WO2024119614A1 - Nouveau marqueur microbien pour prédire le risque de cancer colorectal - Google Patents
Nouveau marqueur microbien pour prédire le risque de cancer colorectal Download PDFInfo
- Publication number
- WO2024119614A1 WO2024119614A1 PCT/CN2023/076160 CN2023076160W WO2024119614A1 WO 2024119614 A1 WO2024119614 A1 WO 2024119614A1 CN 2023076160 W CN2023076160 W CN 2023076160W WO 2024119614 A1 WO2024119614 A1 WO 2024119614A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- rna
- dna
- seq
- unique
- specifically
- Prior art date
Links
- 208000001333 Colorectal Neoplasms Diseases 0.000 title claims abstract description 140
- 206010009944 Colon cancer Diseases 0.000 title claims abstract description 139
- 239000003550 marker Substances 0.000 title claims abstract description 96
- 230000000813 microbial effect Effects 0.000 title abstract description 5
- 201000002758 colorectal adenoma Diseases 0.000 claims abstract description 66
- 108020004414 DNA Proteins 0.000 claims description 281
- 239000003153 chemical reaction reagent Substances 0.000 claims description 200
- 108090000623 proteins and genes Proteins 0.000 claims description 199
- 241001215965 Cloacibacillus porcorum Species 0.000 claims description 136
- 239000000523 sample Substances 0.000 claims description 115
- 241000605909 Fusobacterium Species 0.000 claims description 100
- 241000125969 Lachnoclostridium Species 0.000 claims description 84
- 241000801600 Bacteroides clarus Species 0.000 claims description 77
- 102000040430 polynucleotide Human genes 0.000 claims description 69
- 108091033319 polynucleotide Proteins 0.000 claims description 69
- 239000002157 polynucleotide Substances 0.000 claims description 69
- 150000007523 nucleic acids Chemical group 0.000 claims description 67
- 241001674997 Hungatella hathewayi Species 0.000 claims description 66
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 52
- 238000000034 method Methods 0.000 claims description 49
- 239000003155 DNA primer Substances 0.000 claims description 42
- 239000013615 primer Substances 0.000 claims description 41
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 40
- 238000011529 RT qPCR Methods 0.000 claims description 26
- 101150115599 nusG gene Proteins 0.000 claims description 26
- 230000002550 fecal effect Effects 0.000 claims description 25
- 238000012360 testing method Methods 0.000 claims description 25
- 241000193403 Clostridium Species 0.000 claims description 23
- 238000003752 polymerase chain reaction Methods 0.000 claims description 23
- 241000894006 Bacteria Species 0.000 claims description 21
- 108091034117 Oligonucleotide Proteins 0.000 claims description 21
- 238000012163 sequencing technique Methods 0.000 claims description 20
- 230000003321 amplification Effects 0.000 claims description 19
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 230000002068 genetic effect Effects 0.000 claims description 14
- 230000000984 immunochemical effect Effects 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 238000003757 reverse transcription PCR Methods 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000003247 decreasing effect Effects 0.000 claims 1
- 102000053602 DNA Human genes 0.000 description 229
- 229920002477 rna polymer Polymers 0.000 description 224
- 102000004169 proteins and genes Human genes 0.000 description 133
- 208000003200 Adenoma Diseases 0.000 description 114
- 206010001233 Adenoma benign Diseases 0.000 description 95
- 230000001580 bacterial effect Effects 0.000 description 35
- 238000003745 diagnosis Methods 0.000 description 26
- 241000605986 Fusobacterium nucleatum Species 0.000 description 21
- 150000001413 amino acids Chemical class 0.000 description 21
- 206010028980 Neoplasm Diseases 0.000 description 20
- 201000011510 cancer Diseases 0.000 description 16
- 230000035945 sensitivity Effects 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 14
- 238000001514 detection method Methods 0.000 description 14
- 108090000765 processed proteins & peptides Proteins 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 9
- 125000003729 nucleotide group Chemical group 0.000 description 9
- 229920001184 polypeptide Polymers 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 239000002987 primer (paints) Substances 0.000 description 7
- 238000011002 quantification Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000000090 biomarker Substances 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 244000005709 gut microbiome Species 0.000 description 5
- 238000007477 logistic regression Methods 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 240000006606 bacterium M-3 Species 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 238000003127 radioimmunoassay Methods 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 238000002052 colonoscopy Methods 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- COCMHKNAGZHBDZ-UHFFFAOYSA-N 4-carboxy-3-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]benzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(C([O-])=O)=CC=C1C(O)=O COCMHKNAGZHBDZ-UHFFFAOYSA-N 0.000 description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 2
- 241000192035 Peptostreptococcus anaerobius Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 210000001072 colon Anatomy 0.000 description 2
- 230000004732 colorectal carcinogenesis Effects 0.000 description 2
- 238000013211 curve analysis Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 230000000951 immunodiffusion Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 201000002313 intestinal cancer Diseases 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 229940074571 peptostreptococcus anaerobius Drugs 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- VGONTNSXDCQUGY-RRKCRQDMSA-N 2'-deoxyinosine Chemical group C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 VGONTNSXDCQUGY-RRKCRQDMSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 108700029181 Bacteria lipase activator Proteins 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 241000606125 Bacteroides Species 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 241000390531 Cloacibacillus Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 208000019399 Colonic disease Diseases 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 238000000729 Fisher's exact test Methods 0.000 description 1
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QEFRNWWLZKMPFJ-ZXPFJRLXSA-N L-methionine (R)-S-oxide Chemical compound C[S@@](=O)CC[C@H]([NH3+])C([O-])=O QEFRNWWLZKMPFJ-ZXPFJRLXSA-N 0.000 description 1
- QEFRNWWLZKMPFJ-UHFFFAOYSA-N L-methionine sulphoxide Natural products CS(=O)CCC(N)C(O)=O QEFRNWWLZKMPFJ-UHFFFAOYSA-N 0.000 description 1
- 238000000585 Mann–Whitney U test Methods 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000122116 Parvimonas Species 0.000 description 1
- 241000605894 Porphyromonas Species 0.000 description 1
- 206010038063 Rectal haemorrhage Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 244000000001 Virome Species 0.000 description 1
- 241000193450 [Clostridium] symbiosum Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- UHBYWPGGCSDKFX-UHFFFAOYSA-N carboxyglutamic acid Chemical compound OC(=O)C(N)CC(C(O)=O)C(O)=O UHBYWPGGCSDKFX-UHFFFAOYSA-N 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000005081 chemiluminescent agent Substances 0.000 description 1
- 238000000546 chi-square test Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- LSDPWZHWYPCBBB-UHFFFAOYSA-O methylsulfide anion Chemical compound [SH2+]C LSDPWZHWYPCBBB-UHFFFAOYSA-O 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108091005601 modified peptides Proteins 0.000 description 1
- 238000010198 multi-cohort analysis Methods 0.000 description 1
- 238000000491 multivariate analysis Methods 0.000 description 1
- 239000000101 novel biomarker Substances 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000037425 regulation of transcription Effects 0.000 description 1
- 230000009712 regulation of translation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- 238000007473 univariate analysis Methods 0.000 description 1
- 235000003563 vegetarian diet Nutrition 0.000 description 1
Classifications
-
- 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
- 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
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the present application relates to the identification of bacterial species that help improve the diagnosis of colorectal adenomas and the development of new bacterial marker combinations for the diagnosis of CRC and adenomas.
- the present application relates to reagents that specifically and quantitatively identify DNA, RNA or proteins that are unique to Cloacibacillus porcorum.
- CRC Colorectal cancer
- Most CRCs begin as small polyps. Some polyps, especially adenomas, may develop into cancers. Early detection of tumors can promote successful treatment, and early detection of adenomas can prevent and reduce the incidence of CRC. Although currently available non-invasive tests for CRC screening perform well in detecting CRC, they have limited sensitivity for adenomas. The incidence of CRC is higher in more developed regions compared with less developed regions, and the increase in CRC incidence is thought to be attributed to changes in diet1,2 . Recent evidence suggests that changes in the microbial environment in the intestine are associated with the occurrence of colorectal tumors.
- Bacteria such as Fn13 , Clostridium symbiosum14 , and species within the genera Parvimonas, Porphyromonas, and Parabacteroides15 have been shown to be potential biomarkers for the diagnosis of CRC patients.
