CN110791551A - Method for establishing carrier-free chloramphenicol aptamer signal amplification sensor - Google Patents
Method for establishing carrier-free chloramphenicol aptamer signal amplification sensor Download PDFInfo
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
Abstract
An establishing method of a carrier-free chloramphenicol aptamer signal amplification sensor belongs to the field of detection of substances such as antibiotics. The invention aims to establish an aptamer sensor without a carrier, reduce the experiment cost as much as possible and realize the rapid and high-sensitivity detection of antibiotic residues. Taking chloramphenicol aptamer as an example, the invention realizes the release and cyclic amplification of a fluorescence signal by means of an auxiliary short chain with two ends respectively modified with quenching and fluorescent groups to form a complementary double chain, and adds exonuclease I (EXO I) to establish a carrier-free chloramphenicol aptamer signal amplification sensor. The invention decomposes the aptamer chain in the aptamer-target compound obtained by competitive affinity by means of the enzyme digestion activity of EXO I enzyme and releases the target, thereby leading the target to enter into a circulating reaction to obtain an amplified signal.
Description
Technical Field
The invention belongs to the field of detection of substances such as antibiotics and the like, and particularly relates to a method for detecting chloramphenicol by using an exonuclease I (EXO I) mediated signal amplification sensor in a carrier-free solution system. Most of the existing sensors for detecting chloramphenicol by using aptamers need to fix the aptamers by means of carriers such as graphene and nanogold. The method can provide a new idea for establishing the carrier-free aptamer sensor.
Background
Chloramphenicol (CAP) is a broad-spectrum antibiotic, is the first choice for typhoid fever and paratyphoid fever, is also used for treating anaerobic bacterial infection, and has very obvious effect and extremely strong bactericidal power. However, the chloramphenicol medication has serious adverse reactions: the food additive expert committee of the food and agricultural organization/world health organization of the united nations at present recommends that chloramphenicol is prohibited from being used for edible animals, the food and animal derived animals are prohibited from being used in China, and the ministry of agriculture of the people's republic of China bulletin No. 193 is listed in the forbidden list. However, because the method is low in price and simple and easy to obtain, in order to prevent the harm to human health caused by the fact that illegal merchants use the method as veterinary drugs, the establishment of a high-efficiency and rapid detection method for chloramphenicol is a very urgent and important task. The traditional detection methods of chloramphenicol include microbial detection, chromatographic detection, enzyme-linked immunosorbent assay and the like, but the detection methods have certain defects, such as low detection sensitivity or high instrument cost and the like.
An Aptamer (Aptamer) is a DNA or RNA nucleic acid chain capable of specifically recognizing and combining with a target, and has more obvious advantages than an antibody, such as wider target range, more convenient preparation, good chemical stability, easy modification and the like. Therefore, the aptamer is widely applied to biosensors for detecting drugs, toxins, pesticides, proteins, viruses, bacteria and the like, and is applied to the fields of food safety control, environmental monitoring, clinical diagnosis and treatment and the like.
An important component of aptamer sensors is the specific aptamer to a target, which has high properties, high affinity. The aptamer is competitively displaced from the auxiliary probe chain by utilizing the action of high affinity force of the aptamer and the target, so that a series of chemical, electrochemical, optical and other reactions are initiated, signal output related to the target concentration is generated, and a specific relation formula of the original target concentration and the output signal is established.
Most of the existing chloramphenicol sensors based on aptamers need carrier media such as magnetic nano microspheres, nano gold, gold electrodes and the like, and the aptamers need to be covalently connected with groups on corresponding carriers through auxiliary groups such as sulfydryl, biotin groups, amino groups and the like. The invention takes chloramphenicol aptamer as an example, establishes a carrier-free antibiotic biosensor for amplifying signals mediated by EXO I enzyme, and can realize simple and rapid detection.
Disclosure of Invention
The invention aims to establish an aptamer sensor without a carrier, reduce the experiment cost as much as possible and realize the rapid and high-sensitivity detection of antibiotic residues. Taking chloramphenicol aptamer as an example, the invention realizes the release and cyclic amplification of a fluorescence signal by means of an auxiliary short chain with two ends respectively modified with quenching and fluorescent groups to form a complementary double chain, and adds exonuclease I (EXO I) to establish a carrier-free chloramphenicol aptamer signal amplification sensor.
