CN110777213A - Latent gastric cancer diagnostic kit - Google Patents
Latent gastric cancer diagnostic kit Download PDFInfo
- Publication number
- CN110777213A CN110777213A CN201911068996.9A CN201911068996A CN110777213A CN 110777213 A CN110777213 A CN 110777213A CN 201911068996 A CN201911068996 A CN 201911068996A CN 110777213 A CN110777213 A CN 110777213A
- Authority
- CN
- China
- Prior art keywords
- gastric cancer
- latent
- detecting
- diagnostic kit
- streptococcus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000005718 Stomach Neoplasms Diseases 0.000 title claims abstract description 31
- 206010017758 gastric cancer Diseases 0.000 title claims abstract description 31
- 201000011549 stomach cancer Diseases 0.000 title claims abstract description 31
- 238000009007 Diagnostic Kit Methods 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 101710112752 Cytotoxin Proteins 0.000 claims abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 14
- 231100000599 cytotoxic agent Toxicity 0.000 claims abstract description 14
- 239000002619 cytotoxin Substances 0.000 claims abstract description 14
- 244000005700 microbiome Species 0.000 claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 241000590002 Helicobacter pylori Species 0.000 claims description 12
- 229940037467 helicobacter pylori Drugs 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- 241000186000 Bifidobacterium Species 0.000 claims description 8
- 241000193468 Clostridium perfringens Species 0.000 claims description 8
- 241000194032 Enterococcus faecalis Species 0.000 claims description 8
- 241000605909 Fusobacterium Species 0.000 claims description 8
- 241001134638 Lachnospira Species 0.000 claims description 8
- 241000186660 Lactobacillus Species 0.000 claims description 8
- 241000194017 Streptococcus Species 0.000 claims description 8
- 241001148134 Veillonella Species 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 229940039696 lactobacillus Drugs 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 241000588722 Escherichia Species 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 210000003608 fece Anatomy 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 201000008827 tuberculosis Diseases 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 2
- 238000002965 ELISA Methods 0.000 claims description 2
- 108091005804 Peptidases Proteins 0.000 claims description 2
- 239000004365 Protease Substances 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 2
- 230000009089 cytolysis Effects 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims 1
- 239000003480 eluent Substances 0.000 claims 1
- 239000013615 primer Substances 0.000 claims 1
- 239000002987 primer (paints) Substances 0.000 claims 1
- 239000000523 sample Substances 0.000 description 10
- 101100476480 Mus musculus S100a8 gene Proteins 0.000 description 8
- 230000000968 intestinal effect Effects 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 206010058314 Dysplasia Diseases 0.000 description 3
- 206010054949 Metaplasia Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 231100000517 death Toxicity 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 238000002575 gastroscopy Methods 0.000 description 3
- 244000005709 gut microbiome Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 101150114014 cagA gene Proteins 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000012468 concentrated sample Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 208000004300 Atrophic Gastritis Diseases 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 208000036649 Dysbacteriosis Diseases 0.000 description 1
- 208000027244 Dysbiosis Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 208000036495 Gastritis atrophic Diseases 0.000 description 1
- 241000589989 Helicobacter Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000016644 chronic atrophic gastritis Diseases 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 230000007140 dysbiosis Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 201000011591 microinvasive gastric cancer Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- ZRHANBBTXQZFSP-UHFFFAOYSA-M potassium;4-amino-3,5,6-trichloropyridine-2-carboxylate Chemical compound [K+].NC1=C(Cl)C(Cl)=NC(C([O-])=O)=C1Cl ZRHANBBTXQZFSP-UHFFFAOYSA-M 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 235000013324 preserved food Nutrition 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 208000018556 stomach disease Diseases 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001018 virulence Effects 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/20—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
Abstract
The invention provides a latent gastric cancer diagnostic kit, which comprises a reagent for detecting the expression level of human cytotoxin-related protein A and a reagent capable of detecting microorganisms.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a latent gastric cancer diagnostic kit.
