CN109988813A - The method and culture solution of Resistance detection for external living cells - Google Patents
The method and culture solution of Resistance detection for external living cells Download PDFInfo
- Publication number
- CN109988813A CN109988813A CN201811290781.7A CN201811290781A CN109988813A CN 109988813 A CN109988813 A CN 109988813A CN 201811290781 A CN201811290781 A CN 201811290781A CN 109988813 A CN109988813 A CN 109988813A
- Authority
- CN
- China
- Prior art keywords
- living cells
- detected
- detection
- culture solution
- drug resistance
- 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.)
- Granted
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims description 35
- 150000001875 compounds Chemical class 0.000 claims abstract description 60
- 206010059866 Drug resistance Diseases 0.000 claims abstract description 33
- 238000011534 incubation Methods 0.000 claims abstract description 24
- 239000003550 marker Substances 0.000 claims abstract description 13
- 238000012360 testing method Methods 0.000 claims description 33
- 230000012010 growth Effects 0.000 claims description 25
- 125000001424 substituent group Chemical group 0.000 claims description 18
- 238000000338 in vitro Methods 0.000 claims description 15
- 125000000623 heterocyclic group Chemical group 0.000 claims description 12
- 230000005764 inhibitory process Effects 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 9
- 125000003831 tetrazolyl group Chemical group 0.000 claims description 9
- 125000003118 aryl group Chemical group 0.000 claims description 6
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 6
- 125000000542 sulfonic acid group Chemical group 0.000 claims description 6
- 125000000217 alkyl group Chemical group 0.000 claims description 4
- 125000002843 carboxylic acid group Chemical group 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 239000007822 coupling agent Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 231100000419 toxicity Toxicity 0.000 claims description 3
- 230000001988 toxicity Effects 0.000 claims description 3
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical compound O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 claims description 2
- 240000001462 Pleurotus ostreatus Species 0.000 claims description 2
- 235000001603 Pleurotus ostreatus Nutrition 0.000 claims description 2
- 229910006074 SO2NH2 Inorganic materials 0.000 claims description 2
- 229910006069 SO3H Inorganic materials 0.000 claims description 2
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 2
- 239000000022 bacteriostatic agent Substances 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 125000005843 halogen group Chemical group 0.000 claims description 2
- 150000002367 halogens Chemical class 0.000 claims description 2
- 125000005842 heteroatom Chemical group 0.000 claims description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 229910052717 sulfur Inorganic materials 0.000 claims description 2
- 235000007685 Pleurotus columbinus Nutrition 0.000 claims 1
- 150000002333 glycines Chemical class 0.000 claims 1
- 238000001802 infusion Methods 0.000 claims 1
- 229930195732 phytohormone Natural products 0.000 claims 1
- 210000000582 semen Anatomy 0.000 claims 1
- 210000001082 somatic cell Anatomy 0.000 abstract 5
- 230000001413 cellular effect Effects 0.000 abstract 2
- 210000004027 cell Anatomy 0.000 description 65
- 239000003814 drug Substances 0.000 description 47
- 229940079593 drug Drugs 0.000 description 30
- 239000007788 liquid Substances 0.000 description 23
- 241000894006 Bacteria Species 0.000 description 22
- 239000000243 solution Substances 0.000 description 22
- 229960000723 ampicillin Drugs 0.000 description 18
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 18
- 230000035945 sensitivity Effects 0.000 description 17
- 239000002609 medium Substances 0.000 description 16
- 238000002474 experimental method Methods 0.000 description 14
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000010261 cell growth Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 210000004881 tumor cell Anatomy 0.000 description 6
- 231100000252 nontoxic Toxicity 0.000 description 5
- 230000003000 nontoxic effect Effects 0.000 description 5
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 4
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 4
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 238000009631 Broth culture Methods 0.000 description 3
- 241000222122 Candida albicans Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- -1 methyl PMS Chemical compound 0.000 description 3
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 3
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 3
- VSIVTUIKYVGDCX-UHFFFAOYSA-M sodium;4-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].COC1=CC([N+]([O-])=O)=CC=C1[N+]1=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=NN1C1=CC=C([N+]([O-])=O)C=C1 VSIVTUIKYVGDCX-UHFFFAOYSA-M 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 206010036790 Productive cough Diseases 0.000 description 2
- 238000012339 Real-time fluorescence quantitative polymerase chain reaction Methods 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 229940095731 candida albicans Drugs 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 229940121657 clinical drug Drugs 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- AEUTYOVWOVBAKS-UWVGGRQHSA-N ethambutol Chemical compound CC[C@@H](CO)NCCN[C@@H](CC)CO AEUTYOVWOVBAKS-UWVGGRQHSA-N 0.000 description 2
- 238000002795 fluorescence method Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229960003350 isoniazid Drugs 0.000 description 2
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 2
- 229960001225 rifampicin Drugs 0.000 description 2
- 238000011896 sensitive detection Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 210000003802 sputum Anatomy 0.000 description 2
- 208000024794 sputum Diseases 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010093965 Polymyxin B Proteins 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 229940069765 bean extract Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 229960003669 carbenicillin Drugs 0.000 description 1
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 1
- 229960001139 cefazolin Drugs 0.000 description 1
- MLYYVTUWGNIJIB-BXKDBHETSA-N cefazolin Chemical compound S1C(C)=NN=C1SCC1=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CN3N=NN=C3)[C@H]2SC1 MLYYVTUWGNIJIB-BXKDBHETSA-N 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229960000285 ethambutol Drugs 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 229940107698 malachite green Drugs 0.000 description 1
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229960000210 nalidixic acid Drugs 0.000 description 1
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000017095 negative regulation of cell growth Effects 0.000 description 1
- 238000004848 nephelometry Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229920000024 polymyxin B Polymers 0.000 description 1
- 229960005266 polymyxin b Drugs 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- JUJBNYBVVQSIOU-UHFFFAOYSA-M sodium;4-[2-(4-iodophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=N1 JUJBNYBVVQSIOU-UHFFFAOYSA-M 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001550 time effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 229960001082 trimethoprim Drugs 0.000 description 1
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 230000000007 visual effect 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to cellular drug resistance field of fast detection, disclose a kind of drug resistance rapid sensitive for active somatic cell accurately detection method, comprising the following specific steps detecting the concentration of marker after being incubated for before being detected active somatic cell to be detected to the active somatic cell to be detected, the detection compound that can be utilized by the active somatic cell to be detected is added before incubation.It is an advantage of the current invention that can rapidly be detected to active somatic cell, the detection including its cellular drug resistance, application value with higher.