- current knowledge about biomarkers for the detection of colorectal adenomas is limited.
- Bacterial markers for the non-invasive diagnosis of CRC and adenoma have been previously identified and validated by metagenomic sequencing and targeted qPCR. Specifically, a qPCR test has been developed for the diagnosis of CRC and adenoma for four gene markers from four bacteria, including Fusobacterium nucleatum (Fn), Hungatella hathewayi (formerly known as Clostridium hathewayi, Ch), the Lachnoclostridium bacterial marker m3, and Bacteroides clarus (Bc). Three of the bacteria (Fn, Ch, and m3) were enriched in the feces of patients with CRC or adenoma, while Bc was enriched in healthy subjects. Although the test involving these four bacterial markers showed superior performance to currently available tests for the non-invasive diagnosis of CRC and adenoma, However, the sensitivity for adenoma needs to be further improved.
- Fusobacterium nucleatum Fn
- Hungatella hathewayi previously
- the present application provides a kit for detecting colorectal cancer or colorectal adenoma in a subject, comprising: a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum.
- the DNA or RNA specific to Cloacibacillus porcorum comprises the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum comprises oligonucleotide primers, which comprise the nucleic acid sequences shown in SEQ ID NO:2 and SEQ ID NO:3.
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum comprises a polynucleotide probe shown in SEQ ID NO:4.
- the reagent for specifically and quantitatively identifying a DNA, RNA, or protein unique to Cloacibacillus porcorum comprises an antibody that specifically binds to the protein.
- the kit further comprises a standard control that provides an average amount of Cloacibacillus porcorum in a stool sample.
- the kit further comprises one or more reagents selected from the group consisting of:
- the DNA or RNA specific to the genus Fusobacterium includes the Fusobacterium nusG gene shown in SEQ ID NO:5.
- the DNA or RNA unique to Lachnoclostridium bacterium m3 comprises the genetic marker m482585.
- the DNA or RNA unique to Clostridium hassawayi comprises genetic marker m2736705.
- the DNA or RNA unique to Bacteroides clarus comprises the gene marker m370640.
- DNA that is unique to the genus Fusobacterium is specifically and quantitatively identified.
- the RNA or protein reagent comprises an oligonucleotide primer comprising the nucleic acid sequence shown in SEQ ID NO:6 and SEQ ID NO:7.
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Lachnoclostridium bacteria m3 comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:10 and SEQ ID NO:11.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Hungatella hathewayi comprises an oligonucleotide primer comprising a nucleic acid sequence shown in SEQ ID NO:14 and SEQ ID NO:15.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Bacteroides clarus comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:18 and SEQ ID NO:19.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to the genus Fusobacterium comprises a polynucleotide probe shown in SEQ ID NO:8.
- the reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Lachnoclostridium bacteria m3 comprises a polynucleotide probe shown in SEQ ID NO:12.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Hungatella hathewayi comprises a polynucleotide probe shown in SEQ ID NO:16.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Bacteroides clarus comprises a polynucleotide probe shown in SEQ ID NO:20.
- the agent comprises a detectable moiety.
- the kit further comprises an operating manual.
- the kit further comprises reagents for a fecal immunochemical test (FIT).
- FIT fecal immunochemical test
- the kit further comprises a standard control that provides an average amount in a stool sample of one or more selected from:
- the present application provides the use of a reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum in the preparation of a kit for detecting colorectal cancer or colorectal adenoma in a subject.
- the DNA or RNA unique to Cloacibacillus porcorum comprises The nucleic acid sequence shown in SEQ ID NO: 1 or 21.
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum comprises oligonucleotide primers, which comprise the nucleic acid sequences shown in SEQ ID NO:2 and SEQ ID NO:3.
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum comprises a polynucleotide probe shown in SEQ ID NO:4.
- the reagent for specifically and quantitatively identifying a DNA, RNA, or protein unique to Cloacibacillus porcorum comprises an antibody that specifically binds to the protein.
- the kit further comprises a standard control that provides an average amount of Cloacibacillus porcorum in a stool sample.
- the kit further comprises one or more reagents selected from the group consisting of:
- the DNA or RNA specific to the genus Fusobacterium includes the Fusobacterium nusG gene shown in SEQ ID NO:5.
- the DNA or RNA unique to Lachnoclostridium bacterium m3 comprises the genetic marker m482585.
- the DNA or RNA unique to Clostridium hassawayi comprises genetic marker m2736705.
- the DNA or RNA unique to Bacteroides clarus comprises the gene marker m370640.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to the genus Fusobacterium comprises an oligonucleotide primer comprising a nucleic acid sequence shown in SEQ ID NO:6 and SEQ ID NO:7.
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Lachnoclostridium bacteria m3 comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:10 and SEQ ID NO:11.
- a reagent kit for specifically and quantitatively identifying DNA, RNA, or protein unique to Hungatella hathewayi (formerly known as Clostridium hathewayi) is provided.
- a reagent kit for specifically and quantitatively identifying DNA, RNA, or protein unique to Hungatella hathewayi (formerly known as Clostridium hathewayi) is provided.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Bacteroides clarus comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:18 and SEQ ID NO:19.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to the genus Fusobacterium comprises a polynucleotide probe shown in SEQ ID NO:8.
- the reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Lachnoclostridium bacteria m3 comprises a polynucleotide probe shown in SEQ ID NO:12.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Hungatella hathewayi comprises a polynucleotide probe shown in SEQ ID NO:16.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Bacteroides clarus comprises a polynucleotide probe shown in SEQ ID NO:20.
- the agent comprises a detectable moiety.
- the kit further comprises reagents for a fecal immunochemical test (FIT).
- FIT fecal immunochemical test
- the kit further comprises a standard control that provides an average amount in a stool sample of one or more selected from:
- the reagent quantitatively identifies the DNA or RNA by RT-PCR, real-time quantitative PCR, or metagenomic sequencing.
- the reagent quantitatively identifies the protein by Western blot, radioimmunoassay, enzyme-linked immunosorbent assay, immunofluorescence, or protein chip.
- the present application provides a method for assessing the risk of colorectal cancer or colorectal adenoma in a subject, comprising the following steps:
- step (b) comparing the level obtained in step (a) with a standard control
- step (c) determining the level obtained in step (a) as an increase or decrease relative to a standard control
- step (a) comprises determining the level of DNA or RNA specific to Cloacibacillus porcorum.
- step (a) includes determining the level of the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- step (a) further comprises quantitatively determining the level of at least one of Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi, and Bacteroides clarus in a stool sample taken from the subject.
- step (a) comprises:
- step (a) comprises:
- the level of gene marker m370640 was determined.
- step (a) comprises a polynucleotide amplification reaction.
- the polynucleotide amplification reaction is a polymerase chain reaction (PCR).
- the present application provides a method for detecting an increase or decrease in the level of Cloacibacillus porcorum in a stool sample, comprising the following steps:
- step (b) comparing the level obtained in step (a) with a standard control
- step (c) determining the level obtained in step (a) as an increase or decrease relative to a standard control.
- the stool sample is obtained from a human subject.
- step (a) comprises determining the level of DNA or RNA specific to Cloacibacillus porcorum.
- step (a) includes determining the level of the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- step (a) further includes quantitatively determining the level of at least one of Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi, and Bacteroides clarus in the stool sample.
- step (a) comprises:
- step (a) comprises:
- the level of gene marker m370640 was determined.
- step (a) comprises a polynucleotide amplification reaction.
- the polynucleotide amplification reaction is a polymerase chain reaction (PCR).
- Embodiment 1 A kit for detecting colorectal cancer or colorectal adenoma in a subject, comprising:
- Embodiment 2 The kit according to embodiment 1, wherein
- the DNA or RNA unique to Cloacibacillus porcorum contains the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- Embodiment 3 A kit as described in Embodiment 1, wherein the reagent for specifically and quantitatively identifying DNA or RNA unique to Cloacibacillus porcorum comprises an oligonucleotide primer, and the oligonucleotide primer comprises a nucleic acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3; or
- the reagent for specifically and quantitatively identifying DNA or RNA unique to Cloacibacillus porcorum comprises a polynucleotide probe shown in SEQ ID NO:4.
