the environment may be absorbed directly into the body and directly affect cancer development, and prostatic hyperplasia and prostate cancer are difficult to diagnose early because symptoms are difficult, so effective treatment is difficult.
  • metastatic genome analysis predicts causative factors in patients diagnosed with prostatic hyperplasia or prostate cancer, thereby preventing prostate hypertrophy and prostate cancer, or preventing recurrence.
  • Figure 1 is for evaluating the distribution of bacteria-derived extracellular vesicles in the body
  • Figure 1a after the administration of oral intestinal bacteria (Bacteria) and bacteria-derived vesicles (EV) in the mouth hourly (0, 5min, 3h, 6h, and 12h) is a photograph taken of their distribution
  • Figure 1b is a 12 hours after oral administration of intestinal bacteria (Bacteria) and bacteria-derived extracellular vesicles (EV) to the urine and various organs (heart, Lung, liver, kidney, spleen, adipose tissue, and muscles), and the photographs of the distribution of the bacterial and extracellular vesicles.
  • Figure 2 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the phylum level by separating the bacteria-derived vesicles in prostate cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • Figure 3 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the class level by separating bacteria-derived vesicles from prostate cancer patients and normal urine, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • Figure 4 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the order (order) level by separating the bacteria-derived vesicles in prostate cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • Figure 5 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level by separating bacteria-derived vesicles from prostate cancer patients and normal urine, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • Figure 6 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level after the isolation of bacteria-derived vesicles in prostate cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • FIG. 7 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the phylum level by separating the bacteria-derived vesicles in the urine of patients with prostate cancer and enlarged prostate gland .
  • EVs bacteria-derived vesicles
  • FIG. 8 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the class level by separating the bacteria-derived vesicles in the urine of patients with prostate cancer and enlarged prostate gland. .
  • EVs bacteria-derived vesicles
  • FIG. 9 is a result showing the distribution of bacteria-derived vesicles (EVs) of significant diagnostic performance at the order level by separating the bacteria-derived vesicles in the urine of patients with prostate cancer and enlarged prostate .
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • FIG. 11 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level after separating the bacteria-derived vesicles in the urine of patients with prostate cancer and enlarged prostate gland .
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • FIG. 13 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the class level after isolation of bacteria-derived vesicles from prostatic hyperplasia cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • FIG. 14 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the neck level after isolation of bacteria-derived vesicles from prostatic hypertrophy cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • FIG. 15 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level after isolation of bacteria-derived vesicles from prostatic hypertrophy cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • FIG. 16 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level after isolation of bacteria-derived vesicles from prostatic hypertrophy cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • the present invention relates to a method for diagnosing prostate diseases such as prostate hyperplasia and prostate cancer through bacterial metagenomic analysis, the present inventors extract a gene from a bacterial-derived extracellular vesicles using a sample derived from the subject and metagenomic analysis And extracellular vesicles derived from bacteria that can act as a causative agent of prostate disease.
  • the present invention comprises the steps of (a) extracting DNA from the extracellular vesicles isolated from the subject sample;
  • an information providing method for diagnosing prostate disease comprising the step of comparing the increase and decrease of the bacterial-derived extracellular vesicles in the sample derived from normal and prostatic hypertrophy patients by sequencing the PCR product.
  • the term "diagnosis of prostate cancer” means to determine whether a patient is likely to develop prostate cancer, a relatively high probability of developing prostate cancer, or whether prostate cancer has already occurred.
  • the method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing prostate cancer for any particular patient.
  • the methods of the present invention can be used clinically to determine treatment by early diagnosis of prostate cancer and selection of the most appropriate treatment regimen.
  • the term "diagnosis of prostatic hyperplasia” means determining whether there is a possibility of developing prostatic hyperplasia, a relatively high probability of developing prostatic hyperplasia, or whether an enlarged prostate has already occurred.
  • the method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing prostatic hyperplasia for any particular patient.
