LU505139B1 - Fluorescent biosensor for detecting tetracycline based on crispr-cas system - Google Patents
Fluorescent biosensor for detecting tetracycline based on crispr-cas system Download PDFInfo
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- 239000004098 Tetracycline Substances 0.000 title claims abstract description 46
- 229960002180 tetracycline Drugs 0.000 title claims abstract description 46
- 229930101283 tetracycline Natural products 0.000 title claims abstract description 46
- 235000019364 tetracycline Nutrition 0.000 title claims abstract description 46
- 150000003522 tetracyclines Chemical class 0.000 title claims abstract description 45
- 108091023037 Aptamer Proteins 0.000 claims abstract description 30
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 239000000523 sample Substances 0.000 claims description 29
- 150000001875 compounds Chemical class 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 239000003292 glue Substances 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000011218 binary composite Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000002105 nanoparticle Substances 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
- 239000002245 particle Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- OFVLGDICTFRJMM-WESIUVDSSA-N tetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O OFVLGDICTFRJMM-WESIUVDSSA-N 0.000 description 1
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention relates to a fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system, belonging to the technical field of biosensors. The fluorescent biosensor comprises tetracycline-specific aptamer aptamer76 chain, trigger chain, Cas14a protein, sgRNA and FQ chain. The detection range of the biosensor is as wide as 50 μg/ml to 50 fg/ml, the detection time is short, the experimental results are stable, and it is portable and suitable for on-site instant detection.
Description
DESCRIPTION 17509139
FLUORESCENT BIOSENSOR FOR DETECTING TETRACYCLINE
BASED ON CRISPR-CAS SYSTEM
The invention belongs to the technical field of biosensors, in particular to a fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system.
According to the national food safety standard (GB 31650-2019), the allowable residue of tetracycline in milk is 100 ug kg", and that in honey is 20 pg-kg! At present, traditional methods for detecting tetracycline residues include liquid chromatography and liquid chromatography-tandem mass spectrometry. The traditional detection method needs large instruments and takes a long time, and the detection line is high. Biosensor methods mainly include fluorescence aptamer sensor, colorimetric aptamer sensor and so on. The fluorescent dye of fluorescent aptamer sensor has poor photobleaching resistance, and its fluorescence performance is easily affected by external factors. The gold nanoparticles of colorimetric aptamer sensor are easily affected by salt particles, and the phenomenon of non-specific aggregation and discoloration shows the unstable experimental results. In the prior art, such as high performance liquid chromatography, the detection limit of tetracycline is 0.125 mg/kg, and the relative standard deviation is within 5%; under the optimal conditions, the detection linear range of tetracycline was 0.01-100 ng/ml, and the detection limit (LOD) was 0.0062 ng/ml.
In the detection of tetracycline, there is an urgent need for a biosensor with wide detection range, short detection time, portable instrument and stable experimental results.
SUMMARY LU505139
The purpose of the present invention is to provide a fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system, which has wide detection range (50 pg/ml - 50 fg/ml), short detection time, portable instrument, and stable experimental results.
In order to achieve the above purpose, the invention adopts the following technical scheme.
A fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system comprises: tetracycline-specific aptamer 76 chain, trigger chain, Casl4a protein, sgRNA and FQ chain; the base sequence of the aptamer 76 chain is: 5S’-CGTACGGAATTCGCTAGCCCCCCGGCAGGCCACGGCTTGGGTTGGT
CCCACTGCGCGTGGATCCGAGCTCCACGTG-3’ (SEQ ID NO: 1); the base sequence of the trigger chain is: 5’-CACGTGGAGCTCGAATTCCGTACG-3’ (SEQ ID NO: 2), the base sequence of the sgRNA is:
S'-UUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUU
AGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUG
UCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUG
AAAGAAUGAAGGAAUGCAAC CGU ACG GAA UUC GCU AGU CCG AGC
UCC ACG UG-3' (SEQ ID NO: 3); the base sequence of the FQ chain is:
S’-FAM-TTTTTTTTTTTT-BHQ1-3° (SEQ ID NO: 4).