- Embodiment 4 A kit as described in Embodiment 1, which also comprises a standard control, which provides an average amount of Cloacibacillus porcorum in a stool sample.
- Embodiment 5 The kit according to embodiment 1, wherein the kit further comprises one or more reagents selected from the following:
- Embodiment 6 The kit according to embodiment 5, wherein
- the DNA or RNA unique to the genus Fusobacterium comprises the DNA or RNA shown in SEQ ID NO:5 Fusobacterium genus nusG gene;
- m3 is a DNA or RNA unique to the genus Lachnoclostridium, comprising the gene marker m482585;
- a DNA or RNA unique to Clostridium hassawayi comprises the genetic marker m2736705; or
- the DNA or RNA unique to Bacteroides clarus comprises the gene marker m370640.
- Embodiment 7 The kit according to embodiment 5, wherein
- a reagent for specifically and quantitatively identifying DNA or RNA unique to the genus Fusobacterium comprises an oligonucleotide primer comprising a nucleic acid sequence shown in SEQ ID NO: 6 and SEQ ID NO: 7;
- the reagent for specifically and quantitatively identifying DNA or RNA unique to Lachnoclostridium bacteria m3 comprises oligonucleotide primers, wherein the oligonucleotide primers comprise the nucleic acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11;
- a reagent for specifically and quantitatively identifying DNA or RNA unique to Hungatella hathewayi comprises an oligonucleotide primer comprising a nucleic acid sequence shown in SEQ ID NO: 14 and SEQ ID NO: 15; or
- a reagent for specifically and quantitatively identifying DNA or RNA unique to Bacteroides clarus comprises oligonucleotide primers, wherein the oligonucleotide primers comprise the nucleic acid sequences shown in SEQ ID NO: 18 and SEQ ID NO: 19;
- a reagent for specifically and quantitatively identifying DNA or RNA unique to the genus Fusobacterium comprises a polynucleotide probe shown in SEQ ID NO:8;
- the reagent for specifically and quantitatively identifying DNA or RNA unique to Lachnoclostridium bacteria m3 comprises a polynucleotide probe shown in SEQ ID NO:12;
- a reagent for specifically and quantitatively identifying DNA or RNA unique to Hungatella hathewayi comprises a polynucleotide probe shown in SEQ ID NO:16; or
- a reagent for specifically and quantitatively identifying DNA or RNA unique to Bacteroides clarus comprises a polynucleotide probe shown in SEQ ID NO:20.
- Embodiment 8 The kit of Embodiment 1 or 5, wherein the reagent comprises a detectable portion.
- Embodiment 9 The kit as described in embodiment 1 or 5 further comprises an operation manual.
- Embodiment 10 The kit of embodiment 1 or 5, further comprising a reagent for a fecal immunochemical test (FIT).
- FIT fecal immunochemical test
- Embodiment 11 The kit of embodiment 5, further comprising a standard control providing an average amount of one or more selected from the following in a stool sample:
- Embodiment 12 Use of a reagent for specifically and quantitatively identifying DNA or RNA unique to Cloacibacillus porcorum in the preparation of a kit for detecting colorectal cancer or colorectal adenoma in a subject.
- Embodiment 13 The use according to embodiment 12, wherein
- the DNA or RNA unique to Cloacibacillus porcorum contains the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- Embodiment 14 The use as described in Embodiment 12, wherein the reagent for specifically and quantitatively identifying DNA or RNA unique to Cloacibacillus porcorum comprises an oligonucleotide primer, and the oligonucleotide primer comprises the nucleic acid sequence shown in SEQ ID NO:2 and SEQ ID NO:3; or
- the reagent for specifically and quantitatively identifying DNA or RNA unique to Cloacibacillus porcorum comprises a polynucleotide probe shown in SEQ ID NO:4.
- Embodiment 15 The use as described in Embodiment 12, wherein the kit further comprises a standard control, which provides the average amount of Cloacibacillus porcorum in the stool sample.
- Embodiment 16 The use according to embodiment 12, wherein the kit further comprises one or more reagents selected from the following:
- Embodiment 17 The use according to embodiment 16, wherein
- the DNA or RNA specific to the genus Fusobacterium comprises the Fusobacterium nusG gene shown in SEQ ID NO:5;
- m3 is a DNA or RNA unique to the genus Lachnoclostridium, comprising the gene marker m482585;
- a DNA or RNA unique to Clostridium hassawayi comprises the genetic marker m2736705; or
- the DNA or RNA unique to Bacteroides clarus comprises the gene marker m370640.
- Embodiment 18 The use according to embodiment 16, wherein
- the reagent for specifically and quantitatively identifying DNA or RNA unique to the genus Fusobacterium comprises an oligonucleotide primer comprising the nucleotide sequence shown in SEQ ID NO: 6 and SEQ ID NO: 7. Acid sequence;
- the reagent for specifically and quantitatively identifying DNA or RNA unique to Lachnoclostridium bacteria m3 comprises oligonucleotide primers, wherein the oligonucleotide primers comprise the nucleic acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11;
- a reagent for specifically and quantitatively identifying DNA or RNA unique to Hungatella hathewayi comprises an oligonucleotide primer comprising a nucleic acid sequence shown in SEQ ID NO: 14 and SEQ ID NO: 15; or
- a reagent for specifically and quantitatively identifying DNA or RNA unique to Bacteroides clarus comprises oligonucleotide primers, wherein the oligonucleotide primers comprise the nucleic acid sequences shown in SEQ ID NO: 18 and SEQ ID NO: 19;
- a reagent for specifically and quantitatively identifying DNA or RNA unique to the genus Fusobacterium comprises a polynucleotide probe shown in SEQ ID NO:8;
- the reagent for specifically and quantitatively identifying DNA or RNA unique to Lachnoclostridium bacteria m3 comprises a polynucleotide probe shown in SEQ ID NO:12;
- a reagent for specifically and quantitatively identifying DNA or RNA unique to Hungatella hathewayi comprises a polynucleotide probe shown in SEQ ID NO:16; or
- a reagent for specifically and quantitatively identifying DNA or RNA unique to Bacteroides clarus comprises a polynucleotide probe shown in SEQ ID NO:20.
- Embodiment 19 The use of embodiment 12 or 16, wherein the agent comprises a detectable portion.
- Embodiment 20 The use according to embodiment 12 or 16, wherein the kit further comprises reagents for fecal immunochemical test (FIT).
- FIT fecal immunochemical test
- Embodiment 21 The use according to embodiment 16, wherein the kit further comprises a standard control, the standard control providing an average amount of one or more selected from the following in the stool sample:
- Embodiment 22 The use according to embodiment 12, wherein
- the reagent quantitatively identifies the DNA or RNA by RT-PCR, real-time quantitative PCR or metagenomic sequencing.
- Embodiment 23 A method for assessing a subject's risk of colorectal cancer or colorectal adenoma, comprising the steps of:
- step (b) comparing the level obtained in step (a) with a standard control
- step (c) determining the level obtained in step (a) as an increase or decrease relative to a standard control
- Embodiment 24 A method as described in Embodiment 23, wherein step (a) includes determining the level of DNA or RNA that is specific to Cloacibacillus porcorum.
- Embodiment 25 A method as described in Embodiment 24, wherein step (a) includes determining the level of the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- Embodiment 26 A method as described in Embodiment 23, wherein step (a) further includes quantitatively determining the level of at least one of Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi and Bacteroides clarus in a stool sample taken from the subject.
- Embodiment 27 The method of embodiment 26, wherein step (a) comprises:
- Embodiment 28 The method of embodiment 27, wherein step (a) comprises:
- the level of gene marker m370640 was determined.
- Embodiment 29 A method as described in Embodiment 23, wherein step (a) comprises a polynucleotide amplification reaction.
- Embodiment 30 A method as described in Embodiment 29, wherein the polynucleotide amplification reaction is a polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- Embodiment 31 A method for detecting an increase or decrease in the level of Cloacibacillus porcorum in a stool sample, comprising the following steps:
- step (b) comparing the level obtained in step (a) with a standard control
- step (c) determining the level obtained in step (a) as an increase or decrease relative to a standard control.
- Embodiment 32 The method of embodiment 31, wherein the stool sample is obtained from a human subject.