  • the methods of the present invention can be used clinically to determine treatment by early diagnosis of prostatic hyperplasia and selecting the most appropriate treatment regimen.
  • metagenome used in the present invention, also referred to as “metagenome”, refers to the total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal intestine, It is mainly used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms which are not cultured.
  • metagenome does not refer to one species of genome or genome, but refers to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species.
  • rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.
  • metagenome analysis was preferably performed using bacterial-derived extracellular vesicles isolated from serum.
  • metagenome analysis was performed on genes present in bacteria-derived extracellular vesicles in the urine of normal, prostatic hyperplasia, and prostate cancer patients. Analysis at the levels of), family, and genus, respectively, identified bacterial vesicles that could actually contribute to the development of prostate cancer and prostatic hyperplasia.
  • bacteria-derived metagenomes were analyzed at the gate level, and as a result, Tenericutes, Euryarchaeota, Verrucomicrobia, Gemmatimonadetes, Acidobacteria, and Planctomycetes ⁇ bacteria-derived vesicles were significantly different between prostate cancer patients and normal people. (See Example 4).
  • the bacterial-derived metagenome was analyzed at the neck level.
  • Stramenopiles, Alteromonadales, RF39, Rickettsiales, Neisseriales, Methanobacteriales, Verrucomicrobiales, Myxococcales, Acidimicrobiales, Chthoniobacterales, iii1-15, Acidobacteriales, Ellin329 Vesicle-derived vesicles derived from,, and Pedosphaerales were significantly different between prostate cancer patients and normal individuals (see Example 4).
  • the bacteria-derived metagenome was analyzed at an excessive level.
  • Rikenellaceae, Weeksellaceae, Streptomycetaceae, Helicobacteraceae, Chthoniobacteraceae, and Bacterial-derived vesicles were significantly different between prostate cancer patients and normal individuals (see Example 4).
  • the bacteria-derived metagenome at the genus level Rhizobium, Tetragenococcus, Proteus, Morganella, Exiguobacterium, Oribacterium, Porphyromonas, Actinomyces, Cellulomonas, Jeotgalicoccus, Acinetobacter, Fusobacterium, Enterobacter , Adlercreutzia, SMB53, Parabacteroides, Faecalibacterium, Catenibacterium, Roseburia, Akkermansia, Methanobrevibacter, Clostridium, Klebsiella, Chryseobacterium, Halomonas, Aggregatibacter, Rhodoplanes, Thermoanaerobacterium, and Candidatus Kosibacterium genus There was a difference (see Example 4).
  • metagenome analysis of extracellular vesicles derived from bacteria in the urine of prostatic hyperplasia and prostate cancer patients was carried out, and phylum, class, order, family ), And genus levels were analyzed to identify bacterial vesicles that could actually cause prostate cancer in patients with enlarged prostate.
  • the vesicles derived from Verrucomicrobiae, Acidimicrobiia, Saprospirae, and Pedosphaerae river bacteria showed a significant difference between patients with prostate cancer and hypertrophy. (See Example 5).
  • metagenome analysis was performed on bacteria-derived extracellular vesicles in the urine of normal and prostatic hypertrophy patients, including phylum, class, order, family, Analyzes at the genus and genus levels, respectively, identified bacterial vesicles that can actually cause prostate cancer in asthma patients.
  • the vesicles derived from Euryarchaeota, and Acidobacteria gate bacteria showed a significant difference between patients with prostatic hyperplasia and normal people (see Example 6).
  • the vesicles derived from Methanobacteria, Acidobacteria, and Acidobacteriia strong bacteria had a significant difference between patients with prostatic hyperplasia and normal people (Example 6 Reference).
  • the bacterial-derived metagenome was analyzed at the neck level.
  • stramenopiles, RF39, Saprospirales, Pseudomonadales, Methanobacteriales, and Acidobacteriales-derived vesicle-derived vesicles were significantly different between prostate hyperplasia and normal people (See Example 6).
