CN116426611A - Bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a - Google Patents
Bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a Download PDFInfo
- Publication number
- CN116426611A CN116426611A CN202310509369.4A CN202310509369A CN116426611A CN 116426611 A CN116426611 A CN 116426611A CN 202310509369 A CN202310509369 A CN 202310509369A CN 116426611 A CN116426611 A CN 116426611A
- Authority
- CN
- China
- Prior art keywords
- cas12a
- dpa
- dnazyme
- crispr
- bacillus anthracis
- 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
- 108700004991 Cas12a Proteins 0.000 title claims abstract description 60
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 51
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 51
- 238000001514 detection method Methods 0.000 title claims abstract description 47
- 241000193738 Bacillus anthracis Species 0.000 title claims abstract description 40
- 229940065181 bacillus anthracis Drugs 0.000 title claims abstract description 39
- 108091027757 Deoxyribozyme Proteins 0.000 title claims abstract description 19
- 239000003550 marker Substances 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 34
- 239000010949 copper Substances 0.000 claims description 74
- 238000003776 cleavage reaction Methods 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 29
- JADVWWSKYZXRGX-UHFFFAOYSA-M thioflavine T Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C1=[N+](C)C2=CC=C(C)C=C2S1 JADVWWSKYZXRGX-UHFFFAOYSA-M 0.000 claims description 28
- 230000007017 scission Effects 0.000 claims description 22
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 20
- 238000011534 incubation Methods 0.000 claims description 19
- 238000002156 mixing Methods 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 18
- 108090000790 Enzymes Proteins 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 230000003197 catalytic effect Effects 0.000 claims description 11
- 235000010323 ascorbic acid Nutrition 0.000 claims description 10
- 229960005070 ascorbic acid Drugs 0.000 claims description 10
- 239000011668 ascorbic acid Substances 0.000 claims description 10
- 230000009920 chelation Effects 0.000 claims description 10
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 9
- 238000009739 binding Methods 0.000 claims description 9
- 229910052802 copper Inorganic materials 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 8
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 8
- 239000000758 substrate Substances 0.000 claims description 8
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 7
- 239000005457 ice water Substances 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000008399 tap water Substances 0.000 claims description 6
- 235000020679 tap water Nutrition 0.000 claims description 6
- 239000013522 chelant Substances 0.000 claims description 5
- 239000011535 reaction buffer Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007995 HEPES buffer Substances 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 2
- 230000009849 deactivation Effects 0.000 claims description 2
- 230000003321 amplification Effects 0.000 abstract description 16
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 16
- 239000007850 fluorescent dye Substances 0.000 abstract description 15
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 238000004445 quantitative analysis Methods 0.000 abstract description 7
- 235000013305 food Nutrition 0.000 abstract description 6
- 230000009977 dual effect Effects 0.000 abstract description 5
- 238000004451 qualitative analysis Methods 0.000 abstract description 4
- 238000001215 fluorescent labelling Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 9
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 8
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 7
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 7
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 230000005284 excitation Effects 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- CABMTIJINOIHOD-UHFFFAOYSA-N 2-[4-methyl-5-oxo-4-(propan-2-yl)-4,5-dihydro-1H-imidazol-2-yl]quinoline-3-carboxylic acid Chemical compound N1C(=O)C(C(C)C)(C)N=C1C1=NC2=CC=CC=C2C=C1C(O)=O CABMTIJINOIHOD-UHFFFAOYSA-N 0.000 description 5
- 108091081406 G-quadruplex Proteins 0.000 description 5
- 108020004682 Single-Stranded DNA Proteins 0.000 description 5
- 238000007405 data analysis Methods 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- 239000005711 Benzoic acid Substances 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 235000010233 benzoic acid Nutrition 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229960005190 phenylalanine Drugs 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000002194 synthesizing effect Effects 0.000 description 4
- WJJMNDUMQPNECX-UHFFFAOYSA-N Dipicolinic acid Natural products OC(=O)C1=CC=CC(C(O)=O)=N1 WJJMNDUMQPNECX-UHFFFAOYSA-N 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 3
- 229930064664 L-arginine Natural products 0.000 description 3
- 235000014852 L-arginine Nutrition 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 235000001968 nicotinic acid Nutrition 0.000 description 3
- 229960003512 nicotinic acid Drugs 0.000 description 3
- 239000011664 nicotinic acid Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000000956 alloy Substances 0.000 description 2
- 229910045601 alloy Inorganic materials 0.000 description 2
- 208000022338 anthrax infection Diseases 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 108010040467 CRISPR-Associated Proteins Proteins 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 108010061951 Methemoglobin Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- ZVQOOHYFBIDMTQ-UHFFFAOYSA-N [methyl(oxido){1-[6-(trifluoromethyl)pyridin-3-yl]ethyl}-lambda(6)-sulfanylidene]cyanamide Chemical compound N#CN=S(C)(=O)C(C)C1=CC=C(C(F)(F)F)N=C1 ZVQOOHYFBIDMTQ-UHFFFAOYSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- ZFTFAPZRGNKQPU-UHFFFAOYSA-N dicarbonic acid Chemical compound OC(=O)OC(O)=O ZFTFAPZRGNKQPU-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 1
- 229940025294 hemin Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000012621 metal-organic framework Substances 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- KDMWFFHKQUJBLB-UHFFFAOYSA-N n-methyl-1,1-diphenylpropan-2-amine;hydrochloride Chemical compound Cl.C=1C=CC=CC=1C(C(C)NC)C1=CC=CC=C1 KDMWFFHKQUJBLB-UHFFFAOYSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 150000002910 rare earth metals Chemical class 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
Images
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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
-
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of bacillus anthracis detection, and particularly relates to a bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12 a. The invention provides a bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a, which utilizes Cu 2+ The label-free fluorescent biosensor is constructed by a specific DNAzyme and CRISPR/Cas12a dual signal amplification strategy, so that the sensitivity of the system is remarkably improved, and the system has better specificity. Meanwhile, the G-quadruplex-thioflavin T is taken as a fluorescent probe, and compared with the traditional fluorescent labeling mode, the fluorescent probe is simple and label-freeThe cost is saved. In addition, the method disclosed by the invention is simple to operate and wide in application range, can be used for measuring trace bacillus anthracis markers DPA in food, realizes qualitative and quantitative analysis of the DPA, and provides a new technical support for food safety.