- Embodiment 33 The method of embodiment 31, wherein step (a) comprises determining the level of DNA or RNA specific for Cloacibacillus porcorum.
- Embodiment 34 A method as described in Embodiment 33, wherein step (a) includes determining the level of the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- Embodiment 35 A method as described in Embodiment 31, wherein step (a) further includes quantitatively determining the level of at least one of Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi, and Bacteroides clarus in the stool sample.
- Embodiment 36 The method of embodiment 35, wherein step (a) comprises:
- Embodiment 37 The method of embodiment 36, wherein step (a) comprises:
- the level of gene marker m370640 was determined.
- Embodiment 38 A method as described in Embodiment 31, wherein step (a) comprises a polynucleotide amplification reaction.
- Embodiment 39 A method as described in embodiment 38, wherein the polynucleotide amplification reaction is a polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- Figure 1 Abundance of C. porcorum identified by metagenomic sequencing.
- N represents normal control;
- A represents adenoma;
- CRC represents colorectal cancer.
- Figure 2 Correlation between metagenomic sequencing and qPCR quantification.
- Figure 3 qPCR validation of identified bacterial markers.
- A Correlation between the abundance of candidate markers and disease progression from normal to adenoma and further to CRC.
- B Bacterial abundance detected by qPCR. Where N represents normal control; A represents adenoma; CRC represents colorectal cancer.
- Figure 4 Fecal levels and detection rates of C. porcorum assessed by metagenomic sequencing (A) and qPCR (B).
- A represents adenoma
- CRC represents colorectal cancer
- nAA represents non-advanced adenoma
- AA represents advanced adenoma.
- Figure 5 ROC curve analysis of the performance comparison of single bacterial markers in the diagnosis of CRC and adenoma.
- Fn represents Fusobacterium nucleatum
- Ch represents Clostridium hassawayii
- m3 represents Lachnoclostridium m3
- Cp represents C. porcorum
- N represents normal control
- A represents adenoma.
- Figure 6 ROC curve analysis of the performance of the bacterial marker group in the diagnosis of CRC and adenoma.
- Fn represents Fusobacterium nucleatum
- Ch represents Clostridium hassawayii
- m3 represents Lachnoclostridium m3
- Cp represents C. porcorum
- Bc Bacteroides clarithromyces
- N represents normal control
- A represents adenoma.
- Figure 7 C. porcorum significantly improved the diagnosis of adenoma by the “4Bac” test or m3.
- 4Bac Fusobacterium nucleatum + Clostridium hassawayi + Lachnoclostridium m3 + Bacteroides clarus; Cp represents C. porcorum; N represents normal control; A represents adenoma; AUC represents the area under the receiver operating characteristic curve.
- Figure 8 Combining fecal immunochemical tests (FIT) significantly improved the diagnostic performance of CRC and advanced adenomas.
- 4Bac Fusobacterium nucleatum + Clostridium hassawayii + Lachnoclostridium m3 + Bacteroides clarus; 5Bac: 4Bac + C. porcorum; AA indicates advanced adenoma; nAA indicates non-advanced adenoma; N indicates normal control; AUC indicates area under the receiver operating characteristic curve.
- Figure 9 Effect of fecal immunochemical test (FIT) and C. porcorum on the detection of CRC and adenoma.
- 4Bac Fusobacterium nucleatum + Clostridium hassawayii + Lachnoclostridium m3 + Bacteroides clarus; 5Bac: 4Bac + C. porcorum.
- Figure 10 Comparison of the sensitivity of fecal immunochemical test (FIT), bacterial markers and their combination in detecting CRC according to TNM staging subgroups.
- 4Bac Fusobacterium nucleatum + Clostridium hassawayii + Lachnoclostridium m3 + Bacteroides clarus; 5Bac: 4Bac + C. porcorum; PPV indicates positive predictive value; NPV indicates negative predictive value.
- SEQ ID NO:1 to SEQ ID NO:4 are, respectively, a gene marker fragment (SEQ ID NO:1) for Cloacibacillus porcorum, a forward primer (Cp-F, SEQ ID NO:2) and a reverse primer (Cp-R, SEQ ID NO:3) for detecting this gene marker, and a probe (Cp-probe, SEQ ID NO:4) for detecting this gene marker.
- SEQ ID NO:5 to SEQ ID NO:8 are the nusG gene of Fusobacterium (SEQ ID NO:5), the forward primer (Fn-F, SEQ ID NO:6) and reverse primer (Fn-R, SEQ ID NO:7) for detecting this gene, and the probe for detecting this gene (Fn-probe, SEQ ID NO:8).
- SEQ ID NO:9 to SEQ ID NO:12 are respectively the gene marker m482585 (SEQ ID NO:9) for Lachnoclostridium bacteria m3, the forward primer (m3-F, SEQ ID NO:10) and reverse primer (m3-R, SEQ ID NO:11) for detecting this gene marker, and the probe (m3-probe, SEQ ID NO:12) for detecting this gene marker.
- SEQ ID NO:13 to SEQ ID NO:16 are, respectively, the gene marker m2736705 (SEQ ID NO:13) for Hungatella hathewayi (formerly known as Clostridium hathewayi), the forward primer (Ch-F, SEQ ID NO:14) and reverse primer (Ch-R, SEQ ID NO:15) for detecting this gene marker, and the probe (Ch-probe, SEQ ID NO:16) for detecting this gene marker.
- SEQ ID NO: 17 to SEQ ID NO: 20 are respectively directed against Bacteroides clarus
- the invention also provides a gene marker m370640 (SEQ ID NO: 17), a forward primer (Bc-F, SEQ ID NO: 18) and a reverse primer (Bc-R, SEQ ID NO: 19) for detecting the gene marker, and a probe (Bc-probe, SEQ ID NO: 20) for detecting the gene marker.
- SEQ ID NO:21 is a genetic marker for Cloacibacillus porcorum.
- colonal cancer CRC
- Colorectal cancer cells are colorectal epithelial cells that have characteristics of colorectal cancer, and include precancerous cells, i.e., cells that are in the early stages of transformation into cancer cells or are prone to transformation into cancer cells. Such cells may exhibit one or more phenotypic traits characteristic of cancerous cells.
- nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids including known analogs of natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs) and complementary sequences thereof and sequences explicitly indicated.
- variants e.g., degenerate codon substitutions
- SNPs single nucleotide polymorphisms
- degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues.
- nucleic acid can be used interchangeably with genes, cDNAs, and mRNAs encoded by genes.
- gene refers to a DNA segment involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) that are involved in the transcription/translation of the gene product and the regulation of transcription/translation, as well as intervening sequences (introns) between individual coding segments (exons).
- polypeptide polypeptide
- peptide protein
- polymers of amino acid residues The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
- the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked by covalent peptide bonds.
- amino acid refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
- Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
- amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium.
- amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but A chemical compound that functions in a manner similar to a naturally occurring amino acid.
- the amino acids can include those with non-naturally occurring D-chirality, which can improve the stability (eg, half-life), bioavailability, and other characteristics of polypeptides comprising one or more such D-amino acids.
- Amino acids may be referred to herein by their commonly known three letter symbols, or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.
- primer refers to an oligonucleotide that can be used in an amplification method (such as polymerase chain reaction (PCR)) to amplify a nucleotide sequence based on a polynucleotide sequence corresponding to a target gene (e.g., a DNA or RNA sequence of a related bacterial species).
- amplification method such as polymerase chain reaction (PCR)
- PCR polymerase chain reaction
- at least one PCR primer for amplifying a polynucleotide sequence is sequence-specific for the polynucleotide sequence. The exact length of a primer depends on multiple factors, including temperature, primer source, and the method used.
- an oligonucleotide primer typically contains at least 10, at least 15, at least 20, or at least 25 or more nucleotides, although it may contain fewer nucleotides or more nucleotides.
- the factors involved in determining the appropriate length of a primer are well known to those skilled in the art.
- a “label,””detectablemarker,” or “detectable moiety” is a moiety that can be detected by spectrophotometric, photochemical, biochemical, immunochemical, chemical, or other physical means.
- useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, digoxigenin, or haptens and proteins that can be made detectable (e.g., by incorporating a radioactive component into the peptide) or used to detect antibodies that react specifically with the peptide.