Background
Gastric cancer is an important cancer worldwide, with over 1,000,000 new cases in 2018, of which about 783,000 deaths (corresponding to 1 per 12 deaths worldwide) make it the fifth most common cancer and the third most deaths. Wherein the morbidity and mortality of men are 1.5-2 times higher than those of women. Since patients with early-stage gastric cancer often have no symptoms or only slight symptoms, and when clinical symptoms are obvious, lesions belong to middle and late stages, the early-stage symptoms of gastric cancer need to be very vigilant to avoid delaying diagnosis and treatment.
The common method for examining stomach diseases is gastroscopy, and if abnormality is found, pathological examination of tissue sections is performed. Gastroscopy causes certain pain to patients, requires precise operation and strict disinfection, otherwise causes damage and cross infection to the patients, and has high cost, so most latent gastric cancer patients do not select to carry out the gastroscopy for a plurality of times regularly when no obvious symptoms exist, and treatment delay and life danger can be caused.
The pathogenesis of gastric cancer is not known at present, but the harmful environment, the intake of preserved food and the intake of low fruit increase the risk of gastric cancer, and drinking and smoking are established risk factors, wherein the infection of Helicobacter pylori (Helicobacter pylori) is the main risk factor for gastric cancer, nearly 90% of cases of non-cardiac gastric cancer are attributed to the bacterium, and the world health organization has defined Helicobacter pylori as a class I carcinogen. The balance of intestinal microbial flora is influenced by factors such as diet, medicines, environment and the like, and plays an important role in stabilizing the environment in the intestinal tract. Beneficial bacteria and pathogenic bacteria in the intestinal tract of a human body compete with each other to maintain the balance in the intestinal tract, and when the pathogenic bacteria are increased greatly, the balance of the intestinal mucosa immune system is broken, so that intestinal inflammation is possibly caused. The imbalance of the intestinal flora is considered to be an important factor influencing the occurrence and development of various diseases, so that the diseases can be prevented, the health is promoted, the symptoms are improved, and the disease course is shortened by regulating the intestinal flora.
Through the research on the correlation of several specific genes of helicobacter pylori, the cytotoxin-related gene A (cagA) is considered to be a main virulence gene causing cell canceration, and after the cytotoxin-related protein A coded by the cagA gene enters gastric epithelial cells, the cytotoxin-related protein A can react with a plurality of proteins in the cells to cause the functional disorder of the cells and even cause the pathological change of the gastric epithelial cells, thereby causing diseases. Various studies have shown that infection with CagA-positive H.pylori increases the risk of atrophic gastritis and gastric cancer.
Disclosure of Invention
In view of the above, the invention provides a latent gastric cancer diagnostic kit, which can detect the expression level of human cytotoxin-related protein A and the diversity of intestinal microflora, so that the risk of gastric cancer can be judged more accurately.
The technical scheme of the invention is realized as follows:
in a first aspect, the present invention provides a latent gastric cancer diagnostic kit comprising an agent for detecting the expression level of human cytotoxin-associated protein a and an agent capable of detecting microorganisms including one or more of Fusobacterium (Fusobacterium tuberculosis), Lachnospira (Lachnospira Bryant and Small), Veillonella (Veillonella), Clostridium perfringens (Clostridium perfringens), helicobacter pylori (helicobacter), Escherichia (Escherichia), Streptococcus (Streptococcus), Bifidobacterium (Bifidobacterium), Lactobacillus (Lactobacillus), and Streptococcus faecalis (Streptococcus faecalis).
On the basis of the above technical solution, preferably, the reagent for detecting the expression level of human cytotoxin-associated protein a is a reagent for an ELISA detection method.
Further, preferably, the reagent for detecting the expression level of the human cytotoxin-associated protein A comprises an enzyme conjugate, a washing solution, a sample diluent, a color developing agent and a sealing solution.
In addition to the above technical means, preferably, the reagent capable of detecting microorganisms includes a reagent capable of extracting bacterial genomic DNA from feces of a subject.
Further, preferably, the reagent capable of detecting microorganisms includes a lysis solution, an elution solution, a protease, a primer, a DNA polymerase and dNTPs.