A method for preparing a fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system comprises: (1) constructing a binary compound probe: the binary compound probe is formed by hybridizing an aptamer76 chain with a trigger chain; (2) tetracycline reacts with binary compound probe: evenly mixing and incubating the binary compound probe constructed in step (1) and tetracycline;
(3) homogeneous reaction: adding the mixed solution of Cas14a protein, sgRNA, 17505158
FQ chain, tetracycline and binary complex probe into homogeneous phase, and incubating after uniform mixing; (4) detecting the chemiluminescence intensity by fluorescence instrument.
In the preparation method of the fluorescent biosensor, the concentration ratio of aptamer 76 chain to trigger chain in step (1) is 1.5:1.
An application of the fluorescent biosensor for detecting tetracycline based on
CRISPR-Cas system in detecting tetracycline.
The technical principle of the fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system is as follows:
CRISPR-Cas14a can recognize ssDNA and activate the nonspecific cleavage activity of Casl4a. The ssDNA identified in this invention is trigger chain, and the cut single chain is FQ chain; aptamer76 chain is an aptamer that can specifically recognize tetracycline. The trigger chain is ssDNA that binds to aptamer76 chain according to the principle of base complementary pairing. When tetracycline (TC) exists, tetracycline specifically binds to aptamer 76 chain, which leads to the release of trigger chain. sgRNA can recognize trigger, which leads to the activation of non-specific single-strand cleavage activity of Casl4a, and the FQ chain is cleaved at 483 nm-525 nm.
The invention has the advantages that:
The invention provides a sensor for detecting tetracycline based on CRISPR-Cas system. The biosensor is used for detecting tetracycline, with a wide detection range of 50 pg/ml - 50 fg/ml, short detection time, portable instrument and stable experimental results.
Fig. 1 is a technical roadmap of the present invention.
Fig. 2 shows the determination results of tetracycline samples (positive samples).
The left picture shows the graph of tetracycline sample (positive sample) determined by real-time fluorescence PCR instrument, and the right picture shows the picture 17505158 taken by this sample under blue light glue cutter.
Fig. 3 is a graph showing the measurement results of UP water as a sample (negative sample). The left picture shows a graph with UP water as a sample (negative sample) determined by real-time fluorescence PCR, and the right picture shows a picture taken by this sample under a blue light glue cutter.
Fig. 4 1s a nucleic acid electrophoresis diagram of aptamer76 chain combined with trigger chain in different proportions. 1-6 lanes correspond to different concentration ratios numbered 1-6 in Table 1.
Fig. 5 shows the determination results of standard tetracycline aqueous solution with different concentrations. The above picture is taken under the blue light glue cutter; The picture below shows the picture processed by Image J after shooting under the blue light glue cutter. From left to right: eleven samples correspond to different samples in Table 2, 1-11.
Fig. 6 is a bar chart showing the fluorescence endpoint values of standard tetracycline aqueous solutions with different concentrations after 2 hours.
The technical scheme of the present invention will be further explained by specific examples. The present invention is not limited to the following examples. The raw materials used in the invention can be purchased in the market or can be synthesized by methods known in the field.
Embodiment 1
A fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system comprises: tetracycline-specific aptamer 76 chain, trigger chain, Casl4a protein, sgRNA and FQ chain; the base sequence of the aptamer 76 chain is: 5S’-CGTACGGAATTCGCTAGCCCCCCGGCAGGCCACGGCTTGGGTTGGT
CCCACTGCGCGTGGATCCGAGCTCCACGTG-3’ (SEQ ID NO: 1); the base sequence of the trigger chain is:
5’-CACGTGGAGCTCGAATTCCGTACG-3’ (SEQ ID NO: 2), 17905158 the base sequence of the sgRNA 1s:
S'-UUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUU
AGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUG
5 UCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUG
AAAGAAUGAAGGAAUGCAAC CGU ACG GAA UUC GCU AGU CCG AGC
UCC ACG UG-3' (SEQ ID NO: 3); the base sequence of the FQ chain is:
S’-FAM-TTTTTTTTTTTT-BHQ1-3° (SEQ ID NO: 4).