- the detectable label is linked to a probe or molecule with defined binding properties (e.g., a polypeptide or polynucleotide with known binding specificity) to allow easy detection of the presence of the probe (and its bound target).
- standard control refers to a polynucleotide sequence or polypeptide (e.g., DNA, RNA or protein of related bacteria) present in an established disease-free stool sample (e.g., stool samples from average healthy individuals who have not been diagnosed with CRC or are known to have an increased risk of developing into CRC).
- Standard control values are suitable for the inventive method as the basis for the amount of DNA, RNA or protein of related bacteria present in the comparison test sample.
- an average amount of DNA, RNA or protein of related bacteria common in stool samples of average healthy people who do not suffer from any colon disease (especially CRC) defined conventionally is provided.
- Standard control values can vary according to the properties of the sample and other factors, such as the sex, age, race of the individual as the basis for establishing the control value.
- the term "average” refers to certain characteristics in human stool samples that can represent a randomly selected healthy population that does not suffer from any colorectal disease (especially CRC) and has no known risk of developing the disease, especially the levels of DNA, RNA or proteins of certain related bacteria.
- the selected population should include a sufficient number of people so that the average level or amount of DNA, RNA or protein of the related bacteria found in the feces of these individuals can reflect the corresponding level or amount of these DNA, RNA or proteins in the general healthy population with reasonable accuracy.
- the selected The population of people selected is generally of similar age to the subject for whom the stool sample is to be tested for colorectal cancer indications.
- other factors such as sex, race, medical history, etc. should also be considered, and preferably the test subject is closely matched in characteristics to the selected group of individuals for which the "average" value is established.
- the term “amount” refers to the amount of a polynucleotide or polypeptide of interest (e.g., DNA, RNA or protein of a related bacterium) present in a sample. Such an amount can be expressed as an absolute value, i.e., the total amount of the polynucleotide or polypeptide in the sample, or as a relative value, i.e., the concentration of the polynucleotide or polypeptide in the sample.
- an absolute value i.e., the total amount of the polynucleotide or polypeptide in the sample
- a relative value i.e., the concentration of the polynucleotide or polypeptide in the sample.
- the present application provides a kit for detecting colorectal cancer or colorectal adenoma in a subject, comprising: a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum.
- the adenoma is a non-advanced adenoma or an advanced adenoma.
- the reagent specifically and quantitatively identifies DNA or RNA that is unique to Cloacibacillus porcorum.
- the DNA or RNA specific to Cloacibacillus porcorum comprises the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- the reagent comprises a set of two oligonucleotide primers
- the set of two oligonucleotide primers comprises the nucleic acid sequence shown in SEQ ID NO:2 and SEQ ID NO:3.
- the reagent comprises a polynucleotide probe shown in SEQ ID NO:4.
- the kit further comprises a standard control that provides an average amount of Cloacibacillus porcorum in a stool sample.
- the kit further comprises one or more reagents selected from the group consisting of:
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- the kit comprises (1) a reagent for specifically and quantitatively identifying DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent for specifically and quantitatively identifying DNA, RNA or protein that is unique to the genus Fusobacterium; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is non-advanced In some cases, this combination may be abbreviated as LR2' or Fn+Cp.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Lachnoclostridium bacteria m3; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma. In some cases, this combination may be abbreviated as "LR2" or m3+Cp.
- the kit includes (1) a reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum, and (2) a reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Hungatella hathewayi; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma.
- the kit includes (1) a reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum, and (2) a reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Bacteroides clarus; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Lachnoclostridium bacteria m3, and (3) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Fusobacterium; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma. In some cases, this combination can be abbreviated as LR3 or Fn+m3+Cp.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Hungatella hathewayi, and (3) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to the genus Fusobacterium; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma. In some cases, this combination can be abbreviated as LR3" or Fn+Ch+Cp.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA, or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA, or protein that is unique to Bacteroides clarus, and (3) a reagent that specifically and quantitatively identifies DNA, RNA, or protein that is unique to the genus Fusobacterium; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject.
- the adenoma is a non-advanced adenoma or an advanced adenoma.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Hungatella hathewayi, and (3) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Lachnoclostridium bacteria m3; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma. In some cases, this combination can be abbreviated as LR3"' or Ch+m3+Cp.
- the kit includes (1) a reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum, and (2) a reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Lachnoclostridium m3, and (3) a reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Bacteroides clarus; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Hungatella hathewayi, and (3) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Bacteroides clarus; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Hungatella hathewayi, and (3) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to the genus Fusobacterium, and (4) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Lachnoclostridium bacteria m3; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma. In some cases, this combination can be abbreviated as LR4' or Fn+m3+Cp+Ch.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Hungatella hathewayi, and (3) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Bacteroides clarus, and (4) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Lachnoclostridium bacteria m3; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is non-advanced Adenoma or advanced adenoma. In some cases, this combination may be abbreviated as LR4" or Ch+m3+Cp+Bc.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Hungatella hathewayi, and (3) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to the genus Fusobacterium, and (4) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Bacteroides clarus; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma. In some cases, this combination can be abbreviated as LR4"' or Fn+Ch+Cp+Bc.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Bacteroides clarus, and (3) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to the genus Fusobacterium, and (4) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Lachnoclostridium bacteria m3; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma. In some cases, this combination can be abbreviated as LR4 or Fn+m3+Cp+Bc.
- the kit includes (1) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Cloacibacillus porcorum, and (2) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Hungatella hathewayi, and (3) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Fusobacterium, and (4) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Lachnoclostridium m3, and (5) a reagent that specifically and quantitatively identifies DNA, RNA or protein that is unique to Bacteroides clarus; optionally, the kit is used to detect colorectal cancer and/or colorectal adenoma in a subject, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma. In some cases, this combination can be abbreviated as LR5
- Fn+m3+Ch+Cp+Bc can be used to diagnose CRC.
- Fn+m3+Ch+Cp+Bc can be used to diagnose adenoma, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma.
- the DNA or RNA specific to the genus Fusobacterium includes the Fusobacterium nusG gene shown in SEQ ID NO:5.
- the DNA or RNA unique to Lachnoclostridium bacterium m3 comprises the genetic marker m482585.
- the DNA or RNA unique to Clostridium hassawayi comprises genetic marker m2736705.
- the DNA or RNA unique to Bacteroides clarus comprises the gene marker m370640.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to the genus Fusobacterium comprises an oligonucleotide primer comprising a nucleic acid sequence shown in SEQ ID NO:6 and SEQ ID NO:7.
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Lachnoclostridium bacteria m3 comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:10 and SEQ ID NO:11.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Hungatella hathewayi comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:14 and SEQ ID NO:15.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Bacteroides clarus comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:18 and SEQ ID NO:19.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to the genus Fusobacterium comprises a polynucleotide probe shown in SEQ ID NO:8.
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- the reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Lachnoclostridium bacteria m3 comprises a polynucleotide probe shown in SEQ ID NO:12.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Hungatella hathewayi comprises a polynucleotide probe shown in SEQ ID NO:16.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Bacteroides clarus comprises a polynucleotide probe shown in SEQ ID NO:20.
- a set of two oligonucleotide primers SEQ ID NO:6 and SEQ ID NO:7 are used to detect the nusG gene; in some embodiments, a polynucleotide probe SEQ ID NO:8 is used to detect the nusG gene.
- a set of two oligonucleotide primers SEQ ID NO:10 and SEQ ID NO:11 are used to detect gene marker m482585; in some embodiments, a polynucleotide probe SEQ ID NO:12 is used to detect gene marker m482585.
- a set of two oligonucleotide primers SEQ ID NO: 14 and SEQ ID NO: 15 are used to detect gene marker m2736705; in some embodiments, a polynucleotide probe SEQ ID NO: 16 is used to detect gene marker m2736705.
- a set of two oligonucleotide primers SEQ ID NO:18 and SEQ ID NO:19 are used to detect the gene marker m370640; in some embodiments, the polynucleotide probe SEQ ID NO:20 is used to detect the gene marker m370640.
- the agent comprises a detectable moiety.
- the reagent for specifically and quantitatively identifying the unique DNA or RNA of the target bacteria generally includes at least one oligonucleotide that can be used for specific hybridization with at least one segment of the target DNA or RNA sequence or its complementary sequence. In some embodiments, this oligonucleotide is labeled with a detectable portion.