Description
Technical Field
The invention relates to the field of rapid detection of cell drug resistance, in particular to a rapid, sensitive and accurate detection method for drug resistance of living cells.
Background
Currently, the detection method commonly used in laboratories mainly comprises three major parts, namely bacteriology, immunology and molecular biology. The modern clinical microorganism drug sensitivity experiment in China at present has a plurality of problems, for example, the detection time is long, the result is not accurate enough, the diagnosis result is influenced, some identification equipment is expensive, and some small and medium-sized hospitals are incapable of using the method.
At present, the bacteria drug sensitivity detection method mainly comprises a paper diffusion method (K-B method) for qualitative determination, a dilution method for quantitative determination, an E method and a full-automatic drug sensitivity instrument method, and generally 1 day is needed for obtaining a result.
At present, various methods can be used for detecting tubercle bacillus in laboratories and clinics, and the most common methods mainly comprise sputum smear microscopy, Roche solid culture, BACTEC MGIT960 system culture, real-time fluorescence quantitative Polymerase Chain Reaction (PCR) and the like.
The existing drug sensitive detection method for mycobacterium tuberculosis has the following problems: sputum smear has high requirement on film production, and the microscopic examination needs to be completed by manpower, which greatly increases the workload of clinical laboratory staff and is easy to omit; due to the characteristics of growth of the mycobacterium tuberculosis, the solid culture method takes long time; the rapid culture instrument for mycobacteria and the real-time fluorescence quantitative Polymerase Chain Reaction (PCR) can shorten the culture time, and are convenient to operate, but are not live bacteria, have low accuracy and high price, and are not beneficial to popularization.
The problems in the prior art are partly caused by the limitation of the detection means, but the characteristics of the marker which needs to be cultured to obtain the required concentration in the process of detecting the drug resistance of the cells cause the defects of long time, low accuracy and the like of the detection means.
Therefore, a better detection means needs to be established by combining the characteristics of cell drug sensitivity detection on the basis of the prior art.
Disclosure of Invention
Aiming at the defect of long drug sensitivity detection time of cells in the prior art, the invention provides a rapid, sensitive and accurate detection method for drug resistance of living cells. In addition, in practical application, the rapid detection method also has the advantages of high detection accuracy, capability of carrying out quantitative detection and the like.
In order to achieve the purpose, the invention can adopt the following technical scheme:
a rapid, sensitive and accurate detection method for drug resistance of living cells comprises the following specific steps: incubating the living cells to be detected before detecting the living cells to be detected, and detecting the concentration of the marker after incubation to realize the detection; adding a detection compound capable of being utilized by the living cells to be detected and a test compound for drug resistance detection before incubation; the incubation refers to adding the living cells to be detected into a culture solution with a determined concentration for culture. Viable cells that can be used include fungi, bacteria, and parts of human cells, but cells that have died cannot be detected under the procedures described in the method because the number of dead cells is zero and no detection compound is available; in addition, although the detection instrument used in the embodiments of the present application includes an instrument that can detect the absorbance of a solution or the like and can detect the color of a solution, these instruments may be existing instruments or instruments having a detection function as claimed in the present application. The incubation refers to the culture of the living cells to be detected through a culture solution, and the number of the living cells to be detected is increased as a general culture result, but the technical scheme described in the application does not require that the living cells living at the moment of adding the detection compound before the incubation are continuously alive after the incubation is finished, and does not require that the number of the living cells after the incubation is absolutely increased. Generally, the technical scheme of the application only requires that the detection compound can be utilized by living cells, and the aim of rapid detection can be achieved by detecting products after the cells are utilized. The culture solution used in the incubation may be any mixed solution that allows the growth of the living cells to be detected, for example, MH broth for bacteria, RPMI for fungi, and 7H9 medium for tubercle bacillus. The test compound used in the examples of the present application is EZMTT, and other water-soluble tetrazolium disulfonate WST series WST8, WST1, and the like can be selected, so that although EZMTT is non-toxic or substantially non-toxic to cells, those compounds that are toxic to cells can still be used as test compounds, but obviously, due to the toxicity to cell production, growth is inhibited but not to the extent that it is unable to grow, the methods described herein can still be used, and provide certain IC50 values despite deviations in the results obtained. The test compound used in the present application is a compound that requires drug sensitivity (drug resistance) test on a specific cell, in the examples of the present application, ampicillin, streptomycin, isoniazid, rifampicin, ethambutin and the like are used as test compounds, and the living cells used are escherichia coli, mycobacterium tuberculosis, gram-negative bacteria pseudomonas aeruginosa, candida albicans, mammalian cells, gram-positive bacteria, fungal cells, tumor cells, blood cells, and particularly, various drug-resistant and drug-intolerant cells. The living cells, the test compound, can be determined by one skilled in the art according to the needs of the cell, the compound to be subjected to the drug sensitivity test.