Description
Technical Field
The invention belongs to the technical field of bacillus anthracis detection, and particularly relates to a bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12 a.
Background
Bacillus anthracis (Bacillus anthracis), which is a gram-positive aerobic bacterium, is a dangerous strain that can cause anthrax acute infections in domestic animals, wild animals and humans. 2, 6-pyridinedicarboxylic acid (DPA) is a chemical substance released by bacillus anthracis spores during germination, hydrolysis and heating, accounting for 5-15% of the total dry weight of the spores, and is absent from other natural and synthetic materials and can be used as a biomarker for bacillus anthracis presence. Therefore, the dynamic level of bacillus anthracis can be indirectly measured by accurately detecting the DPA content in a complex sample, and the method has profound significance for preventing and controlling anthracis. Currently, methods for analytical determination of bacillus anthracis mainly include whole genome sequencing, antibody detection and DPA assay.
Wherein, the method for detecting bacillus anthracis by measuring DPA content comprises surface enhanced Raman spectroscopy, liquid chromatography, gas chromatography-mass spectrometry and the like. However, these methods have disadvantages of long time, high cost, complicated operation, expensive equipment and professional operations. In addition, DPA analysis is achieved by synthesizing different fluorescent probes. According to the invention patent CN114957679A, DPA is analyzed and measured by synthesizing a metal organic framework material as a fluorescent probe; CN115109584a realizes the analysis and determination of DPA by synthesizing a fluorescent nano-probe composite material; CN106959290a realizes analysis and measurement of DPA by synthesizing a ratio-type rare earth fluorescent probe. Although the detection speed is high, the synthesis of the fluorescent probe is complex, and the operation difficulty is high. Therefore, it is necessary to develop a method for performing the analytical measurement of DPA that is easy to operate, has high sensitivity and good selectivity, and has no fluorescence labeled.
Deoxyribozymes (dnazymes) are a class of single-stranded DNA or RNA molecules obtained by screening that have catalytic and structural recognition functions and produce a catalytic activity similar to that of proteases under the action of cofactors. DNAzyme mainly comprises two binding sequences at two ends and a middle catalytic active center, and the two binding sequences are hybridized through a base complementary pairing principle; the middle catalytic active center is in a ring-shaped or hairpin structure, and the cofactor is combined to generate the catalytic capability. In the presence of cofactors, the catalytically active sites are activated, intoAnd the substrate chain is cut, and the released enzyme chain enters the next round of cutting reaction, so that the amplification of a target signal is realized. Wherein Cu is 2+ The specific DNAzyme has the advantages of simple preparation, high metal ion selectivity, good programmability, excellent stability, easy modification, stability under severe conditions and the like, and becomes ideal Cu 2+ The platform is identified. DPA and Cu are reported 2+ Has strong chelation, thus Cu can be utilized 2+ The DPA is quantitatively detected by specific DNAzyme. However, considering that a single signal amplification method cannot meet the requirement of micro marker detection, a novel amplification technology needs to be added to further improve the detection sensitivity.
The regularly spaced clustered short palindromic repeats (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR)/CRISPR-associated protein (CRISPR-Associated Protein, cas) system is an adaptive immune system found in most archaea and many bacteria. Wherein the CRISPR/Cas12a system may exhibit trans-cleaving activity on non-target ssDNA after activation by target single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Therefore, the CRISPR/Cas12a system can be used as a high-efficiency signal amplification technology, has shorter reaction time and stronger cutting efficiency, and is used for detecting biomarkers. Currently, most fluorescence analysis methods using CRISPR/Cas12a directly utilize the trans-cleavage property of Cas12a protein, and after target DNA binds to Cas12a-crRNA complex, the ssDNA probe (Reporter) labeled with fluorophore and quencher is trans-cleaved for fluorescence signal output. Chinese patent No. CN112522429A constructs a fluorescent biosensor for detecting bacillus anthracis based on a Recombinase Polymerase Amplification (RPA) technology and CRISPR/Cas12 a. After RPA amplification is carried out on bacillus anthracis genome, the product is combined with Cas12a-crRNA complex to cut a Reporter in a trans-form, and analysis and determination of bacillus anthracis are realized by measuring the fluorescence intensity in a system. The method has higher sensitivity, the lowest concentration of the detection template reaches 1 copy/mu L, but the RPA amplification operation adopted by the method is complex and is easy to generate false positive results, and the problem of higher cost, harsh storage conditions and poor stability exists in a Reporter. Therefore, it is of great importance to develop a label-free fluorescence detection method.