1. A method for establishing a carrier-free chloramphenicol aptamer signal amplification sensor is characterized by comprising the following steps:
1) the following sequences, i.e. chloramphenicol aptamers and helper probes, were synthesized:
CAP-aptamer:
5’-CAATAAGCGATGCGCCCTCGCCTGGGGGCCTAGTCCTCT-3’
probe Probe:
5’FAM-AGAGGACTAGGC-3’BHQ1
2) establishment of Chloramphenicol detection System
2.1.1B & W buffer solution or PBS buffer solution is selected as the environment buffer solution of the established system respectively, wherein the NaCl concentration in the buffer solution is 0.2M-0.8M, CAP-aptamer and auxiliary Probe Probe with the final molar concentration ratio of 0.8:1-1.2:1 are respectively added into a centrifuge tube, wherein the final concentration of the Probe is 0.25 mu M, the heating treatment is carried out for 5min at 95 ℃, and the centrifuge tube is placed at room temperature for cooling;
2.1.2 after cooling to room temperature, placing the centrifuge tube in a condition of 10-15 ℃ lower than the annealing temperature of the auxiliary probe chain, and performing shake incubation for 1h in an incubator at the rotating speed of 120 rpm;
2.1.3 adding chloramphenicol and EXO I enzyme with final concentration of 0.1-5ng/mL into the centrifuge tube after finishing the previous step, wherein the final concentration of the EXO I enzyme is 0.5U/μ L, placing the centrifuge tube in an incubator at 10 ℃ under the condition of 120rpm, shaking and incubating, and selecting incubation time of 20min-80 min;
2.1.4 after the steps are finished, placing the centrifuge tube in a water bath at 85 ℃, heating for 15min, and inactivating the EXO I enzyme.
Further optimization scheme:
2.1 adding CAP-aptamer with the molar concentration of 0.20 mu M and 0.25 mu M auxiliary Probe Probe into PBS buffer solution with the NaCl concentration of 0.4M in a centrifuge tube, heating at 95 ℃ for 5min, and placing at room temperature for cooling;
2.2 after cooling to room temperature, placing the centrifuge tube in an incubator with the optimal incubation temperature of 10 ℃ and shaking and incubating for 1h at the rotating speed of 120 rpm;
2.3 adding chloramphenicol with a final concentration of 0.1-5ng/mL and EXO I enzyme into the centrifuge tube after the last step, wherein the final concentration of the EXO I enzyme is 0.5U/μ L, and placing the centrifuge tube in an incubator with 10 ℃ and shaking and incubating the centrifuge tube for 70min at the condition of 120 rpm;
2.4 after the above steps, placing the centrifuge tube in a water bath at 85 ℃, heating for 15min, and inactivating the EXO I enzyme.
The above purpose is realized by the following technical scheme:
1. the following sequences, i.e., chloramphenicol aptamer and helper probe, were synthesized:
CAP-aptamer:
5’-CAATAAGCGATGCGCCCTCGCCTGGGGGCCTAGTCCTCT-3’
probe Probe:
5’FAM-AGAGGACTAGGC-3’BHQ1
2. establishment of chloramphenicol detection system
Buffers used for the experiments:
Tris-HCl buffer: tris 40mM, magnesium chloride (MgCl)2)67mM, Dithiothreitol (DTT)1mM, pH 9.5.