Further, the detection method described in the present application is mainly used in the case where the marker is a product produced by cell utilization, in other words, the detection compound is utilized by living cells, and then the detectable marker is continuously produced and the production rate of the marker is correlated with the cell growth rate or the inhibition rate of the detection compound on the cells; therefore, if a detection reagent can directly react with a compound existing in a cell or on a cell membrane without affecting the growth of a living cell or with little effect, it is not necessary to use the procedure described in the present application.
Further as an optional solution, in the embodiment of the present application, an incubation concentration adjustment step is further included: the concentration of the culture medium used for incubation is adjusted before incubation so that the living cells to be detected reach approximately the optimal growth rate, where the growth rate is defined as the time period during which the incubation is carried out. The optimized growth rate refers to the concentration of the culture solution, under the condition that other incubation conditions are given, the living cells to be detected have the maximum or the culture solution concentration close to the maximum growth rate in a certain incubation time.
Further as an alternative, in the embodiment of the present application, a pre-culture step is further included: before incubating the living cells to be detected, culturing the living cells to be detected and determining the concentration of a culture solution capable of obtaining the optimal growth rate of the living cells to be detected, wherein the culture solution used in the step is the same as the culture solution used in the step. This step is generally used to determine the concentration of the culture medium that will allow the living cells to be detected to achieve approximately the optimal growth rate. No test compound is added in this step. In addition, this step has the effect that the concentration of bacteria in the experiment (relative to the culture medium concentration) can be selected according to the specificity of the cells.
Further as an optional solution, in the embodiment of the present application, the method further includes the step of accelerating the culture: after the concentration of the culture solution capable of obtaining the optimal growth rate of the living cells to be detected is obtained, the concentration of the culture solution is increased in the incubation process. Compared with the pre-culture step, the method has the advantages that the cell growth rate in a short time is improved by increasing the concentration of the culture solution, the required detection time is shortened, and the detection speed is increased.
As a further alternative, in the embodiments of the present application, the utilization refers to the fact that the detection compound can enter the living cell to be detected, and generally, the reaction with the components located on the cell membrane is also included. In addition, whether the product after utilization can stay in living cells or be released by the cells in vitro or released into the culture solution during incubation due to cell death does not affect the use of the method described herein. As a further alternative, in the embodiments of the present application, the utilization means that the detection compound is capable of reacting with at least a part of the components of the cells to be detected.
Further alternatively, in the examples of the present application, the marker is a product having a color, where the color refers to a color having a certain wavelength range on a spectrum, and thus white is not the color described in the present application.
As a further alternative, in the embodiments of the present application, the detection compound is a compound that can be oxidized or reduced to a product that can develop color in the living cells to be detected, and the detection compound is different according to the detection compound used. In the case of EZMTT, the product is orange-yellow formazan.
As a further alternative, in the examples of the present application, the detection compound is a compound capable of reacting with nad (p) H in the living cells to be detected and being reduced to a product capable of developing a color in the presence of an electron coupling agent.
Further as an optional scheme, in an embodiment of the present application, the method further includes a step of detecting an inhibition rate: adding an equal amount of living cells to be detected into a culture solution with the same concentration while incubating the living cells to be detected to prepare a control group, adding a control compound with the same amount and the same concentration as the compound to be detected before incubating the control group, detecting the concentration of a marker after incubating the control group, comparing the concentration of the marker, and obtaining the inhibition rate of the detection compound on the living cells to be detected by using detection compounds with different toxicity.
In addition, the invention also discloses a culture solution for incubating the living cells, which comprises a culture medium and a detection compound; the detection compound has the following general structural formula:
wherein,
a1 is as defined aboveA linked aromatic or heterocyclic group;
a2 is as defined aboveA linked aromatic or heterocyclic group;
a3 is as defined aboveA linked phenyl group;
r1 is H or at least one R1 substituent attached to said a1, said R1 substituents each independently selected from-NO 2, -SO3H, -SO2NH2, halogen elements, and heterocyclic groups fused to said a1, alkyl; especially the R1 substituent located para to a 1;
r2 is H or at least one R2 substituent attached to said a2, said R2 substituents each being independently selected from NO2, methoxy, alkyl, phenyl and its derivatives linked through azido, halogen atoms, sulfonic acid groups and their salts, and phenyl or heterocyclic groups fused to said a 1; especially the R2 substituent located ortho or para to a 2.
R3 is the R3 substituent attached to the A3; the R3 substituent is respectively and independently selected from sulfonic acid group, carboxylic acid group, substituted sulfonic acid group and substituted carboxylic acid group; in particular toFrom Especially the R3 substituent located ortho to a 3.
The heterocyclic group includes 1-2 heteroatoms each independently selected from Se, N, S or O.
The above aromatic or heterocyclic groups are each independently selected from
In addition, additional sensitizers, such as PMS (commonly known as phenazine methosulfate) derivatives, especially methyl or ethyl derivatives thereof, may also be added to the above-mentioned culture broth.
The culture solution can be prepared by selecting the following culture media in proportion: LB, MH broth was used for gram-negative, positive bacteria; (0.5-2 times) suton (Sauton) medium, Middle brook7H9, Middle brook7H 10, Middle brook7H11, Middle brook7H 12, kunkel medium, Proskauer medium, sodium pyruvate cytochrome C medium, BACTEC MGIT960 medium for tubercle bacillus; RPMI medium is used for fungi; basel medium Eagle, RPMI, DMEM/F12, RPMI-HEPES, MEM, IMDM, HamF10, M199, L15, McCoy5A and the like, and the above medium is usually diluted 0.5 to 2 times. For the sake of clarity, a considerable part of the above names are trade names for commercially available media.