G-quadruplexes are stable, non-B-type DNA secondary structures formed by guanine-rich DNA sequences through Hoogsteen hydrogen bonding, and are widely used in the construction of label-free fluorescent biosensors because of their unique fluorescence enhancement properties upon binding to certain fluorescent dyes such as thioflavin T (ThT). Chinese patent No. CN113444816B constructs a colorimetric biosensor based on the activity of the trans-cleavage G-quadruplex of RPA and CRISPR/Cas12a, and is used for detecting bacillus anthracis. The method uses the genome DNA of bacillus anthracis as a template to carry out RPA amplification, and after the RPA amplification product is combined with a Cas12a-crRNA complex, the G-quadruplex is trans-cut, and after the G-quadruplex is combined with methemoglobin (Hemin), the G-quadruplex has peroxidase activity and is expressed in H 2 O 2 The ABTS substrate, which is capable of catalyzing colorless in the presence, appears green. Therefore, the bacillus anthracis can be visually detected according to the color change of the reaction liquid. The method is simple and portable, is suitable for rapid detection of on-site and clinical specimens, but only realizes qualitative detection of bacillus anthracis, and does not further carry out quantitative analysis and determination on bacillus anthracis. Based on the research foundation, the construction of a fluorescent biosensing platform for detecting bacillus anthracis by utilizing the trans-cleavage activity of CRISPR/Cas12a on the G-quadruplex has certain feasibility.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention is based on Cu 2+ The dual signal amplification strategy of the specific DNAzyme and the CRISPR/Cas12a uses G-quadruplex-ThT as a fluorescent probe to construct a label-free fluorescent biosensor, can be used for qualitative and quantitative analysis and detection of DPA in food, and has the advantages of simplicity and convenience in operation, high sensitivity, good selectivity and the like.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a method for detecting bacillus anthracis markers based on deoxyribozyme (DNAzyme) and CRISPR/Cas12a, which comprises the following steps:
s1, 2, 6-pyridinedicarboxylic acid (DPA) and Cu 2+ The solution is evenly mixed and then incubated, and DPA-Cu is formed through chelation reaction 2+ A chelate compound;
s2, formation of DNAzyme: mixing the enzyme chain (Cu_Enz) shown in SEQ ID No.1 and the substrate chain (Cu_sub) shown in SEQ ID No.2 in a reaction buffer solution, and incubating to form Cu 2+ A specific DNAzyme;
s3, DPA-Cu of the step S1 2+ Chelate and Cu of step S2 2+ Mixing the specific DNAzyme, and adding Ascorbic Acid (AA) to make Cu 2+ Is reduced to Cu + And Cu is obtained by copper enzyme catalytic cutting reaction 2+ Specific DNAzyme cleavage products;
s4, uniformly mixing the Cas12a with crRNA shown in SEQ ID No.3, and incubating to form a Cas12a-crRNA complex;
s5, cu in the step S3 2+ Uniformly mixing the specific DNAzyme cleavage product with the Cas12a-crRNA complex in the step S4, adding a cleavage buffer solution, and continuously incubating a guanine (G-rich) rich Sequence (G-rich Sequence) and thioflavin T (ThT) shown in SEQ ID No.4 to perform a CRISPR/Cas12a cleavage reaction and a binding reaction of the G-rich Sequence and the ThT; in the absence of DPA, cu 2+ Specific DNAzyme is activated to cleave cu_sub, cleaved cu_sub cannot bind to Cas12a-crRNA complex, cas12a protein cannot be activated, G-rich Sequence remains intact to form G-quadruplex-ThT complex with ThT, showing a distinct fluorescence intensity at 490 nm; after DPA addition, cu 2+ Chelation with DPA results in deactivation of DNAzyme, binding of intact cu_sub to Cas12a-crRNA complex, activation of trans cleavage activity of Cas12a protein, cleavage of G-rich Sequence, failure to form complex with ThT, and significant decrease in fluorescence intensity.
The invention provides a Cu-based alloy 2+ And a specific DNAzyme and CRISPR/Cas12a dual signal amplification technology, and a G-quadruplex-ThT is used as a fluorescent probe to construct a label-free fluorescent biosensor for the analysis and determination of DPA. The specific principle is as follows (fig. 1): cu consisting of a substrate chain (Cu_sub) and an enzyme chain (Cu_Enz) when no DPA is present in the system 2+ Specific DNAzyme in Cu 2+ Under the action of catalytic cleavage of Cu_sub, the cleaved Cu_sub cannot be complementary to crRNAPairing, resulting in the Cas12a protein trans-cleavage activity not being activated, the G-rich Sequence within the system remains intact, and thus binds to ThT to form a G-quadruplex-ThT complex, yielding a strong fluorescent signal. When DPA is added into the reaction system, since DPA can be mixed with Cu 2+ Chelating reactions occur, resulting in Cu 2+ The specific DNAzyme cannot catalyze cleavage of Cu_sub, the complete Cu_sub specifically binds to Cas12a-crRNA complex, and disruption of trans-cleavage of G-rich Sequence, results in failure to form a complex with ThT, thereby significantly reducing fluorescence intensity. Therefore, DPA can be detected by qualitative or quantitative analysis by measuring the fluorescence intensity in the reaction system.