- the reagent may include at least two oligonucleotide primers that can be used for at least one segment of the target bacteria DNA or RNA amplification by PCR (including by RT-PCR).
- the reagent may include at least one oligonucleotide probe that can be used for binding at least one segment of the target bacteria DNA or RNA. In some embodiments, a detectable portion of a labeled primer or probe is used.
- the reagent for specifically and quantitatively identifying a protein unique to a target bacterium generally includes at least one antibody that can be used for specific binding to a protein specific to the target bacterium.
- the antibody is labeled with a detectable portion.
- the antibody can be a monoclonal antibody or a polyclonal antibody.
- the reagent can include at least two different antibodies, one for specific binding to a protein specific to the target bacterium (i.e., a primary antibody), and the other for detection of a primary antibody (i.e., a secondary antibody), and the secondary antibody is generally connected to a detectable portion.
- probes or primers are directly conjugated to labels such as fluorophores, chemiluminescent agents, and enzymes.
- labels such as fluorophores, chemiluminescent agents, and enzymes. The choice of detectable moiety depends on the sensitivity required, the ease of conjugation to the probe or primer, the stability requirements, and the available instrumentation.
- the probe carries a 5' reporter dye FAM (6-carboxyfluorescein) or VIC (4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein) and a 3' quencher dye TAMRA (6-carboxytetramethyl-rhodamine).
- FAM 6-carboxyfluorescein
- VIC 4,7,2'-trichloro-7'-phenyl-6-carboxyfluorescein
- TAMRA 6-carboxytetramethyl-rhodamine
- the kit further comprises an operating manual.
- the kit further comprises reagents for a fecal immunochemical test (FIT).
- FIT fecal immunochemical test
- Fn+m3+Ch+Cp+Bc+FIT can be used to diagnose CRC.
- Fn+m3+Ch+Cp+Bc+FIT can be used to diagnose adenoma, preferably, the adenoma is a non-advanced adenoma or an advanced adenoma.
- the kit further comprises a standard control that provides an average amount in a stool sample of one or more selected from:
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- the present application provides a method for specifically and quantitatively identifying Cloacibacillus porcorum Use of a reagent that is a specific DNA, RNA or protein in the preparation of a kit for detecting colorectal cancer or colorectal adenoma in a subject.
- the DNA or RNA specific to Cloacibacillus porcorum comprises the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum comprises oligonucleotide primers, which comprise the nucleic acid sequences shown in SEQ ID NO:2 and SEQ ID NO:3.
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Cloacibacillus porcorum comprises a polynucleotide probe shown in SEQ ID NO:4.
- the reagent for specifically and quantitatively identifying a DNA, RNA, or protein unique to Cloacibacillus porcorum comprises an antibody that specifically binds to the protein.
- the kit further comprises a standard control that provides an average amount of Cloacibacillus porcorum in a stool sample.
- the kit further comprises one or more reagents selected from the group consisting of:
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- the DNA or RNA specific to the genus Fusobacterium includes the Fusobacterium nusG gene shown in SEQ ID NO:5.
- the DNA or RNA unique to Lachnoclostridium bacterium m3 comprises the genetic marker m482585.
- the DNA or RNA unique to Clostridium hassawayi comprises genetic marker m2736705.
- the DNA or RNA unique to Bacteroides clarus comprises the gene marker m370640.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to the genus Fusobacterium comprises an oligonucleotide primer comprising a nucleic acid sequence shown in SEQ ID NO:6 and SEQ ID NO:7.
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- the reagent for specifically and quantitatively identifying DNA, RNA or protein unique to Lachnoclostridium bacteria m3 comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:10 and SEQ ID NO:11.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Hungatella hathewayi comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:14 and SEQ ID NO:15.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Bacteroides clarus comprises oligonucleotide primers comprising the nucleic acid sequences shown in SEQ ID NO:18 and SEQ ID NO:19.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to the genus Fusobacterium comprises a polynucleotide probe shown in SEQ ID NO:8.
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- the reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Lachnoclostridium bacteria m3 comprises a polynucleotide probe shown in SEQ ID NO:12.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Hungatella hathewayi comprises a polynucleotide probe shown in SEQ ID NO:16.
- a reagent for specifically and quantitatively identifying DNA, RNA, or protein unique to Bacteroides clarus comprises a polynucleotide probe shown in SEQ ID NO:20.
- the agent comprises a detectable moiety.
- the kit further comprises reagents for a fecal immunochemical test (FIT).
- FIT fecal immunochemical test
- the kit further comprises a standard control that provides an average amount in a stool sample of one or more selected from:
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- the reagent quantitatively identifies the DNA or RNA by RT-PCR, real-time quantitative PCR, or metagenomic sequencing.
- the reagent can also quantitatively identify the DNA or RNA by at least one method selected from the following: in situ hybridization, polymerase chain reaction (PCR), RNase protection assay (RPA), northern blot, microarray and high-throughput sequencing.
- PCR polymerase chain reaction
- RPA RNase protection assay
- the reagent can also be used to quantitatively identify the The proteins were determined by: two-dimensional electrophoresis, immunohistochemistry (IHC), fluorescence activated cell sorter (FACS), radioimmunoassay (RIA), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), radial immunodiffusion, immunoprecipitation, flow cytometry, Ouchterlony two-way immunodiffusion, and complement fixation assay.
- IHC immunohistochemistry
- FACS fluorescence activated cell sorter
- RIA radioimmunoassay
- MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
- the reagent quantitatively identifies the protein by Western blot, radioimmunoassay, enzyme-linked immunosorbent assay, immunofluorescence, or protein chip.
- the nucleic acid sequence shown in SEQ ID NO: 1 or 21 and the levels of one or more DNA or RNA selected from m482585, nusG gene, m2736705 and m370640 are quantitatively identified by RT-PCR, real-time quantitative PCR or metagenomic sequencing.
- the nucleic acid sequence shown in SEQ ID NO: 1 or 21 and the level of a DNA or RNA selected from m482585, nusG gene, m2736705 and m370640 are quantitatively identified by RT-PCR, real-time quantitative PCR or metagenomic sequencing.
- the nucleic acid sequence shown in SEQ ID NO: 1 or 21 and the levels of two DNAs or RNAs selected from m482585, nusG gene, m2736705 and m370640 are quantitatively identified by RT-PCR, real-time quantitative PCR or metagenomic sequencing.
- the nucleic acid sequence shown in SEQ ID NO: 1 or 21 and the levels of three DNA or RNA selected from m482585, nusG gene, m2736705 and m370640 are quantitatively identified by RT-PCR, real-time quantitative PCR or metagenomic sequencing.
- the nucleic acid sequence shown in SEQ ID NO: 1 or 21 and the levels of DNA or RNA of m482585, nusG gene, m2736705 and m370640 are quantitatively identified by RT-PCR, real-time quantitative PCR or metagenomic sequencing.
- colorectal cancer or colorectal adenoma is diagnosed by comparing the level of the DNA, RNA or protein to a standard control.
- the present application provides a method for assessing the risk of colorectal cancer or colorectal adenoma in a subject, comprising the following steps:
- step (b) comparing the level obtained in step (a) with a standard control
- step (c) determining the level obtained in step (a) as an increase or decrease relative to a standard control
- step (a) comprises determining the level of DNA, RNA or protein specific to at least one of Cloacibacillus porcorum, Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi and Bacteroides clarus.
- step (a) includes determining the level of DNA unique to at least one of Cloacibacillus porcorum, Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi, and Bacteroides clarus.
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- step (a) includes determining the level of the nucleic acid sequence shown in SEQ ID NO:1 or 21, the nusG gene of the Fusobacterium genus, or the gene markers m482585, m2736705 or m370640.
- step (a) when the subject is determined to have an increased risk of colorectal cancer or colorectal adenoma, further comprising repeating step (a) at a subsequent time using another stool sample from the subject at a subsequent time.
- the present application provides a method for detecting an increase or decrease in the level of at least one of Cloacibacillus porcorum, Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi, and Bacteroides clarus in a fecal sample, comprising the following steps:
- step (b) comparing the level obtained in step (a) with a standard control
- step (c) determining the level obtained in step (a) as an increase or decrease relative to a standard control.