In addition, a growth accelerator, a bacteriostatic agent and a bacteriostatic synergist can be added into the culture solution; various types of blood, hormone, oyster mushroom liquid, bean extract, OADC and the like can be selected, and trace malachite green, penicillin, nalidixic acid, polymyxin B, carbenicillin C, amphotericin B, trimethoprim or mgit Pant antibiotics for inhibiting infectious microbes are used for adding the mycobacterium tuberculosis culture medium; serum-free medium such as 1-20% BSA or B-27 is added to the culture medium of mammalian cells;
the selected detection compound EZMTT is a novel synthetic tetrazolium monosulfonate, the detection principle is based on that in living cells, NAD (P) H can enable EZMTT to be reduced into a soluble orange-yellow formazan product in the presence of an electron coupling agent, the light absorption value of the formazan product can be measured at 450nm by using an enzyme labeling instrument, and dead cells do not have the effect. The bacteria survival condition is indirectly reflected by detecting the absorbance through a microplate reader, and the color variation of EZMTT is in direct proportion to the bacteria amount in a certain range and is non-toxic. It is therefore possible to establish a rapid drug sensitisation method.
The invention has the following remarkable technical effects:
the invention can select the concentration of bacteria in a drug sensitivity experiment according to the specificity of the bacteria. The invention has no lethal influence on bacteria, can continuously detect and has reliable data. The invention can realize short drug sensitivity detection time (3-4 hours is needed for detecting EZMTT by clinical fast-growing bacteria, 1 day is needed for the tradition, 4-6 days is needed for tubercle bacillus EZMTT, 4-10 days is needed for the tradition and 1 month is solid), can detect partial inhibition (the EZMTT obtains percent inhibition rate, but the tradition visual detection method can only qualitatively 'have/not have' and can quickly obtain accurate results. The invention can detect a plurality of bacteria and a plurality of antibiotics in a large scale with high flux and can detect the bacteria and the antibiotics quickly. The invention can provide the optimal matching of the medicines for clinic and realize accurate treatment. Compared with other detection reagents, the detection reagent provided by the invention is low in price, easy to popularize and capable of reducing economic burden.
Drawings
Fig. 1 is a graph of the signal for 24 hours following e.coli growth using different tetrazolium salts; the water-soluble tetrazolium salts all have signals; similar results were obtained in LB medium.
FIG. 2 is a graph of signals following the growth of Mycobacterium tuberculosis using different tetrazolium salts for seven days; among them, the signal obtained by the EZMTT test is most preferable.
FIG. 3 is a graph of signals following the growth of Mycobacterium tuberculosis using different media for seven days. The best signal was obtained after growth of M.tuberculosis using medium Middlebrook7H 9.
FIG. 4 is a graph of signals following the growth of Mycobacterium tuberculosis using different additives for seven days. The use of the growth accelerator OADC signal is preferred.
FIG. 5 shows the results of tracking H22 tumor cells for 5 days using different media.
FIGS. 6-1, 6-2 detection of ampicillin resistance in various cell amounts (from 20-fold to 640-fold unit cell amount) of resistant E.coli (FIG. 6-1) and sensitive E.coli (FIG. 6-2) for testing using EZMTT under optimal conditions; the 4h detection result shows that the IC50 and MIC values are slightly increased (about 2 times) under the condition of higher bacterial concentration, but the drug sensitive detection result is not influenced.
FIG. 7. alignment for testing ampicillin resistance in clinically isolated and characterized E.coli using EZMTT as a test indicator, samples of known resistance (from left to right, in top to bottom series): (drug resistance) testing using EZMTT as the test compound also showed drug resistance; (drug resistance) testing using EZMTT as the test compound also showed drug resistance; (sensitive) detection using EZMTT as the test compound also showed a sensitive IC50 ═ 13 ug/ml; (sensitive) detection using EZMTT as test compound also showed some sensitivity IC50 ═ 14ug/ml, but was not completely inhibitory. Belonging to a partially suppressed state.
FIGS. 8-1 and 8-2 are graphs showing the comparison of the results of drug sensitivity of E.coli (FIG. 8-1) in FIG. 7 using EZMTT as an indicator for detection and the same samples (FIG. 8-2) using conventional nephelometry (24 hours), where the left side is more clear in color, the sensitivity is high, the right side is not sensitive enough, and partial inhibition of cell growth is not detected. The cell growth curve shows that the sensitivity of the EZMTT method is 10 times higher than that of the nephelometric method.
FIGS. 9-1, 9-2, and 9-3 are comparisons of the use of EZMTT as a detection indicator and other soluble tetrazolium salts (WST8/CCK8) where both agents were detectable for resistant bacteria but were toxic to sensitive bacteria, WST8, and were not detectable for bacterial resistance.
FIG. 10 is a graph comparing the detection of clinically resistant bacteria using EZMTT as a detection indicator and the detection of conventional liquid fluorescence after tracking for 4-7 days (R-resistant, S-sensitive, PR-partially resistant) per day. Different cell mass and addition of nutrients OADC can increase the signal while EZMTT can be quantitatively analyzed and the time effect of cell growth measured.