In step 1, cu is used as a preferable embodiment of the present invention 2+ The concentration is 200-400nM; the incubation temperature is 20-30 ℃ and the incubation time is 30-60min; DPA concentration is 0-3000nM.
As a preferred embodiment of the invention, in the step 2, the incubation temperature is 25-35 ℃ and the incubation time is 30-60min; the concentration of the substrate strand is 0.2-1.8nM; the concentration of enzyme chains was 0.4-3.6nM and the reaction buffer contained 50mM HEPES,500mM NaCl,pH 7.4.
As a preferred embodiment of the invention, in step 3, the AA concentration is 40-60. Mu.M; the temperature of the copper enzyme catalytic cutting reaction is 25-35 ℃ and the time is 20-100min.
As a preferred embodiment of the invention, in step 4, the concentration of Cas12a and crRNA is 4-10nM.
As a preferred embodiment of the present invention, in step 5, the concentration of G-rich Sequence is 50-250nM; the cleavage Buffer solution was 10 Xreaction Buffer, which consisted of 100mM Tris-HCl,500mM NaCl,100mM MgCl 2 10mM DTT, pH 8.0; the concentration of ThT is 5-7 mu M; the incubation time of the CRISPR/Cas12a trans-cleavage reaction is 30-50min, and the temperature is 37 ℃; the incubation is continued in an ice-water bath for 40-60min.
As a preferred embodiment of the present invention, in step 5, fluorescence intensity detection is performed by using a fluorescence spectrophotometer, the excitation wavelength at the time of detection is 425nm, the emission wavelength is in the range of 450-600nm, the fluorescence intensity value at 490nm is recorded, and the slit width is 5/10nm.
The second aspect of the invention provides an application of the bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a in bacillus anthracis detection.
As a preferred embodiment of the present invention, the limit of detection of DPA is 30.5nM. Much lower than the DPA (60. Mu.M) released by the anthrax infection dose.
As a preferred embodiment of the present invention, the linear relationship between fluorescence intensity and DPA concentration in quantitative detection is F= 696.6-0.2944C, F means fluorescence intensity at 490nm, and C means DPA concentration.
As a preferred embodiment of the present invention, the samples tested include tap water and juice.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a, which utilizes Cu 2+ The label-free fluorescent biosensor is constructed by a specific DNAzyme and CRISPR/Cas12a dual signal amplification strategy, so that the sensitivity of the system is remarkably improved, and the system has better specificity. Meanwhile, the G-quadruplex-ThT is used as a fluorescent probe, so that the fluorescent probe is simple and has no mark, and compared with the traditional fluorescent marking mode, the fluorescent probe has the advantage of obviously saving the cost. In addition, the method disclosed by the invention is simple to operate and wide in application range, can be used for measuring trace DPA in food, realizes qualitative and quantitative analysis and detection of DPA, and provides a new technical support for food safety.
Compared with the invention patent CN113444816B, the method adopts Cu 2+ The specific DNAzyme isothermal enzyme-free signal amplification technology is simpler to operate, and false positive results are prevented; the method can realize quantitative detection of bacillus anthracis and has a quantitative analysis on the release dosage of bacillus anthracis. Compared with the invention patent CN112522429A, the method not only uses isothermal enzyme-free amplification technology, but also uses G-quadruplex-ThT as a fluorescent probe to replace a double-labeled fluorescent report probe, thereby constructing a label-free fluorescent sensor and remarkably saving modification cost. And invention patent CN114957679A, CN115109584A andcompared with CN106959290A, the method adopts a nucleic acid biosensor to carry out analysis and determination, and avoids a complex chemical synthesis process.
Drawings
FIG. 1 is a Cu-based 2+ Schematic diagram of DPA content detection methods of specific DNAzyme and CRISPR/Cas12 a;
FIG. 2 is a Cu-based 2+ The detection methods of the specific DNAzyme and CRISPR/Cas12a analyze fluorescence spectrograms of different samples (DPA exists or not);
FIG. 3 is a Cu-based 2+ Standard curves of detection methods of specific DNAzyme and CRISPR/Cas12a for different concentration DPA detection;
FIG. 4 is a Cu-based 2+ Specificity verification results of detection methods of specific DNAzyme and CRISPR/Cas12a on DPA detection figures [ note: 1. blank (Blank), 2. Nicotinic Acid (NA), 3. Terephthalic acid (p-PA), 4. Benzoic Acid (BA), 5. Phthalic Acid (PA), 6.Cl - ,7.SO 4 2- ,8.NO 3 - 9.L-Glycine (L-Gly), 10L-phenylalanine (L-Phe), 11L-arginine (L-Arg), 12.DPA,13. The above mixture (mixture) ].
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
Example 1 Cu-based 2+ DPA content detection method of specific DNAzyme and CRISPR/Cas12a
The DPA content detection method specifically comprises the following steps:
(1)Cu 2+ chelation reaction with DPA: 300nM Cu 2+ With 2000nM DPA standard solution [ or DEPC (pyrocarbonic acid)Diethyl ester) treated water (blank) was mixed and incubated at 25℃for 30min to form DPA-Cu 2+ A chelate.