B&W buffer solution: 2M sodium chloride (NaCl), 10mM Tris, disodium EDTA (Na EDTA)2)1mM，pH 7.5。
PBS buffer: 137mM of sodium chloride (NaCl), 2.7mM of potassium chloride (KCl), and disodium hydrogen phosphate (Na)2HPO4)10mM, potassium dihydrogen phosphate (KH)2PO4)2mM，pH 7.4。
2.1 feasibility verification of Carrier-free detection System
2.1.1B & W buffer solution or PBS buffer solution is selected as the environment buffer solution of the established system respectively, wherein the NaCl concentration in the buffer solution is 0.2M-0.8M, CAP-aptamer and auxiliary Probe Probe with the final molar concentration ratio of 0.8:1-1.2:1 are respectively added into a centrifuge tube, wherein the final concentration of the Probe is 0.25 mu M, the heating treatment is carried out for 5min at 95 ℃, and the centrifuge tube is placed at room temperature for cooling;
2.1.2 after cooling to room temperature, placing the centrifuge tube in a condition of 10-15 ℃ lower than the annealing temperature of the auxiliary probe chain, and performing shake incubation for 1h in an incubator at the rotating speed of 120 rpm;
2.1.3 adding chloramphenicol and EXO I enzyme with final concentration of 0.1-5ng/mL into the centrifuge tube after finishing the previous step, wherein the final concentration of the EXO I enzyme is 0.5U/μ L, placing the centrifuge tube in an incubator at 10 ℃ under the condition of 120rpm, shaking and incubating, and selecting incubation time of 20min-80 min;
2.1.4 after the steps are finished, placing the centrifuge tube in a water bath at 85 ℃, heating for 15min, and inactivating the EXO I enzyme.
2.2 method for establishing detection System
2.2.1 adding CAP-aptamer with the molar concentration of 0.20 mu M and 0.25 mu M auxiliary Probe Probe into PBS buffer solution with the NaCl concentration of 0.4M in a centrifuge tube, heating at 95 ℃ for 5min, and cooling at room temperature;
2.2.2 after cooling to room temperature, placing the centrifuge tube in an incubator with the optimum incubation temperature of 10 ℃ and shaking and incubating for 1h at the rotating speed of 120 rpm;
2.2.3 adding chloramphenicol with a final concentration of 0.1-5ng/mL and EXO I enzyme into the centrifuge tube after finishing the previous step, wherein the final concentration of the EXO I enzyme is 0.5U/μ L, and placing the centrifuge tube in an incubator with a temperature of 10 ℃ and shaking and incubating the centrifuge tube for 70min at a speed of 120 rpm;
2.2.4 after the above steps, placing the centrifugal tube in a water bath at 85 ℃, heating for 15min, and inactivating the EXO I enzyme.
2.2.5 when the reaction solution in the above step is cooled to room temperature, 200. mu.L of the reaction solution is taken and transferred to a black 96-well plate, and the fluorescence value is measured by a multifunctional microplate reader.
3. Determination of a Linear Interval
Chloramphenicol at a final concentration of 0.1-5ng/mL was added to the reaction as an experimental group and sterile water was added as a blank group. And (3) taking the concentration of the chloramphenicol as an abscissa and the fluorescence difference value between the experimental group and the blank group as an ordinate to draw a standard curve. Chloromyces variotiiThe standard curve of the concentration of the element and the fluorescence value of △ is that y is 593.86x +851.55, and the correlation coefficient R2Linear detection ranged from 0.1ng/ml to 5ng/ml 0.9963.
4. Determination of detection Limit
Randomly selecting 10 blank groups without chloramphenicol, calculating the standard deviation SD of the fluorescence values of the 10 blank groups, and obtaining the value which is the lowest detection limit by calculating 3SD/k according to the standard curve formula y obtained in the previous step of 593.86x + 851.55. And (3) if the blank group standard deviation SD is 16.424, then 3SD is 49.272, the standard curve y is 593.86x +851.55, and the minimum detection limit of the built carrier-free aptamer detector to chloramphenicol is 0.083 ng/ml.
The invention has the advantages that:
(1) carrier-free aptamer sensor is established
(2) The aptamer free in the system can be fully contacted with the target so as to obtain larger signal value
(3) The aptamer chain in the aptamer-target compound obtained by competitive affinity is decomposed by means of the enzyme digestion activity of the EXO I enzyme, and the target is released, so that the target enters a circulating reaction to obtain an amplified signal.
Drawings
FIG. 1: fluorescence values of experimental groups at different incubation times
FIG. 2: fluorescence variation of system under different NaCl concentrations
FIG. 3: standard curve for chloramphenicol detection
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: synthesis of the following sequences, Chloramphenicol aptamers and helper probes shown
CAP-aptamer:
5’-CAATAAGCGATGCGCCCTCGCCTGGGGGCCTAGTCCTCT-3’
auxiliary Probe Probe:
5’FAM-AGAGGACTAGGC-3’BHQ1
example 2: establishment of chloramphenicol detection system
Reagents used for the experiment:
Tris-HCl buffer: tris 40mM, magnesium chloride (MgCl)2)67mM, Dithiothreitol (DTT)1mM, pH 9.5.