Embodiment 2 Study on the Optimum Combination Ratio of Aptamer 76 and trigger
Step 1: Mix aptamer 76 (10 uM) and trigger (10 uM) according to different concentration ratios (see Table 1), then add 8 ul of 10xBuffer A (NaCl 1 M, Tris 0.5
M, MgCl,-6H,O 20 mm, CaCl, 10 mm, KCI 5 mm) to mix. UP water (18.25 Qu) is replenished to 80 ul at 90°C for 10 min, and then it is slowly cooled for 30 min in a 100 ml beaker filled with boiling water, so that aptamer 76 can fully hybridize with trigger to form aptamer 76-trigger binary compound probe;
Step 2: add 5 pg/ml standard tetracycline aqueous solution and aptamer 76-trigger binary complex probe according to the ratio of 1:1, and react at 37°C for 30 min:
Step 3: Casl4a protein (0.4 uM), sgRNA (0.1 uM), FQ (0.8 uM), buffer B, 5 ul of the reaction solution in Step 2 and DEPC water were replenished to 25 ul and reacted for 2 h at 37°C;
Step 4: Observe the real-time fluorescence change within 2 hours with real-time fluorescence quantitative PCR instrument, take photos with blue-light glue cutter, and process with Image J, so as to obtain a clearer and more intuitive experimental result.
Fig. 2 is a graph showing the determination results of tetracycline samples (positive samples); the picture on the left is a graph of tetracycline samples (positive samples) determined by real-time fluorescence PCR, and the picture on the right is taken by a blue light glue cutter. Fig. 3 is a graph showing the measurement results of
UP water as a sample (negative sample); the picture on the left is a graph with UP 17505158 water as a sample (negative sample) determined by real-time fluorescence PCR, and the picture on the right 1s taken by a blue light glue cutter. Fig. 4 verifies the optimal binding ratio of aptamer 76 to trigger by 15% nucleic acid PAGE electrophoresis, and the trigger is completely blocked when aptamer 76: trigger = 1.5: 1.
Embodiment 3 Detection Sensitivity Test
Step 1: mix aptamer 76 (6 ul, 10 uM) and trigger (4 ul, 10 uM) (that is, the concentration ratio of aptamer 76 to trigger is 1.5:1), and add 8 ul of 10xbuffer A (NaCl 1 M, Tris 0.5 M, MgCl, 6H,0 20 mM, CaCl, 10 mM, KCI 5 mM), UP water (18.25 Qu) make up to 80 pl, then put it in a 100 ml beaker filled with boiling water and slowly cool it for 30 min, so that aptamer 76 can fully hybridize with trigger to form aptamer 76-trigger binary compound probe;
Step 2: add standard tetracycline aqueous solution with different concentrations (Table 2) and aptamer76-trigger binary complex probe according to the ratio of 1:1, and react at 37°C for 30 min;
Step 3: Casl4a protein (0.4 uM), sgRNA (0.1 uM), FQ(0.8 uM), buffer B, 5 ul of reaction solution in Step 2 and DEPC water were replenished to 25 ul and reacted for 2 hat 37°C;
Step 4: Observe the real-time fluorescence change within 2 hours with real-time fluorescence quantitative PCR instrument, take photos with blue-light glue cutter, and process with Image J, so as to obtain a clearer and more intuitive experimental result.
Fig. 5 shows the gray scale image taken by step4 blue light glue cutter, and the eleventh sample 1s the control sample. After being taken by blue light glue cutter and processed by Image J, it can be clearly seen that samples from 50 pg/ml to 50 fg/ml can be detected.