- the stool sample is obtained from a human subject.
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- step (a) includes determining the level of DNA, RNA or protein that is unique to at least one of Cloacibacillus porcorum, Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi and Bacteroides clarus.
- step (a) includes determining the level of DNA unique to at least one of Cloacibacillus porcorum Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi, and Bacteroides clarus.
- the Fusobacterium genus is Fusobacterium nucleatum (Fn).
- step (a) includes determining the level of the nucleic acid sequence shown in SEQ ID NO:1 or 21, the nusG gene, or the gene marker m482585, m2736705, or m370640.
- the present application provides a method for assessing the risk of colorectal cancer or colorectal adenoma in a subject, comprising the following steps:
- step (b) comparing the level obtained in step (a) with a standard control
- step (c) determining the level obtained in step (a) as an increase or decrease relative to a standard control
- step (a) includes determining the level of DNA or RNA specific to Cloacibacillus porcorum.
- step (a) includes determining the level of the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- step (a) further comprises quantitatively determining the level of at least one of Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi, and Bacteroides clarus in a stool sample taken from the subject.
- step (a) comprises determining the level of DNA or RNA specific to the genus Fusobacterium.
- step (a) comprises determining the level of DNA or RNA that is specific to Lachnoclostridium m3.
- step (a) includes determining the level of DNA or RNA specific to Hungatella hathewayi.
- step (a) includes determining the level of DNA or RNA specific to Bacteroides clarus.
- step (a) includes determining the level of the Fusobacterium genus nusG gene shown in SEQ ID NO:5.
- step (a) comprises determining the level of gene marker m482585.
- step (a) comprises determining the level of gene marker m2736705.
- step (a) comprises determining the level of gene marker m370640.
- step (a) comprises a polynucleotide amplification reaction.
- the polynucleotide amplification reaction is a polymerase chain reaction (PCR).
- the present application provides a method for detecting an increase or decrease in the level of Cloacibacillus porcorum in a stool sample, comprising the following steps:
- step (b) comparing the level obtained in step (a) with a standard control
- step (c) determining the level obtained in step (a) as an increase or decrease relative to a standard control.
- the stool sample is obtained from a human subject.
- step (a) comprises determining the level of DNA or RNA specific to Cloacibacillus porcorum.
- step (a) includes determining the level of the nucleic acid sequence shown in SEQ ID NO:1 or 21.
- step (a) further comprises quantitatively determining the The level of at least one of Fusobacterium, Lachnoclostridium m3, Hungatella hathewayi, and Bacteroides clarus.
- step (a) comprises determining the level of DNA or RNA specific to the genus Fusobacterium.
- step (a) comprises determining the level of DNA or RNA that is specific to Lachnoclostridium m3.
- step (a) includes determining the level of DNA or RNA that is specific to Hungatella hathewayi.
- step (a) includes determining the level of DNA or RNA specific to Bacteroides clarus.
- step (a) includes determining the level of the Fusobacterium genus nusG gene shown in SEQ ID NO:5.
- step (a) comprises determining the level of gene marker m482585.
- step (a) comprises determining the level of gene marker m2736705.
- step (a) comprises determining the level of gene marker m370640.
- step (a) comprises a polynucleotide amplification reaction.
- the polynucleotide amplification reaction is a polymerase chain reaction (PCR).
- This study analyzed stool metagenomic sequencing data from 589 subjects (184 CRC patients, 185 adenoma patients, and 220 control subjects) 16 . Written informed consent was obtained from all subjects. The relative abundance of species was analyzed by MetaPhlAn3 17 .
- the FIT test was performed using an automated quantitative OC-Sensor test (Eiken Chemical, Japan) using stool samples.
- the quantitative OC-Sensor test was performed as described previously20 , with a positive cutoff value equivalent to a hemoglobin concentration of 100 ng/mL.
- the combined score of four bacterial markers (4Bac) using a logistic regression model (4Bac score I 1 + ⁇ 1 *Fn + ⁇ 2 *m3 + ⁇ 3 *Bc + ⁇ 4 *Ch) was determined in a previous study 19 .
- the combined scores of 2 to 5 markers with or without FIT using a logistic regression model are shown in Table 2 .
- I represents the intercept
- ⁇ represents the regression coefficient
- marker represents the corresponding Cq value.
- ROC curves were used to evaluate the bacterial markers/models in distinguishing CRC/adenomas from controls. The diagnostic value of ROC curves was determined.
- Cloacibacillus porcorum which has not been reported as a marker for colorectal tumor diagnosis, increased significantly as the disease progressed from normal to adenoma and further to cancer, and was significantly different between patients with adenoma or CRC and control subjects (Table 3; Figure 1). Cloacibacillus porcorum was confirmed to show a significant increasing trend from normal to adenoma to CRC (P ⁇ 0.0001, one-way ANOVA; Figure 1).
- Cloacibacillus porcorum assessed by metagenomic sequencing and qPCR was similar.
- the prevalence of Cloacibacillus porcorum was significantly higher in patients with adenoma and CRC compared with normal subjects, as assessed by metagenomic sequencing (A1 of Figure 4) and qPCR quantification (B1 of Figure 4).
- Cloacibacillus porcorum was present at significantly higher levels in patients with adenoma and CRC than in controls, as assessed by metagenomic sequencing (A2 of Figure 4) and qPCR quantification (B2 of Figure 4), with no differences between non-advanced and advanced adenomas or between different TNM stages of cancer.
- Cloacibacillus porcorum showed an AUC of 0.657 (95% CI: 0.583 to 0.725; P ⁇ 0.0001) for CRC and 0.618 (95% CI: 0.550 to 0.682; P ⁇ 0.0001) for adenoma ( Figure 5).
- Fn, m3, and Ch which are enriched in colorectal cancer and adenoma
- Cloacibacillus porcorum did not differ from Ch in distinguishing CRC patients from control subjects. These markers did not differ significantly from Fn in distinguishing adenoma patients from control subjects, and Cloacibacillus porcorum was significantly superior to Fn. Ch (P ⁇ 0.05) ( Figure 5).
- Fecal Cloacibacillus porcorum was significantly associated with the diagnosis of CRC and adenoma, independent of age
- Cloacibacillus porcorum and other bacterial markers improves the diagnostic performance for CRC and/or adenoma
- the bacterial markers Fn, Ch, Bc, and m3 for diagnosing CRC and adenoma have been previously reported, and the performance of these markers in combination with Cloacibacillus porcorum for diagnosing CRC and/or adenoma was further analyzed in this application (Table 6).
- a logistic regression model was established to distinguish patients with CRC/adenoma from control subjects using all five or fewer markers, removing the least important markers one by one according to their importance in the model.
- the 5-marker model showed no significant difference in performance in distinguishing adenoma from control subjects compared with the 4-marker and 3-marker combinations without Ch and/or Bc ( Figure 6A and Figure 6B).
- Cloacibacillus porcorum significantly improved the diagnostic performance of “4Bac” and m3 for adenoma
- FIT detected only 11.2% of advanced adenomas, but not non-advanced adenomas, with a specificity of 98.7%.
- logistic regression models were further trained by combining bacterial markers and FIT to distinguish patients with CRC/adenomas from normal controls, and then their diagnostic performance for CRC, advanced adenomas, and non-advanced adenomas was evaluated separately ( Figure 8; Table 6).
- ROC curve comparison analysis after combining with FIT, there was no difference in the diagnosis of CRC between 5Bac&FIT and 4Bac&FIT, while both performed significantly better than the corresponding models without FIT (all P ⁇ 0.05).
- the diagnostic sensitivity of FIT, bacterial markers, and their combination in detecting CRC was further compared according to the TNM stage subsets (Figure 10).
- the bacterial marker model had higher sensitivity than FIT for stage I-III cancer.
- the combination of 4Bac/5Bac and FIT had significantly higher sensitivity than FIT for stage I-III cancer, and also improved the detection rate of stage IV cancer.
- Cloacibacillus porcorum increased the sensitivity of 4Bac in detecting stage II-IV cancer, although not significantly.
- the present application further compared the diagnostic performance of Cloacibacillus porcorum with previously identified bacterial markers (including m3, Fn, Ch and Bc).
- the present application also designed a new bacterial marker combination, combined with or without FIT, for the diagnosis of CRC and adenoma.