(2) Formation of DNAzyme: mixing 2nM enzyme chain (Cu_Enz, nucleotide sequence 5'-GGT AAG CCT GGG CCT CTT TCT TTT TAA GAA AGA AC-3', SEQ ID No. 1) with 1nM substrate chain (Cu_Sub, nucleotide sequence 5'-AGC TTC TTT CTA ATA CGG CTT ACC TGC-3', SEQ ID No. 2) in reaction buffer (50mM HEPES,500mM NaCl,pH 7.4), and incubating at 30deg.C for 30min to form Cu 2+ Specific DNAzyme.
(3)Cu 2+ Cleavage of specific DNAzyme: mixing the solution after incubation in step (1) and the solution after incubation in step (2), adding 50 mu M Ascorbic Acid (AA), and mixing to obtain Cu 2+ Is reduced to Cu + Carrying out a copper enzyme catalytic cutting reaction at 30 ℃ for 80min to obtain Cu 2+ Specific DNAzyme cleavage products.
(4) Formation of Cas12a-crRNA complex: while step (3) was ongoing, 2 μl of 0.5 μΜ LbCas12a ((LbCas 12a (Cpf 1) purchased from american biotechnology limited, guangzhou) and 2 μl of 0.5 μΜ crRNA (sequence 5'-UAA UUU CUA CUC UUG UAG AU GCA GGU AAG CCG UAU UAG AA-3', SEQ idno. 3) were mixed and incubated at 37 ℃ for 30min to form Cas12a-crRNA complex.
(5) CRISPR/Cas12a trans-cleaves and binds to ThT with guanine (G) rich Sequence (Sequence 5'-GGG AAG GGA GGG ATG GGA-3', SEQ ID No. 4): combining the Cas12a-crRNA complex obtained in step (4) with the Cu obtained in step (3) 2+ The specific DNAzyme cleavage products were thoroughly mixed, and 10. Mu.L of 10 Xreaction Buffer (100 mM Tris-HCl,500mM NaCl,100mM MgCl) was added thereto 2 10mM DTT, pH 8.0), 150nM G-rich Sequence and 6. Mu.M ThT were mixed and incubated at 37℃for 40min to allow CRISPR/Cas12a to trans-cleave the G-rich Sequence at a final volume of 200. Mu.L. After the reaction is finished, the whole system is placed in an ice water bath for continuous incubation for 45min, so that the combined reaction of the G-rich Sequence and the ThT is continuously carried out under the condition that the CRISPR/Cas12a is inactive.
(6) Fluorescence spectrophotometer detection: and measuring the fluorescence intensity of the reaction system by using a fluorescence spectrophotometer to realize the analysis of DPA. When the excitation wavelength is 425nm and the slit width is 5/10nm, the fluorescence spectrum between 450 and 600nm is scanned, and the fluorescence intensity at 490nm is adopted for data analysis.
As shown in FIG. 2, the system has strong fluorescence intensity at 490nm (curve a) when DPA is not present, mainly because Cu is present in the absence of DPA 2+ The specific DNAzyme catalyzes the cleavage of Cu_Sub, so that the trans-cleavage activity of Cas12a protein cannot be activated, G-rich Sequence in the system remains intact, and the G-rich Sequence is combined with ThT to form a G-quadruplex-ThT complex to generate a strong fluorescent signal. When DPA was added, the fluorescence intensity was significantly decreased (curve b), mainly because when DPA was added to the reaction system, it was able to react with Cu 2+ Chelating reactions occur, resulting in Cu 2+ The specific DNAzyme cannot be activated, cu_Sub specifically binds to the Cas12a-crRNA complex, the trans-cleaved G-rich Sequence, the G-quadruplex-ThT complex cannot be formed, and the fluorescence intensity is significantly reduced. The results show that the invention provides Cu-based alloy 2+ Methods of specific DNAzyme and CRISPR/Cas12a can be used for detection of DPA.
Example 2 Cu-based 2+ Sensitivity analysis of DPA content detection methods of specific DNAzyme and CRISPR/Cas12a
According to example 1, DPA standard solutions of different concentrations (0, 60, 80, 100, 200, 400, 600, 800, 1000, 1500, 2000 and 3000 nM) were used as test samples under optimal experimental conditions, and the sensitivity of the method of the invention for detecting DPA was analyzed as follows:
DPA at different concentrations was combined with 300nM Cu 2+ The solutions were mixed well and incubated at 25℃for 30min for chelation. At the same time, 1nM Cu_sub, 2nM Cu_Enz were incubated at 30℃for 30min to form Cu 2+ Specific DNAzyme. Mixing the above two solutions, adding 50 μm AA, and mixing to obtain Cu 2+ Is reduced to Cu + And (3) reacting at 30 ℃ for 80min to perform a copper enzyme cleavage reaction to obtain an enzyme cleavage product. An additional 30min incubation of 5nM Cas12a with 5nM crRNA at 37℃was performed to form a Cas12a-crRNA complex. Subsequently, the pre-assembled Cas12a-crRNA complex, 10. Mu.L of 10 Xreaction Buffer, 150nM G-rich Sequence and 6. Mu.M ThT plusInto the cleavage product, the final volume was 200 μl, incubated at 37 ℃ for 40min, allowing CRISPR/Cas12a to trans-cleave the G-rich Sequence. After the reaction is finished, the whole system is placed in an ice water bath for further incubation for 45min, and the binding reaction of the G-rich Sequence and the ThT is continued under the condition that CRISPR/Cas12a is inactive. Finally, the fluorescence intensity of the reaction solution at an excitation wavelength of 425nm, an emission wavelength of 490nm and a slit width of 5/10nm was measured for data analysis to prepare a standard curve.