FIG. 11 shows the results of a 4-hour drug susceptibility test using Staphylococcus aureus, a gram-positive bacterium (diluted 40-fold in fresh 0.5 MCF), where A is IC50 ═ 129ng/ml after ampicillin treatment; b is IC50 ═ 872ng/ml after kanamycin drug treatment; c is IC50 ═ 87ng/ml after treatment with cefazolin drug.
FIG. 12 shows the gram-negative bacterium Pseudomonas aeruginosa (diluted 40 times with fresh bacterial liquid of 0.5 MCF) for 4-hour gentamicin susceptibility test (IC)500.17ug/ml) results.
FIG. 13 shows the result of ampicillin resistance detection in 4-hour drug sensitivity test using Candida albicans (diluted 40-fold with fresh bacterial solution of 0.5 MCF).
FIGS. 14-1, 14-2, and 14-3 are graphs showing the detection of drug resistance and partial drug resistance in tumor cells using EZMTT medium. The 5-day drug sensitivity test shows that complete inhibition (FIG. 14-2) and partial inhibition (FIG. 14-3) can be distinguished by the novel EZMTT culture medium and detection method (no drug, dose effect of cell growth) of the patent; this explains the tumor resistance reported by drugs with partial inhibition, and provides guidance for early prediction of tumor resistance.
FIGS. 15-1, 15-2, 15-3 are graphs showing growth inhibition of lentivirus after partial infection of tumor cells HEK293t with the novel EZMTT medium, wherein the effect of viral infection on HEK293t cell growth, and the middle graphs are growth curves. The upper line was 2000 HEK293t cells grown. The next line is the growth of 2000 HEK293t cells after transfection with the virus. Transfected cells are shown on the left and virus-containing transfected cells are shown on the right.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
Susceptibility testing was performed using e.coli: ampicillin liquid medicine (0-12.8 mg/ml; concentration gradient of two-fold dilution) is prepared for standby. And taking 2 mul of ampicillin standby liquid medicine into a 96-well plate by using a discharge gun for standby. Coli strains were adjusted to a concentration of 0.5MCF using MH broth, diluted 20-640 times (preferably 40 times) with MH broth containing EZMTT (EZMTT can be replaced with various tetrazolium salts and PMS derivatives including methyl PMS) detection reagents, and 200 μ l was taken in a 96-well plate. Setting a control hole, wherein 2ul of ampicillin in the control hole is to be used for replacing the liquid medicine with sterilized water, and the rest is consistent with the operation of the administration hole. And (3) putting the 96-well plate into an incubator at 37 ℃ for culture, and detecting OD450 and OD600 values after 4 hours.
Example 2
Drug susceptibility experiments were performed using mycobacterium tuberculosis: preparing streptomycin, isoniazid, rifampicin and ethambutol medicinal liquid for later use. According to the conventional medicine concentration detection, taking the liquid medicine to be used into the screw tube. Freshly cultured Mycobacterium tuberculosis are adjusted to a concentration of 0.5MCF using 7H9 medium, diluted 4-40 fold (preferably 20 fold) with 7H9 medium containing EZMTT (which may also be replaced with other tetrazolium salts and PMS derivatives, including methyl PMS, as described in the examples below) detection reagents, and 4ml are added to the drug-containing tube. And (4) setting a control hole, replacing the liquid medicine to be used by sterilized water in the control hole, and taking a positive control (signal) when bacteria are added and taking a negative control (background) when bacteria are not added. The tube was incubated at 37 ℃ for 5 days, and then the OD450 and OD600 values were measured every day. When the signal/background is greater than 2 times, the inhibition rate is calculated to judge drug resistance. After an additional day, when the signal/background was > 4-fold, the detection was terminated.
Example 3
Drug susceptibility experiments were performed using the gram-negative bacterium pseudomonas aeruginosa (40-fold dilution of fresh bacterial fluid at 0.5 MCF): ampicillin liquid medicine (0-12.8 mg/ml; concentration gradient of two-fold dilution) is prepared for standby. And taking 2 mul of ampicillin standby liquid medicine into a 96-well plate by using a discharge gun for standby. Coli strains were adjusted to a concentration of 0.5MCF using MH broth culture, diluted 20-640 times (preferably 40-fold) with MH broth containing EZMTT detection reagents, and 200 μ l was dispensed into a 96-well plate. Setting a control hole, wherein 2ul of ampicillin in the control hole is to be used for replacing the liquid medicine with sterilized water, and the rest is consistent with the operation of the administration hole. And (3) putting the 96-well plate into an incubator at 37 ℃ for culture, and detecting OD450 and OD600 values after 4 hours.
Example 4
Drug susceptibility experiments were performed using the gram-negative bacterium pseudomonas aeruginosa (40-fold dilution of fresh bacterial fluid at 0.5 MCF): ampicillin liquid medicine (0-12.8 mg/ml; concentration gradient of two-fold dilution) is prepared for standby. And taking 2 mul of ampicillin standby liquid medicine into a 96-well plate by using a discharge gun for standby. Coli strains were adjusted to a concentration of 0.5MCF using MH broth culture, diluted 20-640 times (preferably 40-fold) with MH broth containing EZMTT detection reagents, and 200 μ l was dispensed into a 96-well plate. Setting a control hole, wherein 2ul of ampicillin in the control hole is to be used for replacing the liquid medicine with sterilized water, and the rest is consistent with the operation of the administration hole. And (3) putting the 96-well plate into an incubator at 37 ℃ for culture, and detecting OD450 and OD600 values after 4 hours.