In a second aspect, the invention provides an application of the latent gastric cancer diagnostic kit in screening or identifying drugs for treating gastric cancer.
In a third aspect, the present invention also provides a method of detecting microorganisms including one or more of the genera Fusobacterium (Fusobacterium tuberculosis), Lachnospira (Lachnospira Bryant and Small), Veillonella (Veillonella), Clostridium perfringens (Clostridium perfringens), Helicobacter pylori (Helicobacter pylori), Escherichia (Escherichia), Streptococcus (Streptococcus), Bifidobacterium (Bifidobacterium), Lactobacillus (Lactobacillus), and Streptococcus faecalis (Streptococcus faecalis) for providing information required to predict or diagnose latent gastric cancer.
On the basis of the above technical solution, preferably, the method for detecting microorganisms includes the step of extracting bacterial genomic DNA from feces of a subject.
Compared with the prior art, the latent gastric cancer diagnostic kit has the following beneficial effects:
(1) the kit can detect the expression level of the CagA protein of the helicobacter pylori and the composition of intestinal microbial flora, and can more accurately predict early gastric cancer by detecting whether the intestinal microbial community is dysregulated or not on the basis of judging the CagA to be positive;
(2) the method for identifying the microorganisms commonly used in hospitals adopts a traditional culture method, is long in time consumption, high in culture requirement and multiple in influencing factor, more importantly, is difficult to simulate the conditions of natural environment, so that some bacteria cannot obtain pure cultures, in addition, the culture method only can culture live bacteria, but dead bacteria cannot count, however, the method for identifying the microbial flora by detecting the bacterial genome DNA in the excrement has the advantages of multiple obtained strains, more accurate result, convenience, rapidness and high sensitivity.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Subject information was registered and 25 male and female patients with intestinal metaplasia and dysplasia were selected as a patient group, 50 of which were diagnosed with gastric cancer at a later time, 25 healthy male and female patients were selected as a control group, and the ages of both groups were between 42 and 48 years.
S1 detection of protein A (CagA) associated with cytotoxin
Urine samples of testees of a patient group and a control group are collected, and the content of CagA protein in the urine is detected by using a human cytotoxin associated protein A (CagA) ELISA kit of Wuhan Moishak.
Sample preparation: 10mL of urine is collected by a sterile tube, centrifuged at 3000r/min at 4 ℃ for 20min, and the supernatant is carefully collected, and centrifuged again if a precipitate is generated during the storage.
Dilution of concentrated sample diluent: the concentrated sample dilution was diluted 10-fold with distilled water for use.
Sample adding: and respectively arranging a blank hole, a standard hole and a sample hole to be detected on the enzyme-labeled coated plate, respectively and accurately adding 50 mu L of diluted sample diluent and 50 mu L of standard substance into the blank hole and the standard hole, and adding 40 mu L of diluted sample diluent and then 10 mu L of sample to be detected into the sample hole to be detected. The sample is not contacted with the hole wall as much as possible during sample adding, and the sample is gently shaken to be uniform after sample adding.
And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 min.
Preparing liquid: the 30 times of concentrated washing liquid is diluted by 30 times of distilled water for standby.
Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling each hole with a cleaning solution, standing for 30s, discarding, and patting dry.
Adding an enzyme: 50 μ L of enzyme conjugate was added to each well, except for blank wells.
And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 min.
Washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling each hole with a cleaning solution, standing for 30s, discarding, and patting dry.
Color development: adding 50 μ L of color-developing agent A into each well, adding 50uL of color-developing agent B, shaking gently, mixing, and developing at 37 deg.C in dark for 15 min.
And (4) terminating: the reaction was stopped by adding 50. mu.L of blocking solution to each well, whereupon the color in the wells immediately turned yellow.
And (3) determination: the blank wells were zeroed, and the absorbance (OD) of each well was measured at a wavelength of 450nm, and the measurement should be performed within 15min after the addition of the stop solution.