As a result, as shown in FIG. 3, in the range of 80nM to 2000nM, the fluorescence intensity (F) of the system at 490nM exhibited a good linear relationship with the concentration (C) of DPA. The linear equation is F= 696.6-0.2944C, and the fitting constant of the correlation equation is R 2 =0.997, detection limit as low as 30.5nM (S/n=3), far lower than DPA released by anthrax infection dose (60 μm), indicating that the method of the invention can realize high-efficiency detection of DPA for quantitative analysis of DPA.
Example 3 Cu-based 2+ Specific analysis of DPA content detection methods of specific DNAzyme and CRISPR/Cas12a
The samples to be tested in this example are DPA and DPA structural analogues of Nicotinic Acid (NA), terephthalic acid (p-PA), benzoic Acid (BA) and Phthalic Acid (PA); anionic substance Cl - 、SO 4 2- And NO 3 - The method comprises the steps of carrying out a first treatment on the surface of the Standard solutions of the amino acid molecules L-glycine (L-Gly), L-phenylalanine (L-Phe) and L-arginine (L-Arg) and mixed solutions (mixtures) of the above. Wherein the DPA concentration is 500,1000 and 2000nM, and the other substances are 10. Mu.M. According to example 1, the specific operation is as follows:
different test solutions were combined with 300nM Cu 2+ The solutions were mixed well and incubated at 25℃for 30min for chelation. At the same time, 1nM Cu_sub, 2nM Cu_Enz were incubated at 30℃for 30min to form Cu 2+ Specific DNAzyme. Mixing the above two solutions, adding 50 μm AA, and mixing to obtain Cu 2+ Is reduced to Cu + The reaction was carried out at 30℃for 80min to carry out a cleavage reaction with copper. 5nM Cas12a was incubated with 5nM crRNA for 30min at 37℃to form a Cas12a-crRNA complex. Subsequently, the pre-assembled Cas12a-crRNA complex, 10. Mu.L of 10 Xreaction Buffer150nM G-rich Sequence and 6. Mu.M ThT were added to the digested product and the final volume was 200. Mu.L and incubated at 37℃for 40min to allow CRISPR/Cas12a to cleave the G-rich Sequence in trans. After the reaction is finished, the whole system is placed in an ice water bath for further incubation for 45min, and the binding reaction of the G-rich Sequence and the ThT is continued under the condition that CRISPR/Cas12a is inactive. Finally, the fluorescence intensity of the reaction solution at an excitation wavelength of 425nm, an emission wavelength of 490nm and a slit width of 5/10nm was measured for data analysis.
As a result, as shown in FIG. 4, the fluorescence intensity measured when DPA was added or DPA was present in the system was weak. When other interfering substances are added, the fluorescence intensity of the system is equivalent to that of a group without DPA (Blank), which shows that the method has good specificity for detecting the DPA.
Example 4 Cu-based 2+ Detection of DPA content in actual samples by DPA content detection methods of specific DNAzyme and CRISPR/Cas12a
1) Detecting DPA content in tap water:
in this example, the samples to be tested are running water samples (university of guangdong medical laboratory) containing different concentrations (100, 500,1000 nM) of DPA standard solution, according to example 1, the following procedure is followed:
filtering tap water sample with 0.22 μm microporous membrane, collecting 2 μl sample and 300nM Cu 2+ The solutions were mixed well and incubated at 25℃for 30min for chelation. At the same time, 1nM Cu_sub, 2nM Cu_Enz were incubated at 30℃for 30min to form Cu 2+ Specific DNAzyme. Mixing the above two solutions, adding 50 μm AA, and mixing to obtain Cu 2+ Is reduced to Cu + The reaction was carried out at 30℃for 80min to carry out a cleavage reaction with copper. 5nM Cas12a was incubated with 5nM crRNA for 30min at 37℃to form a Cas12a-crRNA complex. Subsequently, the pre-assembled Cas12a-crRNA complex, 10. Mu.L of 10 Xreaction Buffer, 150nM G-rich Sequence and 6. Mu.M ThT were added to the cleavage product, with a final volume of 200. Mu.L, incubated for 40min at 37℃to allow CRISPR/Cas12a to cleave the G-rich Sequence in trans. After the reaction is finished, the whole system is placed in ice water bath for continuous incubation for 45min, and the combination of G-rich Sequence and ThT is continuously carried out under the condition that CRISPR/Cas12a is inactiveAnd (3) reacting. Finally, the fluorescence intensity of the reaction solution at an excitation wavelength of 425nm, an emission wavelength of 490nm and a slit width of 5/10nm was measured for data analysis.
TABLE 1 results of measurement of DPA content in tap Water (mean.+ -. SD a ,n＝3)
Note that: a mean ± standard deviation, three sets of data were measured in parallel.
The results are shown in Table 1, and the recovery rate was measured to be between 96.9% and 99.7% by the addition of the standard recovery test according to the linear equation in example 2, and the overall recovery rate was good and the standard deviation was less than 8.52%. The method has better overall anti-interference capability in detecting the DPA concentration in the tap water sample.