Fig. 6 is a histogram of the fluorescence endpoint value made by step 3 after setting the parameters on the real-time fluorescence PCR instrument to react at 37°C for 2 hours, and shooting fluorescence once per minute. From this graph, it can be known that the detection range of this biosensor is between 50 pg/ml and 50 fg/ml, and it shows high sensitivity.
Embodiment 4 Method for preparing a fluorescent biosensor for detecting 17505158 tetracycline based on CRISPR-Cas system, comprising:
Step 1: add aptamer 76 (6 ul, 10 uM), trigger (4 ul, 10 uM), 10xbuffer A (8 ul,
NaCl 1m, Tris 0.5 M, MgCl, 6H,O 20mM, CaCl, 10 mM, KCI 5 mM) UP water (18.25 Qu) was added to 80 ul, and then it was put in a 100 ml beaker filled with boiling water and cooled slowly for 30 min, so that aptamer76 could fully hybridize with trigger to form aptamer 76-trigger binary composite probe;
Step 2: add standard tetracycline aqueous solution and aptamer 76-trigger binary compound probe according to the ratio of 1:1, and react at 37°C for 30 min;
Step 3: Casl4a protein (0.4 uM), sgRNA (0.1 uM), FQ(0.8 uM), buffer B, 5 ul of the reaction solution in Step 2 and DEPC water were replenished to 25 ul and reacted for 2 h at 37°C:
Step 4: observe the real-time fluorescence change within 2 hours with real-time fluorescence quantitative PCR instrument, take photos with blue-light glue cutter, and process with Image J, so as to obtain a clearer and more intuitive experimental result.
Claims (4)
1. A fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system, comprising: tetracycline-specific aptamer 76 chain, trigger chain, Casl4a protein, sgRNA and FQ chain; the base sequence of the aptamer 76 chain is: 5S’-CGTACGGAATTCGCTAGCCCCCCGGCAGGCCACGGCTTGGGTTGGT CCCACTGCGCGTGGATCCGAGCTCCACGTG-3’ (SEQ ID NO: 1); the base sequence of the trigger chain is: 5’-CACGTGGAGCTCGAATTCCGTACG-3’ (SEQ ID NO: 2), the base sequence of the sgRNA is: S'-UUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUU AGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUG UCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUG AAAGAAUGAAGGAAUGCAAC CGU ACG GAA UUC GCU AGU CCG AGC UCC ACG UG-3' (SEQ ID NO: 3); the base sequence of the FQ chain is: 5’-FAM-TTTTTTTTTTTT-BHQ1-3’ (SEQ ID NO: 4).
2. A method for preparing a fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system according to claim 1, comprising: (1) constructing a binary compound probe: the binary compound probe is formed by hybridizing an aptamer 76 chain with a trigger chain; (2) tetracycline reacts with binary compound probe: evenly mixing and incubating the binary compound probe constructed in step (1) and tetracycline; (3) homogeneous reaction: adding the mixed solution of Cas14a protein, sgRNA, FQ chain, tetracycline and binary complex probe into homogeneous phase, and incubating after uniform mixing; (4) detecting the chemiluminescence intensity by fluorescence instrument.
3. The preparation method according to claim 2, wherein the concentration ratio of aptamer 76 chain to trigger chain in step (1) is 1.5:1.
. . . LU505139
4. Use of the fluorescent biosensor for detecting tetracycline based on CRISPR-Cas system according to claim 1 in detecting tetracycline.
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| Application Number | Priority Date | Filing Date | Title |
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| LU505139A LU505139B1 (en) | 2023-09-21 | 2023-09-21 | Fluorescent biosensor for detecting tetracycline based on crispr-cas system |
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| LU505139A LU505139B1 (en) | 2023-09-21 | 2023-09-21 | Fluorescent biosensor for detecting tetracycline based on crispr-cas system |
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