The experimental results are as follows:
when the CagA is more than 150mg/L, the judgment is positive, the CagA content of the patient with the intestinal metaplasia and dysplasia is obviously higher than that of a healthy control group, the significant difference is achieved (P is less than 0.05), and the possibility of suffering from gastric cancer is obviously improved due to the high expression of the cytotoxin-related protein A.
S2, detecting intestinal microorganisms
1) 200mg of excrement samples of patients and control group testers are respectively weighed and put into a sterile test tube to be placed on ice, bacterial genome DNA of all samples is extracted by using a DNA extraction kit of the Foregene company, and the extracted DNA samples are frozen in a refrigerator at the temperature of-20 ℃.
2) PCR amplification
The 16S rRNA gene V3 variable region sequence of the flora genome is amplified, and the sequence of the universal primer is as follows: an upstream primer: GC-341F (5 '-GC clamp-CCTACGGGAGGCAGCAG-3'), downstream primer: 518R (5'-ATTACCGCGGCTGG-3') with a "GC clamp" of 40bp and a sequence of CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGG.
PCR amplification was carried out using the PCR kit of Shanghai assist in san Francisco. PCR amplification System (50. mu.L): 8 μ L10 XPCR mix Buffer (Mg)
2+) 5 μ L of 1% BSA, 5 μ L of dNTPs, 1 μ L of each of the upstream and downstream primers, 3 μ L of template DNA, 0.5 μ L of Taq DNA polymerase (5U/. mu.L), and 26.5 μ L of deionized water. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 6 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; 30 cycles; fully extending for 10min at 72 ℃. After the reaction is finished, the product is stored on ice or at 4 ℃. After the PCR was completed, the PCR product was subjected to 1% agarose gel electrophoresis using 5. mu.L of the PCR product to examine whether the PCR was successful or not.
3) Denaturing Gradient Gel Electrophoresis (DGGE)
After 20. mu.L of the PCR product was mixed with 5. mu.L of 6 × Loading Buffer, DGGE electrophoresis analysis was performed using a denaturing gradient gel electrophoresis system. According to the preliminary experiment, the concentration gradient of the denatured gel is set to be 30% -55%, electrophoresis is carried out in a thermostatic water bath at 60 ℃, the high pressure impact is carried out at 140V for 20min, then the low pressure is changed to 60V, and the used electrophoresis Buffer is 1 × TAE Buffer. After electrophoresis, the gel is stained for 10min on a shaking table by using EB staining solution and detected in a gel imaging system.
4) DGGE profiling
Cutting the differential bands into sterile EP tubes, washing 3 times with sterile water, blotting the supernatant, and adding 50 μ L of 3dH
2O-20 ℃ refrigerator overnight storage. The next day, water bath at 90 ℃ for 10min, centrifugation at 10000 Xg for 5min, taking 3 μ L of supernatant as a template, 341F without GC clamp as an upstream primer, and carrying out PCR amplification on the rest components and conditions in the same step 2). The amplified PCR product was subjected to 1% agarose electrophoresis, and the amplified DNA band was recovered by cutting the gel according to the instructions of a general agarose gel DNA recovery kit of TIANGEN. The purified DNA samples were sent to TaKaRa (Wuhan) for sequencing and the sequencing results were compared in NCBI database. And (3) applying Quantity One software to perform strip number, similarity analysis and clustering analysis on the DGGE map.
The experimental results are as follows:
the 16S rDNA V3 region PCR amplification products of the patient group and the control group are detected by 1% agarose gel electrophoresis, and the results show that the PCR products are both 200bp and have no non-specific amplification fragments.
And carrying out DGGE electrophoresis on the PCR products, analyzing the difference bands, and identifying the composition of the intestinal microflora of the patient group and the control group. The differences in the dominant intestinal microbial flora between the patient groups and the control groups by sequencing and comparison with the NCBI database are shown in the following table.