2) Detecting DPA content in the juice:
in this example, the samples to be tested are juice samples containing different concentrations (100, 500,1000 nM) of DPA standard solution, according to example 1, the following procedure is followed:
the lime juice is first centrifuged at 10000rpm/min for 30min, then filtered with a 0.22 μm microporous filter membrane, and the supernatant is diluted 5-fold for later use. 2. Mu.L of the sample to be tested and 300nM Cu were taken 2+ The solutions were mixed well and incubated at 25℃for 30min for chelation. At the same time, 1nM Cu_sub, 2nM Cu_Enz were incubated at 30℃for 30min to form Cu 2+ Specific DNAzyme. Mixing the above two solutions, adding 50 μm AA, and mixing to obtain Cu 2+ Is reduced to Cu + The reaction was carried out at 30℃for 80min to carry out a cleavage reaction with copper. 5nM Cas12a was incubated with 5nM crRNA for 30min at 37℃to form a Cas12a-crRNA complex. Subsequently, the pre-assembled Cas12a-crRNA complex, 10. Mu.L of 10 Xreaction Buffer, 150nM G-rich Sequence and 6. Mu.M ThT were added to the cleavage product, with a final volume of 200. Mu.L, incubated for 40min at 37℃to allow CRISPR/Cas12a to cleave the G-rich Sequence in trans. After the reaction is finished, the whole system is placed in ice water bath for continuous incubation for 45min, and the combination reaction of G-rich Sequence and ThT is continuously carried out under the condition that CRISPR/Cas12a is inactive. Finally, the fluorescence intensity of the reaction solution at an excitation wavelength of 425nm, an emission wavelength of 490nm and a slit width of 5/10nm was measured for data analysis.
TABLE 2 detection of DPA content in fruit juices a (Mean±SD b ,n＝3)
Note that: a diluting the juice sample by 5 times;
note that: b mean ± standard deviation, three sets of data were measured in parallel.
The results are shown in Table 2, and the recovery rate is between 99.5% and 104.9% as measured by the standard recovery experiment according to the linear equation in example 2, and the overall recovery rate is good, with a standard deviation of less than 5.60%. The method has better overall anti-interference capability in detecting the DPA concentration in the juice sample.
In summary, it can be seen that the invention is based on Cu 2+ The dual signal amplification strategy of the specific DNAzyme and CRISPR/Cas12a uses G-quadruplex-ThT as a fluorescent probe to construct a label-free fluorescent biosensor which is used for analyzing and measuring the bacillus anthracis biomarker 2, 6-pyridine dicarboxylic acid (DPA), has the advantages of simple operation, high sensitivity, good selectivity and the like, and can be used for qualitative and quantitative detection of the DPA concentration in foods.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (10)
1. A bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a, which is characterized by comprising the following steps:
s1, DPA and Cu 2+ The solution is evenly mixed and then incubated, and DPA-Cu is formed through chelation reaction 2+ A chelate compound;
s2, shape of DNAzymeThe method comprises the following steps: mixing the enzyme chain (Cu_Enz) shown in SEQ ID No.1 and the substrate chain (Cu_sub) shown in SEQ ID No.2 in a reaction buffer solution, and incubating to form Cu 2+ A specific DNAzyme;
s3, DPA-Cu of the step S1 2+ Chelate and Cu of step S2 2+ Mixing the specific DNAzyme, and adding ascorbic acid to make Cu 2+ Is reduced to Cu + And Cu is obtained by copper enzyme catalytic cutting reaction 2+ Specific DNAzyme cleavage products;
s4, uniformly mixing the Cas12a with crRNA shown in SEQ ID No.3, and incubating to form a Cas12a-crRNA complex;
s5, cu in the step S3 2+ Uniformly mixing the specific DNAzyme cleavage product with the Cas12a-crRNA complex in the step S4, adding a cleavage buffer solution, and continuously incubating a guanine (G-rich) rich Sequence (G-rich Sequence) and thioflavin T (ThT) shown in SEQ ID No.4 to perform a CRISPR/Cas12a cleavage reaction and a binding reaction of the G-rich Sequence and the ThT; in the absence of DPA, cu 2+ Specific DNAzyme is activated to cleave cu_sub, cleaved cu_sub cannot bind to Cas12a-crRNA complex, cas12a protein cannot be activated, G-rich Sequence remains intact to form G-quadruplex-ThT complex with ThT, showing a distinct fluorescence intensity at 490 nm; after DPA addition, cu 2+ Chelation with DPA results in deactivation of DNAzyme, binding of intact cu_sub to Cas12a-crRNA complex, activation of trans cleavage activity of Cas12a protein, cleavage of G-rich Sequence, failure to form complex with ThT, and significant decrease in fluorescence intensity.
2. The method for detecting bacillus anthracis markers based on DNAzyme and CRISPR/Cas12a according to claim 1, wherein in step 1, cu 2+ The concentration is 200-400nM; the incubation temperature is 20-30 ℃ and the incubation time is 30-60min; DPA concentration is 0-3000nM.
3. The method for detecting bacillus anthracis markers based on DNAzyme and CRISPR/Cas12a according to claim 1, wherein in step 2, the incubation temperature is 25-35 ℃ for 30-60min; the concentration of the substrate strand is 0.2-1.8nM; the concentration of enzyme chains was 0.4-3.6nM and the reaction buffer contained 50mM HEPES,500mM NaCl,pH 7.4.
4. The method for detecting bacillus anthracis markers based on DNAzyme and CRISPR/Cas12a according to claim 1, wherein in step 3, the concentration of AA is 40-60 μΜ; the temperature of the copper enzyme catalytic cutting reaction is 25-35 ℃ and the time is 20-100min.