B&W buffer solution: 2M sodium chloride (NaCl), 10mM Tris, disodium EDTA (Na EDTA)2)1mM，pH 7.5。
PBS buffer: 137mM of sodium chloride (NaCl), 2.7mM of potassium chloride (KCl), and disodium hydrogen phosphate (Na)2HPO4)10mM, potassium dihydrogen phosphate (KH)2PO4)2mM，pH 7.4。
2.1 establishment of a Carrier-free detection System
2.1.1B & W buffer solution or PBS buffer solution is selected as the environment buffer solution of the established system respectively, wherein the NaCl concentration in the buffer solution is 0.2M-0.8M, CAP-aptamer and auxiliary Probe Probe with the final molar concentration ratio of 0.8:1-1.2:1 are respectively added into a centrifuge tube, wherein the final concentration of the Probe is 0.25 mu M, the heating treatment is carried out for 5min at 95 ℃, and the centrifuge tube is placed at room temperature for cooling;
2.1.2 after cooling to room temperature, placing the centrifuge tube in a condition of 10-15 ℃ lower than the annealing temperature of the auxiliary probe chain, and performing shake incubation for 1h in an incubator at the rotating speed of 120 rpm;
2.1.3 adding chloramphenicol and EXO I enzyme with final concentration of 0.1-5ng/mL into the centrifuge tube after finishing the previous step, wherein the final concentration of the EXO I enzyme is 0.5U/μ L, placing the centrifuge tube in an incubator at 10 ℃ under the condition of 120rpm, shaking and incubating, and selecting incubation time of 20min-80 min;
2.1.4 after the steps are finished, placing the centrifugal tube in a water bath at 85 ℃, heating for 15min, and inactivating the EXO I enzyme;
2.1.5 when the reaction solution in the above step is cooled to room temperature, 200. mu.L of the reaction solution is taken and transferred to a black 96-well plate, and the fluorescence value is measured by a multifunctional microplate reader.
2.2 Condition optimization of the detection System
2.2.1 selecting B&W buffer solution or PBS buffer solution is respectively used as environmental buffer solution of the system. When using PThe fluorescence range of the BS buffer is larger than that of B&The wide fluorescence range of the W buffer resulted in this result for two reasons, ① due to B&The interaction force between the chloramphenicol aptamer and the complementary strand is increased by the action of the W buffer solution, so that the chloramphenicol can not easily replace the complementary strand from the aptamer, and the ② EXO I enzyme needs Mg in the buffer solution2+And B is&EDTA in the W buffer is a chelating agent combined with divalent metal ions, which can lead to the inhibition of the EXO I enzyme, thereby leading to a smaller final fluorescence range of the system;
2.2.2 taking 0.2-0.8M of NaCl concentration in the buffer solution, and obtaining the best experimental result when the NaCl concentration in the buffer solution system is 0.4M by comparison;
2.2.3 the experiment is carried out under the condition that the final molar concentration ratio of CAP-aptamer to auxiliary Probe Probe is 0.8:1-1.2:1, and a higher fluorescence range can be obtained when the ratio of chloramphenicol aptamer to complementary strand is 0.8: 1;
2.2.4 incubation temperature affects not only the binding between aptamer strand and probe strand but also the efficiency and effect of enzyme digestion. The incubation temperature of 10-15 ℃ is selected for comparison in the experiment, and 10 ℃ is adopted as the incubation temperature according to the experiment result;
2.2.5 when the EXO I enzyme is added into the reaction system, multiple enzyme digestion reactions are involved, the incubation time has great influence on the experimental result, the incubation time of 20-80 min is selected in the experiment, and the optimal incubation time obtained in the experiment is 70 min.
Example 3: determination of a linear detection interval
Adding a chloramphenicol test group with final concentration of 0.1-5ng/ml and a sterile water blank group into a reaction system, drawing a standard curve by taking the chloramphenicol concentration as an abscissa and the fluorescence difference value between the test group and the blank group as an ordinate, wherein the standard curve of the chloramphenicol concentration and the △ fluorescence value is that y is 593.86x +851.55, and the correlation coefficient R is2Linear detection ranged from 0.1ng/ml to 5ng/ml 0.9963.