  • the bacterial-derived metagenome is analyzed at an exaggerated level, and Exiguobacteraceae, Flavobacteriaceae, Actinomycetaceae, Moraxellaceae, Ruminococcaceae, Rikenellaceae, Methanobacteriaceae, and Koribacteraceae are bacteria-derived vesicles between the prostate hypertrophy and the normal There was a significant difference (see Example 6).
  • bacteria-derived metagenome was analyzed at the genus level, and Rhizobium, Proteus, Acinetobacter, SMB53, Halomonas, Ruminococcus, Faecalibacterium, Klebsiella, Roseburia, Leuconostoc, Bilophila, Chromohalobacter, and Methanobrevibacter Bacterial-derived vesicles were significantly different between prostate hypertrophy patients and normal subjects (see Example 6).
  • the bacterial-derived vesicles whose contents were significantly changed in prostate cancer patients compared to normal and prostatic hypertrophy patients by performing metagenomic analysis on bacterial-derived extracellular vesicles isolated from urine.
  • the metagenome analysis confirmed that prostate cancer can be diagnosed by analyzing the increase and decrease of the contents of the bacteria-derived vesicles at each level.
  • the present invention through the results of the above embodiment, by identifying the bacteria-derived extracellular vesicles isolated from urine by metagenomic analysis of bacteria-derived vesicles significantly changed in the content of prostate hypertrophy patients compared to normal people Meta-genomic analysis confirmed that prostatic hyperplasia can be diagnosed by analyzing the increase and decrease of the contents of the bacteria-derived vesicles at each level.
  • Example 1 Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
  • the fluorescently labeled 50 ⁇ g of bacteria and bacteria-derived vesicles were administered in the same manner as above 12 hours.
  • Blood, Heart, Lung, Liver, Kidney, Spleen, Adipose tissue, and Muscle were extracted from mice.
  • the intestinal bacteria (Bacteria) were not absorbed in each organ, whereas the intestinal bacteria-derived extracellular vesicles (EVs) were urine, heart, lung as shown in FIG. And distribution in liver, kidney, spleen, adipose tissue, and muscle.
  • the urine was first placed in a 10 ml tube and centrifuged (3,500 x g, 10 min, 4 ° C.) to settle the suspended solids to recover only the supernatant and then transferred to a new 10 ml tube. After removing the bacteria and foreign substances from the recovered supernatant using a 0.22 ⁇ m filter, transfer to centripreigugal filters (50 kD) and centrifuged at 1500 xg, 4 °C for 15 minutes to discard the material smaller than 50 kD and 10 ml Concentrated until.
  • centripreigugal filters 50 kD
  • PCR was performed using the 16S rDNA primer shown in Table 1 to amplify the gene and perform sequencing (Illumina MiSeq sequencer). Output the result as a Standard Flowgram Format (SFF) file, convert the SFF file into a sequence file (.fasta) and a nucleotide quality score file using GS FLX software (v2.9), check the credit rating of the lead, and window (20 bps) The part with the average base call accuracy of less than 99% (Phred score ⁇ 20) was removed.
  • SFF Standard Flowgram Format
  • the Operational Taxonomy Unit performed UCLUST and USEARCH for clustering according to sequence similarity. Specifically, the clustering is based on 94% genus, 90% family, 85% order, 80% class, and 75% sequence similarity. OTU's door, river, neck, family and genus level classifications were performed, and bacteria with greater than 97% sequence similarity were analyzed using BLASTN and GreenGenes' 16S DNA sequence database (108,453 sequences) (QIIME).