- the results of the present application indicate that combination with Cloacibacillus porcorum significantly improves the diagnostic performance of previously identified bacterial markers for diagnosing CRC and adenomas (including non-advanced and advanced adenomas), while combination with FIT further improves the diagnosis of CRC and advanced adenomas.
- bacterial markers were quantified by qPCR and performed well in the diagnosis of CRC and adenoma.
- the combination of Fn, m3, and Cloacibacillus porcorum showed an AUC of 0.897 (95% CI: 0.844 to 0.937) and 0.770 (95% CI: 0.709 to 0.824) for CRC and adenoma, respectively.
- Further addition of Bc and/or Ch increased the AUC for CRC and adenoma to more than 0.92 and 0.77, respectively.
- Combination with FIT further improved the diagnostic sensitivity of bacterial markers for CRC and advanced adenoma.
- the optimal combination containing all five bacterial markers and FIT showed a sensitivity of 64.3% for adenoma, 96.2% for CRC, and a specificity of 84.6%.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
La présente invention concerne un nouveau marqueur microbien pour évaluer le risque de cancer colorectal ou d'adénome colorectal chez un sujet.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211563402.3 | 2022-12-07 | ||
CN202211563402.3A CN116042827B (zh) | 2022-12-07 | 2022-12-07 | 预测结直肠癌风险的新型微生物标志物 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2024119614A1 true WO2024119614A1 (fr) | 2024-06-13 |
Family
ID=86112315
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2023/076160 WO2024119614A1 (fr) | 2022-12-07 | 2023-02-15 | Nouveau marqueur microbien pour prédire le risque de cancer colorectal |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN116042827B (fr) |
WO (1) | WO2024119614A1 (fr) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016119190A1 (fr) * | 2015-01-30 | 2016-08-04 | Bgi Shenzhen | Biomarqueurs pour maladies liées à un cancer colorectal |
WO2018036503A1 (fr) * | 2016-08-25 | 2018-03-01 | The Chinese University Of Hong Kong | Marqueurs bactériens fécaux pour le cancer colorectal |
CN109943636A (zh) * | 2019-04-11 | 2019-06-28 | 上海宝藤生物医药科技股份有限公司 | 一种结直肠癌微生物标志物及其应用 |
CN112553344A (zh) * | 2021-02-22 | 2021-03-26 | 天津奇云诺德生物医学有限公司 | 一种与结直肠癌相关的生物标志物及其应用 |
TW202237857A (zh) * | 2020-11-19 | 2022-10-01 | 香港中文大學 | 通過非侵入性方法評估結腸直腸腺瘤復發風險 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10325685B2 (en) * | 2014-10-21 | 2019-06-18 | uBiome, Inc. | Method and system for characterizing diet-related conditions |
CN107779505B (zh) * | 2016-08-25 | 2023-06-06 | 香港中文大学 | 结肠直肠癌的粪便细菌标志物 |
AU2017335732A1 (en) * | 2016-09-27 | 2019-04-04 | Board Of Regents, The University Of Texas System | Methods for enhancing immune checkpoint blockade therapy by modulating the microbiome |
AU2019384816A1 (en) * | 2018-11-21 | 2021-06-24 | Board Of Regents, The University Of Texas System | Methods and compositions for treating cancer |
CN111743952A (zh) * | 2019-03-28 | 2020-10-09 | 珠海岐微生物科技有限公司 | 生地在调节肠道微生物中的用途 |
CN114672556A (zh) * | 2020-12-25 | 2022-06-28 | 上海善准生物科技有限公司 | 结直肠癌分子分型及生存风险基因群及诊断产品和应用 |
-
2022
- 2022-12-07 CN CN202211563402.3A patent/CN116042827B/zh active Active
-
2023
- 2023-02-15 WO PCT/CN2023/076160 patent/WO2024119614A1/fr unknown
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016119190A1 (fr) * | 2015-01-30 | 2016-08-04 | Bgi Shenzhen | Biomarqueurs pour maladies liées à un cancer colorectal |
WO2018036503A1 (fr) * | 2016-08-25 | 2018-03-01 | The Chinese University Of Hong Kong | Marqueurs bactériens fécaux pour le cancer colorectal |
CN109943636A (zh) * | 2019-04-11 | 2019-06-28 | 上海宝藤生物医药科技股份有限公司 | 一种结直肠癌微生物标志物及其应用 |
TW202237857A (zh) * | 2020-11-19 | 2022-10-01 | 香港中文大學 | 通過非侵入性方法評估結腸直腸腺瘤復發風險 |
CN112553344A (zh) * | 2021-02-22 | 2021-03-26 | 天津奇云诺德生物医学有限公司 | 一种与结直肠癌相关的生物标志物及其应用 |
Non-Patent Citations (1)
Title |
---|
LIU NING-NING; JIAO NA; TAN JING-CONG; WANG ZILIANG; WU DINGFENG; WANG AN-JUN; CHEN JIE; TAO LIWEN; ZHOU CHENFEN; FANG WENJIE; CHE: "Multi-kingdom microbiota analyses identify bacterial–fungal interactions and biomarkers of colorectal cancer across cohorts", NATURE MICROBIOLOGY, NATURE PUBLISHING GROUP UK, LONDON, vol. 7, no. 2, 27 January 2022 (2022-01-27), London, pages 238 - 250, XP037683704, DOI: 10.1038/s41564-021-01030-7 * |
Also Published As
Publication number | Publication date |
---|---|
CN116042827B (zh) | 2023-12-22 |
CN116042827A (zh) | 2023-05-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2015289758B2 (en) | Methods for evaluating lung cancer status | |
EP2572000B1 (fr) | Procédé pour diagnostiquer le cancer colorectal | |
AU2011279555B2 (en) | Diagnostic for colorectal cancer | |
JP6114035B2 (ja) | 肺癌検出用唾液バイオマーカー | |
JP2016538884A (ja) | 遺伝子型及び表現型バイオマーカーを用いる無症候性血尿を有する患者のトリアージ | |
WO2014085434A1 (fr) | Compositions et procédés de diagnostic de tumeurs de la thyroïde | |
JP2011516077A (ja) | 癌を検出するための方法、薬剤、およびキット | |
US8076077B2 (en) | Compositions and methods for prognosis and therapy of liver cancer | |
WO2010104662A1 (fr) | Identification de marqueurs protéiques pour un diagnostic du cancer gastrique | |
WO2016112488A1 (fr) | Biomarqueurs de maladies liées au cancer colorectal | |
TW201500553A (zh) | 用於黑色素癌之預後預測之裝置的用途 | |
US10793911B2 (en) | Host DNA as a biomarker of Crohn's disease | |
WO2012009382A2 (fr) | Indicateurs moléculaires pour le pronostic du cancer de la vessie et la prédiction de la réponse au traitement | |
Izumi et al. | A genomewide transcriptomic approach identifies a novel gene expression signature for the detection of lymph node metastasis in patients with early stage gastric cancer | |
CN110229899B (zh) | 用于结直肠癌早期诊断或预后预测的血浆标记物组合 | |
Zhang et al. | Leveraging fecal microbial markers to improve the diagnostic accuracy of the fecal immunochemical test for advanced colorectal adenoma | |
CN107208149B (zh) | 结直肠癌相关疾病的生物标志物 | |
WO2002059367A2 (fr) | Jeu de microechantillons de diagnostic pour ibd, maladie de crohn et colite ulcereuse | |
WO2024119614A1 (fr) | Nouveau marqueur microbien pour prédire le risque de cancer colorectal | |
Bian et al. | Genome-wide methylation profiling identified methylated KCNA3 and OTOP2 as promising diagnostic markers for esophageal squamous cell carcinoma | |
CN108064273B (zh) | 结直肠癌相关疾病的生物标志物 | |
CN116377070A (zh) | 预测结直肠癌或结直肠腺瘤风险的新型微生物标志物 | |
CN116356026A (zh) | 预测结直肠癌或结直肠腺瘤风险的新型微生物标志物 | |
CN110982901B (zh) | 用于侵袭性甲状腺乳头状癌诊断的循环环状rna标志物及应用 | |
EP2607494A1 (fr) | Biomarqueurs pour l'évaluation du risque de cancer des poumons |