As shown by the above table, the dysbacteriosis is severe in patients with intestinal metaplasia and dysplasia, with male patients more severe than female patients. Therefore, the risk of gastric cancer can be judged by detecting the diversity of intestinal microflora.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A latent gastric cancer diagnostic kit characterized in that: comprises an agent for detecting the expression level of human cytotoxin-associated protein A and an agent capable of detecting microorganisms including one or more of the genera Fusobacterium (Fusobacterium Nuclear), Lachnospira (Lachnospira Bryant and Small), Veillonella (Veillonella), Clostridium perfringens (Clostridium perfringens), Helicobacter pylori (Helicobacter pylori), Escherichia coli (Escherichia), Streptococcus (Streptococcus), Bifidobacterium (Bifidobacterium), Lactobacillus (Lactobacillus), and Streptococcus faecalis (Streptococcus faecalis).
2. The latent gastric cancer diagnostic kit according to claim 1, wherein: the reagent for detecting the expression level of the human cytotoxin-associated protein A is a reagent for an ELISA detection method.
3. The latent gastric cancer diagnostic kit according to claim 2, wherein: the reagent for detecting the expression level of the human cytotoxin-related protein A comprises an enzyme conjugate, a washing solution, a sample diluent, a color developing agent and a confining solution.
4. The latent gastric cancer diagnostic kit according to claim 1, wherein: the reagent capable of detecting microorganisms includes a reagent capable of extracting bacterial genomic DNA from feces of a subject.
5. The latent gastric cancer diagnostic kit according to claim 4, wherein: the reagent capable of detecting the microorganism comprises lysis solution, eluent, protease, primers, DNA polymerase and dNTPs.
6. Use of the latent gastric cancer diagnostic kit according to claim 1 for screening or identifying a drug for treating gastric cancer.
7. A method of detecting a microorganism, comprising: the method is used to provide information required to predict or diagnose latent gastric cancer, and the microorganisms include one or more of the genera Fusobacterium (Fusobacterium tuberculosis), Lachnospira (Lachnospira Bryant and Small), Veillonella (Veillonella), Clostridium perfringens (Clostridium perfringens), Helicobacter pylori (Helicobacter pylori), Escherichia (Escherichia), Streptococcus (Streptococcus), Bifidobacterium (Bifidobacterium), Lactobacillus (Lactobacillus), and Streptococcus faecalis (Streptococcus faecalis).
8. The method for detecting microorganisms according to claim 7, wherein: comprises the step of extracting bacterial genome DNA in the feces of a tested person.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911068996.9A CN110777213A (en) | 2019-11-05 | 2019-11-05 | Latent gastric cancer diagnostic kit |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911068996.9A CN110777213A (en) | 2019-11-05 | 2019-11-05 | Latent gastric cancer diagnostic kit |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110777213A true CN110777213A (en) | 2020-02-11 |
Family
ID=69388857
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911068996.9A Pending CN110777213A (en) | 2019-11-05 | 2019-11-05 | Latent gastric cancer diagnostic kit |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110777213A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023074869A1 (en) * | 2021-11-01 | 2023-05-04 | 国立大学法人 東京大学 | Method for evaluating risk of stomach cancer onset |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007088537A2 (en) * | 2006-01-31 | 2007-08-09 | Medical Research Fund Of Tel Aviv Sourasky Medical Center | Methods and kits for early detection of cancer or predisposition thereto |
CN102721815A (en) * | 2012-04-18 | 2012-10-10 | 天津天佛罗生物技术有限公司 | Detection kit for helicobacter pylori virulence protein antibody and detection method by using the same |
WO2015151128A1 (en) * | 2014-04-04 | 2015-10-08 | Over S.R.L. | Method for rapid detection of infection from caga positive helicobacter pylori and diagnostic kit therefor |
CN108279302A (en) * | 2017-07-11 | 2018-07-13 | 深圳市伯劳特生物制品有限公司 | A kind of composition and pylori spiral bacilli antibody for enzyme linked immunological kit composes detection kit and preparation method thereof |