  • Example 3 By the method of Example 3, vesicles were isolated from urine of 53 prostate cancer patients and 159 normal humans whose age and sex were matched, followed by metagenome sequencing. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • Rhizobium Tetragenococcus, Proteus, Morganella, Exiguobacterium, Oribacterium, Porphyromonas, Actinomyces, Cellulomonas, Jeotgalicoccus, Acinetobacter, Fusobacterium, Enterobacter, Neissezi, Adissemb, Diagnosis and development of one or more biomarkers for the development of one or more biomarkers for Faecalibacterium, Catenibacterium, Roseburia, Akkermansia, Methanobrevibacter, Clostridium, Klebsiella, Chryseobacterium, Halomonas, Aggregatibacter, Rhodoplanes, Thermoanaerobacterium, Candidatus Koribacter, and Flexispira genus Performance was significant (see Table 6 and FIG. 6).
  • Example 3 By the method of Example 3, the vesicles were isolated from the urine of 53 patients with prostate cancer and 55 patients with enlarged prostate gland, and metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • the diagnostic performance of prostate cancer was significant when developing a diagnostic model with Verrucomicrobia cultivars as a biomarker (see Table 7 and FIG. 7).
  • the diagnostic performance of prostate cancer was significant when developing a diagnostic model with one or more biomarkers in Verrucomicrobiales, Acidimicrobiales, Saprospirales, Pedosphaerales, and Ellin329 throat bacteria. (See Table 9 and FIG. 9).
  • Example 3 By the method of Example 3, the vesicles were isolated from the urine of 55 prostate cancer patients and 159 normal people, and then metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • Predicting the risk of developing prostate cancer and prostatic hyperplasia through metagenomic analysis on genes present in bacteria-derived extracellular vesicles using a human-derived sample according to the present invention by early diagnosis and prediction of risk groups of prostate disease, appropriate management It can delay the onset or prevent the onset of the disease, and can be diagnosed early after the enlargement of the prostate or prostate cancer, thereby reducing the incidence of prostate disease and improving the treatment effect.

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Abstract

La présente invention concerne un procédé de diagnostic d'une maladie prostatique, telle qu'un cancer de la prostate et l'hypertrophie prostatique, par analyse métagénomique bactérienne et, plus particulièrement, un procédé de diagnostic du cancer de la prostate et de l'hypertrophie prostatique par la réalisation d'une analyse métagénomique bactérienne à l'aide d'un échantillon dérivé d'un sujet et par l'analyse d'une augmentation ou d'une diminution de la teneur en une vésicule extracellulaire dérivée d'une bactérie spécifique. Une vésicule extracellulaire sécrétée par une bactérie présente dans l'environnement peut être absorbée dans le corps et influencer directement l'apparition d'une inflammation et d'un cancer et le diagnostic précoce d'une maladie prostatique, telle qu'un cancer de la prostate et l'hypertrophie prostatique, est difficile avant l'apparition d'un quelconque symptôme, ce qui complique un traitement efficace. Ainsi, par l'intermédiaire de l'analyse métagénomique sur un gène présent dans une vésicule extracellulaire dérivée d'une bactérie à l'aide d'un échantillon dérivé du corps humain selon la présente invention, le risque d'apparition d'un cancer de la prostate et d'une hypertrophie prostatique peut être prédit à l'avance, ce qui permet un diagnostic précoce et la prédiction d'un groupe à risque de maladie prostatique et de retarder le moment d'apparition ou de prévenir l'apparition par des soins appropriés et un diagnostic précoce est encore possible même après l'apparition de l'hypertrophie prostatique ou du cancer de la prostate, ce qui peut abaisser le taux d'incidence de la maladie prostatique et améliorer l'effet de traitement.
PCT/KR2017/015576 2016-12-28 2017-12-27 Procédé de diagnostic d'une maladie prostatique par analyse métagénomique bactérienne WO2018124742A1 (fr)

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JP2019535312A JP7084637B2 (ja) 2016-12-28 2017-12-27 細菌メタゲノム分析を通した前立腺疾患の診断方法
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US16/474,543 US11708611B2 (en) 2016-12-28 2017-12-27 Method for diagnosing prostatic disease via bacterial metagenomic analysis
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