Example 5
Drug susceptibility experiments were performed using the fungus candida albicans (40-fold dilution of fresh bacterial fluid at 0.5 MCF): ampicillin liquid medicine (0-12.8 mg/ml; concentration gradient of two-fold dilution) is prepared for standby. And taking 2 mul of ampicillin standby liquid medicine into a 96-well plate by using a discharge gun for standby. Coli strains were adjusted to a concentration of 0.5MCF using MH broth culture, diluted 20-640 times (preferably 40-fold) with MH broth containing EZMTT detection reagents, and 200 μ l was dispensed into a 96-well plate. Setting a control hole, wherein 2ul of ampicillin in the control hole is to be used for replacing the liquid medicine with sterilized water, and the rest is consistent with the operation of the administration hole. And (3) putting the 96-well plate into an incubator at 37 ℃ for culture, and detecting OD450 and OD600 values after 4 hours.
Example 6
Drug susceptibility experiments were performed using tumor cells HCT116 (1000-: preparing the antitumor drug liquid (concentration gradient of two-fold dilution or combined medication) for standby. Take 2 μ l of the liquid medicine into a 96-well plate by a discharge gun for later use. Freshly cultured HCT116 was diluted (1000 wells) with RPMI or the like medium containing serum of EZMTT detection reagent, and 200. mu.l thereof was taken into a 96-well plate. Setting up control holes, replacing 2ul of the liquid medicine to be used in the control holes with sterilized water, and operating the rest holes in accordance with the administration holes. The 96-well plate was cultured in a 37 ℃ incubator for 4 hours, 1 day, 2 days, 3 days, 4 days, and 5 days, and then the OD450 and OD600 values were measured.
Example 7
Viral infection experiments were performed using tumor cells HEK293T (1000-8000): freshly cultured HEK293T was diluted (1000 wells) with RPMI or the like medium containing serum of EZMTT detection reagents and 200. mu.l was taken into a 96-well plate. Adding a large amount of lentivirus, setting up a control hole, replacing the lentivirus in the control hole with sterilized water, and performing the same operation except for the control hole. The 96-well plate was cultured in a 37 ℃ incubator for 4 hours, 1 day, 2 days, 3 days, 4 days, and 5 days, and then the OD450 and OD600 values were measured.
Example 8
In addition to the above examples 1 to 6, the following compounds can be used as the test compounds in place of the EZMTT component in example 1, and the test compounds used in this example can be used in concentrations equivalent to those of EZMTT in example 1, and an approximate concentration range of 0.1 to 50mM is proposed, and the test compounds used in this example are all low-toxic, high-potency compounds, which are low-toxic to the living cells to be tested, and some of the compounds are non-toxic or substantially non-toxic to the living cells.
Further description with respect to the figures
FIGS. 1-15 are graphs showing the results of different experiments, which are based on the experimental procedure described in example 1, and are an alignment of data obtained by performing experiments after substituting the compounds and experimental conditions described in the figure description with the corresponding contents described in example 1. Therefore, the duplicated data in the drawings correspond to a plurality of experiments in which the contents described in example 1 are partially replaced. As shown in fig. 10, the results of detection of drug resistance of clinical drug-resistant bacteria using EZMTT as a detection indicator and the contents described in example 1; for further comparison with the above results, the conventional liquid fluorescence method is used as a reference, and the following table 1 shows the results of detecting clinical drug-resistant bacteria by the conventional liquid fluorescence method:
TABLE 1
In addition to the test compound described in example 1, any combination of groups A1-3 and R1-3 described in the claims of the present application; some of the more important combinations of groups are listed individually here:
using the procedure described in example 1 for the test compound CPD1-51, a drug sensitivity test was performed using e.coli, in which ampicillin was used as the test compound; the rest of the procedure is the same as in example 1; after experiments are carried out one by one, the result is similar to the turbidity 600nm detection result in FIG. 8, but the detection time is greatly shortened, and the sensitivity is higher.
In summary, the above-mentioned embodiments are only preferred embodiments of the present invention, and all equivalent changes and modifications made in the claims of the present invention should be covered by the claims of the present invention.
Claims (19)
1. A method for detecting drug resistance of in vitro living cells is characterized by comprising the following specific steps: incubating the living cells to be detected before detecting the living cells to be detected, and detecting the concentration of the marker after incubation to realize the detection; adding a detection compound capable of being utilized by the living cells to be detected and a test compound for drug resistance detection before incubation; the incubation refers to adding the living cells to be detected into a culture solution with a determined concentration for culture.
2. The method for detecting drug resistance of living cells in vitro according to claim 1, further comprising the step of adjusting incubation concentration: adjusting the concentration of the culture solution for incubation before incubation to approximately achieve an optimal growth rate of the living cells to be detected.
3. The method for detecting drug resistance in living cells in vitro according to claim 2, further comprising a pre-culturing step of: before incubating the living cells to be detected, culturing the living cells to be detected and determining the concentration of a culture solution capable of obtaining the optimal growth rate of the living cells to be detected.
4. The method of claim 3, further comprising the step of accelerating the culturing: after the concentration of the culture solution capable of obtaining the optimal growth rate of the living cells to be detected is obtained, the concentration of the culture solution is increased in the incubation process.
5. The method of claim 1, wherein said utilizing means that said test compound is capable of entering into said living cells to be tested.
6. The method of claim 1, wherein the detecting compound is capable of reacting with at least a portion of the components of the cells to be detected.
7. The method of claim 1, wherein the marker is a colored product.
8. The method of claim 1, wherein the detection compound is a compound that can be oxidized or reduced to a product that develops color in the living cells to be detected.
9. Method for the detection of drug resistance in living cells in vitro according to claim 1, characterized in that the detection compound is a tetrazolium salt capable of reacting with nad (p) H in the living cells to be detected and being reduced to a product that develops color in the presence of an electron coupling agent.