Example 4: determination of detection Limit
10 blank groups without chloramphenicol were randomly selected, and the standard deviation SD of fluorescence values of the 10 groups was calculated. The signal-to-noise ratio is 3:1, and a value calculated by 3SD/k is the lowest detection limit according to the obtained standard curve formula y of 593.86x + 851.55. And (3) if the blank group standard deviation SD is 16.424, then 3SD is 49.272, the standard curve y is 593.86x +851.55, and the minimum detection limit of the built carrier-free aptamer detector to chloramphenicol is 0.083 ng/ml.
Sequence listing
<110> Beijing university of chemical industry
<120> establishment method of carrier-free chloramphenicol aptamer signal amplification sensor
<141>2019-09-16
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>39
<212>DNA
<213> CAP-aptamer (2 Ambystoma laterale x Ambystoma jeffersonanum)
<400>1
caataagcga tgcgccctcg cctgggggcc tagtcctct 39
<210>2
<211>12
<212>DNA
<213> Probe (2 Ambystoma laterale x Ambystoma jeffersonanium)
<400>2
agaggactag gc 12
Claims (2)
1. A method for establishing a carrier-free chloramphenicol aptamer signal amplification sensor is characterized by comprising the following steps:
1) the following sequences, i.e. chloramphenicol aptamers and helper probes, were synthesized:
CAP-aptamer:
5’-CAATAAGCGATGCGCCCTCGCCTGGGGGCCTAGTCCTCT-3’
probe Probe:
5’FAM-AGAGGACTAGGC-3’BHQ1
2) establishment of Chloramphenicol detection System
2.1.1B & W buffer solution or PBS buffer solution is selected as the environment buffer solution of the established system respectively, wherein the NaCl concentration in the buffer solution is 0.2M-0.8M, CAP-aptamer and auxiliary Probe Probe with the final molar concentration ratio of 0.8:1-1.2:1 are respectively added into a centrifuge tube, wherein the final concentration of the Probe is 0.25 mu M, the heating treatment is carried out for 5min at 95 ℃, and the centrifuge tube is placed at room temperature for cooling;
2.1.2 after cooling to room temperature, placing the centrifuge tube in a condition of 10-15 ℃ lower than the annealing temperature of the auxiliary probe chain, and performing shake incubation for 1h in an incubator at the rotating speed of 120 rpm;
2.1.3 adding chloramphenicol and EXO I enzyme with final concentration of 0.1-5ng/mL into the centrifuge tube after finishing the previous step, wherein the final concentration of the EXO I enzyme is 0.5U/μ L, placing the centrifuge tube in an incubator at 10 ℃ under the condition of 120rpm, shaking and incubating, and selecting incubation time of 20min-80 min;
2.1.4 after the steps are finished, placing the centrifuge tube in a water bath at 85 ℃, heating for 15min, and inactivating the EXO I enzyme.
2. The method of claim 1, further comprising:
2.1 adding CAP-aptamer with the molar concentration of 0.20 mu M and 0.25 mu M auxiliary Probe Probe into PBS buffer solution with the NaCl concentration of 0.4M in a centrifuge tube, heating at 95 ℃ for 5min, and placing at room temperature for cooling;
2.2 after cooling to room temperature, placing the centrifuge tube in an incubator with the optimal incubation temperature of 10 ℃ and shaking and incubating for 1h at the rotating speed of 120 rpm;
2.3 adding chloramphenicol with a final concentration of 0.1-5ng/mL and EXO I enzyme into the centrifuge tube after the last step, wherein the final concentration of the EXO I enzyme is 0.5U/μ L, and placing the centrifuge tube in an incubator with 10 ℃ and shaking and incubating the centrifuge tube for 70min at the condition of 120 rpm;
2.4 after the above steps, placing the centrifuge tube in a water bath at 85 ℃, heating for 15min, and inactivating the EXO I enzyme.
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| CN111879738A (en) * | 2020-06-15 | 2020-11-03 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
| CN115389581A (en) * | 2022-04-17 | 2022-11-25 | 北京化工大学 | Method for establishing tetrahedral electrochemical aptamer sensor of targeted chloramphenicol |
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