5. The method for detecting bacillus anthracis markers based on DNAzyme and CRISPR/Cas12a according to claim 1, wherein in the step 4, the concentration of the Cas12a and the concentration of the crRNA are both 4-10nM.
6. The method for detecting bacillus anthracis marker based on DNAzyme and CRISPR/Cas12a according to claim 1, wherein in step 5, the concentration of G-rich Sequence is 50-250nM; the cleavage Buffer solution was 10 Xreaction Buffer, which consisted of 100mM Tris-HCl,500mM NaCl,100mM MgCl 2 10mM DTT, pH 8.0; the concentration of ThT is 5-7 mu M; the incubation time of the CRISPR/Cas12a trans-cleavage reaction is 30-50min, and the temperature is 37 ℃; the incubation is continued in an ice-water bath for 40-60min.
7. Use of a DNAzyme and CRISPR/Cas12 a-based bacillus anthracis marker detection method according to any one of claims 1-6 in bacillus anthracis detection.
8. The use according to claim 7, wherein the limit of detection of DPA is 30.5nM.
9. The use according to claim 7, wherein the linear relationship between fluorescence intensity and DPA concentration in quantitative detection is f= 696.6-0.2944C, F means fluorescence intensity at 490nm, C means DPA concentration.
10. The use of claim 7, wherein the samples tested include tap water and juice.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310509369.4A CN116426611A (en) | 2023-05-08 | 2023-05-08 | Bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310509369.4A CN116426611A (en) | 2023-05-08 | 2023-05-08 | Bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116426611A true CN116426611A (en) | 2023-07-14 |
Family
ID=87079808
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310509369.4A Pending CN116426611A (en) | 2023-05-08 | 2023-05-08 | Bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116426611A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116732211A (en) * | 2023-08-09 | 2023-09-12 | 湖南工程学院 | Probe set and method for detecting mycobacterium bovis based on 8-17 deoxyribozyme and CRISPR-Cas13a trans-cleavage |
-
2023
- 2023-05-08 CN CN202310509369.4A patent/CN116426611A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116732211A (en) * | 2023-08-09 | 2023-09-12 | 湖南工程学院 | Probe set and method for detecting mycobacterium bovis based on 8-17 deoxyribozyme and CRISPR-Cas13a trans-cleavage |
| CN116732211B (en) * | 2023-08-09 | 2023-10-27 | 湖南工程学院 | Probe set and method for detecting mycobacterium bovis based on 8-17 deoxyribozyme and CRISPR-Cas13a trans-cleavage |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ma et al. | DNAzyme biosensors for the detection of pathogenic bacteria | |
| Tian et al. | A new label-free and turn-on strategy for endonuclease detection using a DNA–silver nanocluster probe | |
| L Neo et al. | G-quadruplex based probes for visual detection and sensing | |
| Zhu et al. | Avenues toward microRNA detection in vitro: a review of technical advances and challenges | |
| Xu et al. | Novel rolling circle amplification biosensors for food-borne microorganism detection | |
| Li et al. | High sensitive and label-free colorimetric DNA detection based on nicking endonuclease-assisted activation of DNAzymes | |
| TW201617357A (en) | Polynucleotide probe, method for detecting a target nucleic acid by using the same and kit comprising the same | |
| JPS5811194B2 (en) | Bilirubin-specific fungal enzyme composition, use thereof and method for producing the same | |
| CN116426611A (en) | Bacillus anthracis marker detection method based on DNAzyme and CRISPR/Cas12a | |
| Feng et al. | Label-free optical bifunctional oligonucleotide probe for homogeneous amplification detection of disease markers | |
| Li et al. | Electrochemiluminescence aptasensor based on cascading amplification of nicking endonuclease-assisted target recycling and rolling circle amplifications for mucin 1 detection | |
| Guo et al. | Label-free and enzyme-free sensitive fluorescent detection of human immunodeficiency virus deoxyribonucleic acid based on hybridization chain reaction | |
| Wang et al. | Construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background | |
| CN113640268B (en) | Tobramycin detection system and detection method based on CRISPR-Cas12a | |
| Chen et al. | Nucleic acid-aided molecular amplification techniques for food microorganism detection | |
| Song et al. | Label-free visual detection of nucleic acids in biological samples with single-base mismatch detection capability | |
| Xu et al. | Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene | |
| CN107083437B (en) | Method for simultaneously detecting multiple DNA glycosylases by using inherent fluorescent nucleotide ultrasensitiveness | |
| CN109402128A (en) | Aflatoxin B1Aptamer, the aflatoxin B containing the aptamer1Detection kit and detection method | |
| ZHANG et al. | Highly sensitive fluorescent aptasensor for thrombin detection based on competition triggered rolling circle amplification | |
| Zhou et al. | Strategy of functional nucleic acids-mediated isothermal amplification for detection of foodborne microbial contaminants: A review | |
| CN112080551A (en) | Ampicillin detection aptamer sensor for amplifying double-enzyme mediated cascade signal | |
| Zeng et al. | A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification | |
| JP2004515229A (en) | Methods of using fluorescent energy conversion probes to detect nucleic acid cleavage | |
| Li et al. | Robust and highly specific fluorescence sensing of Salmonella typhimurium based on dual-functional phi29 DNA polymerase-mediated isothermal circular strand displacement polymerization |
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 |