10. The method of claim 1, further comprising the step of detecting the inhibition rate of: adding an equal amount of living cells to be detected into a culture solution with the same concentration while incubating the living cells to be detected to prepare a control group, adding a control compound with the same amount and the same concentration as the compound to be detected before incubating the control group, detecting the concentration of a marker after incubating the control group, comparing the concentration of the marker, and obtaining the inhibition rate of the detection compound on the living cells to be detected by using detection compounds with different toxicity.
11. A culture solution for drug resistance detection of in vitro living cells, which is used for incubating living cells to be detected, comprises a culture medium and a detection compound; the detection compound has the following general structural formula:
wherein,
a1 is as defined aboveA linked aromatic or heterocyclic group;
a2 is as defined aboveA linked aromatic or heterocyclic group;
a3 is as defined aboveA linked phenyl group;
r1 is H or at least one R1 substituent attached to said a1, said R1 substituents each independently selected from-NO 2, -SO3H, -SO2NH2, halogen elements, and heterocyclic groups fused to said a1, alkyl;
r2 is H or at least one R2 substituent attached to said a2, said R2 substituents each being independently selected from NO2, methoxy, alkyl, phenyl and its derivatives linked through azido, halogen atoms, sulfonic acid groups and their salts, and phenyl or heterocyclic groups fused to said a 1;
r3 is the R3 substituent attached to the A3; the R3 substituent is respectively and independently selected from sulfonic acid group, carboxylic acid group, substituted sulfonic acid group and substituted carboxylic acid group;
the heterocyclic group includes 1-2 heteroatoms each independently selected from Se, N, S or O.
12. The culture solution for detecting drug resistance of living cells in vitro according to claim 11, wherein R3 is selected from the group consisting of:
13. the culture solution for detecting drug resistance of living cells in vitro according to claim 11, wherein the R1 comprises at least one R1 substituent at the para position of A1.
14. The culture solution for detecting drug resistance of living cells in vitro according to claim 11, wherein the R2 comprises at least one R2 substituent located at the ortho-position or para-position of A2.
15. The culture solution for detecting drug resistance of living cells in vitro according to claim 11, wherein the R3 includes at least one R3 substituent located at the ortho position of A3.
16. The culture solution for detecting drug resistance of living cells in vitro according to claim 11, wherein the aromatic group or heterocyclic group is independently selected from
17. The culture solution for the detection of drug resistance in living cells in vitro according to claim 11, further comprising a sensitizer; the sensitizer comprises a PMS derivative.
18. The culture solution for detecting drug resistance of living cells in vitro according to claim 11, further comprising a growth accelerator; the growth accelerator comprises blood, phytohormone, Pleurotus Ostreatus solution, semen glycines infusion, and OADC.
19. The culture solution for detecting drug resistance of living cells in vitro according to claim 11, further comprising bacteriostatic agents.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810691378 | 2018-06-28 | ||
| CN2018106913789 | 2018-06-28 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109988813A true CN109988813A (en) | 2019-07-09 |
| CN109988813B CN109988813B (en) | 2023-03-10 |
Family
ID=67128832
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811290781.7A Active CN109988813B (en) | 2018-06-28 | 2018-11-01 | Method and culture solution for detecting drug resistance of in vitro living cells |
| CN201910378609.5A Pending CN111286525A (en) | 2018-06-28 | 2019-05-08 | Method and culture solution for detecting drug resistance of in vitro living cells |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910378609.5A Pending CN111286525A (en) | 2018-06-28 | 2019-05-08 | Method and culture solution for detecting drug resistance of in vitro living cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN109988813B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904185A (en) * | 2019-12-25 | 2020-03-24 | 武汉市农业科学院 | Quick detection kit for drug sensitivity of duck pathogenic bacteria based on iodine nitro tetrazole color development |
| CN111286525A (en) * | 2018-06-28 | 2020-06-16 | 安吉汉菁生物科技有限公司 | Method and culture solution for detecting drug resistance of in vitro living cells |
| CN111304278A (en) * | 2020-02-24 | 2020-06-19 | 杭州伽玛生物科技有限公司 | Drug sensitive kit |
| CN111321195A (en) * | 2020-03-14 | 2020-06-23 | 浙江工业大学 | EZMTT-based method for continuously determining cancer cell proliferation inhibition effect |
| CN111500670A (en) * | 2020-04-20 | 2020-08-07 | 杭州伽玛生物科技有限公司 | High-throughput drug sensitivity detection kit and use method and application thereof |
| CN111705107A (en) * | 2020-07-13 | 2020-09-25 | 浙江工业大学 | Antibiotic susceptibility and compound antibacterial activity detection method based on EZMTT reagent |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114113510B (en) * | 2021-11-23 | 2024-05-03 | 杭州汉菁生物科技有限公司 | Method for determining MIC value of medicine and storage medium |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559844A (en) * | 2012-01-09 | 2012-07-11 | 上海交通大学 | Preparation method of detecting system for minimal inhibitory concentration of mycobacteria |
| CN105418526B (en) * | 2015-11-09 | 2019-02-15 | 杭州伽玛生物科技有限公司 | One kind has compound and its application of single sulphenyl tetrazole structure |