-
2019
- 2019-11-05 CN CN201911068996.9A patent/CN110777213A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007088537A2 (en) * | 2006-01-31 | 2007-08-09 | Medical Research Fund Of Tel Aviv Sourasky Medical Center | Methods and kits for early detection of cancer or predisposition thereto |
CN102721815A (en) * | 2012-04-18 | 2012-10-10 | 天津天佛罗生物技术有限公司 | Detection kit for helicobacter pylori virulence protein antibody and detection method by using the same |
WO2015151128A1 (en) * | 2014-04-04 | 2015-10-08 | Over S.R.L. | Method for rapid detection of infection from caga positive helicobacter pylori and diagnostic kit therefor |
CN108279302A (en) * | 2017-07-11 | 2018-07-13 | 深圳市伯劳特生物制品有限公司 | A kind of composition and pylori spiral bacilli antibody for enzyme linked immunological kit composes detection kit and preparation method thereof |
Non-Patent Citations (2)
Title |
---|
朱燕燕等: "胃癌患者肠道菌群的分布特点分析", 《中国微生态学杂志》 * |
杜雅菊等: "幽门螺杆菌CagA蛋白与胃癌组织中Bcl-2、p53蛋白表达的关系", 《世界华人消化杂志》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023074869A1 (en) * | 2021-11-01 | 2023-05-04 | 国立大学法人 東京大学 | Method for evaluating risk of stomach cancer onset |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Willing et al. | A pyrosequencing study in twins shows that gastrointestinal microbial profiles vary with inflammatory bowel disease phenotypes | |
Andoh et al. | Comparison of the fecal microbiota profiles between ulcerative colitis and Crohn’s disease using terminal restriction fragment length polymorphism analysis | |
Hoshino et al. | PCR detection and identification of oral streptococci in saliva samples using gtf genes | |
Burnens et al. | Association between clinical presentation, biogroups and virulence attributes of Yersinia enterocolitica strains in human diarrhoeal disease | |
CN107430644A (en) | Method for determining gastrointestinal bacterial flora imbalance | |
Könönen et al. | Bacteroides, Porphyromonas, Prevotella, Fusobacterium, and Other Anaerobic Gram‐Negative Rods | |
Açik et al. | Heterogeneity of Campylobacter jejuni and Campylobacter coli strains from healthy sheep | |
Shah et al. | Gastroduodenal “Dysbiosis”: a new clinical entity | |
Jiang et al. | Changes in the intestinal microbiota in patients with stage 5 chronic kidney disease on a low-protein diet and the effects of human to rat fecal microbiota transplantation | |
Abd El-Baky et al. | Antibiotic susceptibility pattern and genotyping of Campylobacter species isolated from children suffering from gastroenteritis | |
CN110777213A (en) | Latent gastric cancer diagnostic kit | |
CN113136446A (en) | Method, primer group and kit for identifying non-tuberculous mycobacteria and detecting drug-resistant gene mutation | |
US20150284779A1 (en) | Determination of a tendency to gain weight | |
CN113584193B (en) | Application of chaetomium as marker for evaluating curative effect of antihistamine for chronic spontaneous urticaria patient | |
CN109055588A (en) | Specific primer, kit and the PCR detection method of the outstanding Bordetella of a pair of detection uncle | |
CN112553344B (en) | Biomarker related to colorectal cancer and application thereof | |
Peng et al. | Endoscopic 13C-urea breath test for the diagnosis of Helicobacter pylori infection | |
CN112410449A (en) | Microbial marker related to colorectal cancer and application thereof | |
Kobayashi et al. | Numerical analyses of intestinal microbiota by data mining | |
Ali et al. | Genotyping of vacA of Helicobacter pylori in patients from Baghdad with gastro-duodenal diseases | |
CN113667767B (en) | Method for rapidly and high-flux detection of drug resistance of helicobacter pylori | |
KR102533533B1 (en) | Information provision method for the treatment of alcoholic cirrhosis using Veillonella dispar strain | |
KR20220024141A (en) | A method for detecting the level of Helicobacter pylori in a fecal sample | |
Také et al. | Impact of liver fibrosis on the relative abundance of a urease‐positive Streptococcus salivarius group from saliva in patients with chronic liver disease | |
Nouri et al. | DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR DETECTION OF BACTERIAL FLORA IN PATIENTS AFTER BARIATRIC SURGERY |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200211 |