| CN107365827A (en) * | 2016-05-11 | 2017-11-21 | 杭州健昵福生物科技有限公司 | A kind of purposes and method for being used to single sulfonic acid tetrazolium detect cell viability |
| CN108179173A (en) * | 2018-03-23 | 2018-06-19 | 武汉轻工大学 | A kind of mycobacterium tuberculosis susceptibility detection color developing agent and its susceptibility detection method |
| CN109988813B (en) * | 2018-06-28 | 2023-03-10 | 杭州汉菁生物科技有限公司 | Method and culture solution for detecting drug resistance of in vitro living cells |
-
2018
- 2018-11-01 CN CN201811290781.7A patent/CN109988813B/en active Active
-
2019
- 2019-05-08 CN CN201910378609.5A patent/CN111286525A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| ALESSANDRO DE LOGU ET AL: "Comparison of the susceptibility testing of clinical isolates of Mycobacterium tuberculosis by the XTT colorimetric method and the NCCLS standards method", 《INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS》 * |
| WEI ZHANG ET AL: "Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays", 《ANALYTICAL BIOCHEMISTRY》 * |
| 张京京等: "新型细胞活力检测EZMTT法的研究", 《发酵科技通讯》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111286525A (en) * | 2018-06-28 | 2020-06-16 | 安吉汉菁生物科技有限公司 | Method and culture solution for detecting drug resistance of in vitro living cells |
| CN110904185A (en) * | 2019-12-25 | 2020-03-24 | 武汉市农业科学院 | Quick detection kit for drug sensitivity of duck pathogenic bacteria based on iodine nitro tetrazole color development |
| CN111304278A (en) * | 2020-02-24 | 2020-06-19 | 杭州伽玛生物科技有限公司 | Drug sensitive kit |
| CN111321195A (en) * | 2020-03-14 | 2020-06-23 | 浙江工业大学 | EZMTT-based method for continuously determining cancer cell proliferation inhibition effect |
| CN111500670A (en) * | 2020-04-20 | 2020-08-07 | 杭州伽玛生物科技有限公司 | High-throughput drug sensitivity detection kit and use method and application thereof |
| CN111705107A (en) * | 2020-07-13 | 2020-09-25 | 浙江工业大学 | Antibiotic susceptibility and compound antibacterial activity detection method based on EZMTT reagent |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111286525A (en) | 2020-06-16 |
| CN109988813B (en) | 2023-03-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109988813B (en) | Method and culture solution for detecting drug resistance of in vitro living cells | |
| Van Belkum et al. | Matrix-assisted laser desorption ionization time-of-flight mass spectrometry in clinical microbiology: what are the current issues? | |
| EP0254771B1 (en) | Detection of microbes in a sample | |
| Dixit et al. | Evaluation of nitrate reduction assay, resazurin microtiter assay and microscopic observation drug susceptibility assay for first line antitubercular drug susceptibility testing of clinical isolates of M. tuberculosis | |
| US20190078144A1 (en) | Dual labeling methods for measuring cellular proliferation | |
| CN114214238B (en) | Multi-drug-resistant Indiananas and application thereof | |
| CN107266463B (en) | Compound and application thereof in mycobacterium tuberculosis detection | |
| CN109790564A (en) | With the method for solvatochromic dye conjugation analyte detection mycobacteria | |
| US11519017B2 (en) | Kit for diagnosing infection with Methicillin-resistant Staphylococcus aureus (MRSA) by detecting magnesium ions | |
| CN101643713B (en) | Legionnella selective separation medium | |
| Jabbar et al. | Extraction, purification and characterization of Pyocyanin pigment from Pseudomonas aeruginosa and testing its biological efficacy. | |
| US20100291591A1 (en) | Novel plating media | |
| Sriram et al. | Evaluation of antimycobacterial and DNA gyrase inhibition of fluoroquinolone derivatives | |
| KR20180136908A (en) | Pathogen detection method | |
| RU2648160C2 (en) | Nutrient medium for the isolation of bacteria yersinia enterocolitica | |
| CN111166750B (en) | Novel antibacterial application of 4-fluoro-2-methylindole compound | |
| JP2005261337A (en) | Method for detecting microorganism in high sensitivity, and reagent kit and system for assaying luminescence for the detection | |
| CN115287371B (en) | Method for detecting genomic DNA of penicillium chrysogenum | |
| Tha et al. | Resazurin based rapid screening and Spectrophotometric analysis of potent antimicrobials from Streptomyces isolated in Nepal | |
| Krämer et al. | Artificial fluorogenic substrates in microfluidic devices for bacterial diagnostics in biotechnology | |
| CN117737183A (en) | Microbial limit detection method of norfloxacin capsule | |
| CN117230221A (en) | Primer group for detecting mycobacterium species, application and kit | |
| CN110938672A (en) | Method for testing microbial drug sensitivity test by using drug sensitivity indicator | |
| CN117925772A (en) | Helicobacter pylori culture selection separation, identification, drug sensitivity detection reagent, kit and application thereof | |
| CN118304316A (en) | Application of cladribine in preparation of drug for inhibiting growth of multi-drug resistant klebsiella pneumoniae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20201228 Address after: 310000 3rd floor, building 13, Fuchun Park, Zhongguo Zhigu, Yinhu street, Fuyang District, Hangzhou City, Zhejiang Province Applicant after: Hangzhou Hanjing Biotechnology Co.,Ltd. Address before: 313300 Sunshine Industrial Park, Dipu street, Anji County, Huzhou City, Zhejiang Province Applicant before: Anji Hanqing Biotechnology Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Method and culture medium for detecting drug resistance in live cells in vitro Granted publication date: 20230310 Pledgee: Shuangpu sub branch of Hangzhou United Rural Commercial Bank Co.,Ltd. Pledgor: Hangzhou Hanjing Biotechnology Co.,Ltd. Registration number: Y2024980012005 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |