CN111751524A - Method for establishing antibiotic double detection sensor based on aptamer - Google Patents
Method for establishing antibiotic double detection sensor based on aptamer Download PDFInfo
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Abstract
An establishment method of an aptamer-based antibiotic double detection sensor belongs to the field of antibiotic and other substance detection. The invention establishes an aptamer sensor which can detect two antibiotics, namely chloramphenicol and kanamycin, reduces the experiment cost as much as possible, and realizes the rapid and high-sensitivity detection of antibiotic residues. The invention takes chloramphenicol and kanamycin aptamers as an example, an auxiliary short chain of biotin is modified at one end to form a partially complementary complex chain structure and is fixed on magnetic beads, after target substance antibiotics are added, the aptamers are separated from the magnetic beads into a solution through high affinity between the antibiotics and the aptamers, the content determination of the corresponding aptamers is realized by utilizing a real-time fluorescence quantitative PCR method, and an aptamer biological detector for jointly detecting the antibiotics is established. The invention quantitatively analyzes the content of the competitive free aptamer by a q-PCR method by virtue of the high specificity affinity action between the antibiotics and the aptamer, thereby respectively obtaining the content of the two antibiotics.
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 two antibiotics, namely chloramphenicol and kanamycin, by using a biosensor established based on an aptamer. Most of the sensors for detecting antibiotics using aptamers currently detect a single target substance. The method can provide a new idea for the establishment of double detection aptamer sensors or even multiple detection sensors.
Background
Chloramphenicol (CAP) is a broad-spectrum antibiotic with 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 currently recommends that chloramphenicol is prohibited from being used for eating animals, and the ministry of agriculture of the people's republic of china, publication No. 193, is already listed in the forbidden list. Because the chloramphenicol has low price and simple and easily obtained preparation, the establishment of a high-efficiency and rapid detection method for chloramphenicol is a very urgent and important task in order to prevent the harm to human health caused by the use of the chloramphenicol as a veterinary drug by illegal merchants. Kanamycin (Kanamycin) is an aminoglycoside antibiotic used primarily for aerobic gram-negative bacterial infections such as pseudomonas, acinetobacter and enterobacter. The kanamycin antibacterial mechanism is as follows: binds to the bacterial ribosome 30S subunit, inhibiting bacterial protein synthesis. However, kanamycin has some degree of nephrotoxicity, neurotoxicity and ototoxicity. The traditional detection methods for antibiotics comprise 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.
Aptamers (aptamers) are a class of artificially synthesized short-chain Ribonucleotides (RNAs) or single-chain deoxyribonucleotides (ssdnas) having specific binding targets, and have more obvious advantages than antibodies, such as wider target range, more convenient preparation, good chemical stability, easy modification, and the like. Therefore, the aptamer serving as a specific recognition element of the biosensor has great potential in the fields of food safety control, environmental monitoring, clinical diagnosis and treatment and the like.
As an important component of aptamer sensors, aptamers have high specificity and high affinity for a specific target substance. According to this feature, most of the existing aptamer-based biosensors use some kind of immobilization matrix as a carrier, and when the target substance exists, the aptamer can be competitively eluted from the immobilization matrix, thereby inducing a series of signal changes.
Most of the existing aptamer sensors can detect a single target substance, and the invention takes chloramphenicol and kanamycin aptamers as an example to establish a biosensor capable of detecting two antibiotics simultaneously, thereby realizing efficient and rapid antibiotic detection.
Disclosure of Invention
The invention aims to establish an aptamer sensor for simultaneously detecting chloramphenicol and kanamycin, reduce the detection cost and realize the rapid and high-sensitivity detection of antibiotic residues. The invention takes chloramphenicol aptamer and kanamycin aptamer as examples, two aptamer chains are fixed by an auxiliary short chain which can be complementarily paired with partial bases of the two aptamer chains, and one end of the complementary chain is modified by biotin to fix the complementary chain on streptavidin modified magnetic beads. When both antibiotics are present, the corresponding aptamers are competitively eluted due to their specific affinity interaction with the corresponding specific aptamers. Two groups of different upper and lower primers are used for respectively detecting the contents of two aptamers in the supernatant by a real-time fluorescent quantitative PCR method, and the content of the corresponding antibiotic is determined according to the amount of aptamer nucleic acid chains.
Chloramphenicol aptamer signal amplification sensor:
1. the establishment of antibiotic double detection sensor based on aptamer is characterized in that:
1) the following sequences, i.e. chloramphenicol and kanamycin aptamers, were synthesized:
chloramphenicol aptamer (Apt-CAP):
5’-AGCAGCACAGAGGTCAGATGACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAGCCTATGCGTGCTACCGTGAA-3’
kanamycin aptamer (Apt-KANA):
5’-CACCTAATACGACTCACTATAGCGGATCCGTGTCCAAGTGGTCTTGAGGTTCTGGCTCGAACAAGCTTGC-3’
2) establishment of Dual detection System
2.1 respectively putting 2 mu L of Apt-CAP, Apt-KANA and complementary strand C-DNA with the concentration of 1 mu M into a 200 mu L tubule, adding 34 mu L of F2 buffer solution, mixing uniformly, heating at 95 ℃ for 5min, putting on ice for 15min, finally putting in a 20 ℃ shaking table, and shaking for 1h at the rotating speed of 150 rpm;
2.2 vortex and mix the magnetic beads modified with streptavidin for 20s, take 75 μ L of magnetic beads in a centrifuge tube, separate the magnetic beads on a magnetic separation rack, and remove the supernatant. Adding F2 buffer solution, mixing, magnetically separating again, removing supernatant, and repeating the washing operation for 3 times. Finally, resuspending the suspension in F2 buffer solution for later use;
2.3 Add the solution of the complex strand from step 2.1.1 to the suspension of magnetic beads from the previous step and add F2 buffer to the final incubation system at 625. mu.L. Placing the mixture in a shaking table at 20 ℃ and shaking the mixture for 1h at 150 rpm. Then placing the mixture in a refrigerator at 4 ℃ for overnight;
2.4 put the small tubes in the above step on a magnetic separation rack and remove the supernatant. Adding 1 XB buffer solution, blowing, beating and mixing uniformly, then carrying out magnetic separation again, removing supernatant, and repeating the operation and washing for 3 times. Then 2 XB buffer is added for resuspension.
The above purpose is realized by the following technical scheme:
1. the following sequences, i.e. chloramphenicol and kanamycin aptamers, were synthesized:
chloramphenicol aptamer (Apt-CAP):
5’-AGCAGCACAGAGGTCAGATGACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAGCCTATGCGTGCTACCGTGAA-3’
kanamycin aptamer (Apt-KANA):
5’-CACCTAATACGACTCACTATAGCGGATCCGTGTCCAAGTGGTCTTGAGGTTCTGGCTCGAACAAGCTTGC-3’
Apt-CAP corresponds to the up-lead (C1): 5'-AGCAGCACAGAGGTCAGATG-3'
Apt-CAP corresponds to the drop down (C2): 5'-CCTATGCGTGCTACCGTGAA-3'
Apt-KANA corresponds to the up-lead (K1): 5'-CACCTAATACGACTCACTATA-3'
Apt-KANA corresponds to the drop (K2): 5'-CTGGCTCGAACAAGCTTGC-3'
2. Establishment of antibiotic double detection system
Buffers used for the experiments:
f2 buffer: 20mM of Tris (hydroxymethyl) aminomethane (Tris), 200mM of sodium chloride (NaCl), and disodium ethylenediaminetetraacetate (EDTA Na)2)1mM, 0.02% by mass of Triton X-100, and pH 7.8.
1 × B buffer Tris 50mM, magnesium chloride (MgCl)2)1mM, 200mM of sodium chloride (NaCl), 5mM of potassium chloride (KCl), 0.02 mass percent of Triton (Triton X-100) and pH 7.4.
2 × B buffer Tris 100mM, magnesium chloride (MgCl)2)2mM, 400mM of sodium chloride (NaCl), 10mM of potassium chloride (KCl), 0.02 mass percent of Triton (Triton X-100) and pH 7.4.
2.1 double-detector establishment basis and feasibility verification experiment
2.1.1 respectively simulating and predicting the secondary structures of two aptamers through mfold, determining the complementary region sequences of two aptamer chains Apt-CAP, Apt-KANA and a complementary chain C-DNA by consulting data, and determining the complementary chain C-DNA according to the complementary region sequences;
2.1.2 sequence alignment is carried out on the pair primers (C1/C2 and K1/K2) of the two aptamer chains respectively, and in order to avoid non-specific binding between the two groups, the specificity of the two groups of primers to the aptamer sequences in the mixed system is verified through design experiments.
2.1.3 prepare Apt-CAP, Apt-Kana and C-DNA nucleic acid chain solutions with the same concentration of 1nM as templates, respectively add two pairs of primers C1/C2 and K1/K2, and perform real-time fluorescence quantitative PCR experiment.
2.1.4, measuring the performance of the magnetic nanoparticles, taking a magnetic bead suspension, washing the magnetic bead suspension for three times by using an F2 buffer solution, sequentially adding C-DNA, Apt-CAP and Apt-KANA into the magnetic bead suspension, and comparing the content of the aptamer in a supernatant before and after the addition to determine the content of the loadable aptamer of the magnetic bead.
2.2 method for establishing detection System
2.2.1 respectively putting 2 μ L of Apt-CAP, Apt-KANA and complementary strand C-DNA with the concentration of 1 μ M into a 200 μ L tubule, adding 34 μ L of F2 buffer solution, mixing, heating at 95 ℃ for 5min, putting on ice for 15min, finally putting in a 20 ℃ shaking table, and shaking at 150rpm for 1 h;
2.2.2 vortex and mix the magnetic beads modified with streptavidin for 20s, take 75 μ L of magnetic beads into a centrifuge tube, separate the magnetic beads on a magnetic separation rack, and remove the supernatant. Adding F2 buffer solution, mixing, magnetically separating again, removing supernatant, and repeating the washing operation for 3 times. Finally, resuspending the suspension in F2 buffer solution for later use;
2.2.3 Add the solution of the complex strand from step 2.1.1 to the suspension of magnetic beads from the previous step and add F2 buffer to the final incubation system at 625. mu.L. Placing the mixture in a shaking table at 20 ℃ and shaking the mixture for 1h at 150 rpm. Then placing the mixture in a refrigerator at 4 ℃ for overnight;
2.2.4 Place the tubules from the previous step on a magnetic separation rack and remove the supernatant. Adding 1 XB buffer solution, blowing, beating and mixing uniformly, then carrying out magnetic separation again, removing supernatant, and repeating the operation and washing for 3 times. Then 2 XB buffer is added for resuspension.
2.2.5 put 10. mu.L of sterilized water, chloramphenicol and kanamycin solution into three 200. mu.L vials, and add 100. mu.L of the magnetic bead suspension from the above step and mix them well. Triplicate were made for each sample.
2.2.6 the sample is then placed in a shaker at 20 ℃ and shaken for 1h at 150 rpm. After the reaction, the suspension was placed on a magnetic separation rack and the supernatant carefully pipetted into small tubes for use.
2.2.7 taking supernatant to do real-time fluorescence quantitative PCR and analyzing the experimental result.
3. Determination of detector linear interval and detection limit
3.1 adding chloramphenicol and antibiotic with gradient final concentration into the reaction system as experimental group, and adding sterile water as blank group. And (3) taking the antibiotic concentration as an abscissa and the 2^ -delta Ct values of the experimental group and the blank group as an ordinate to draw a standard curve.
3.2 standard curve of chloramphenicol concentration and 2^ -delta Ct value is 0.0125X +1.0135, correlation coefficient R2Linear detection ranged from 0ng/ml to 20ng/ml 0.9867. The standard deviation SD of the fluorescence values of 10 groups was calculated from 10 blank groups without chloramphenicol, and the value calculated from 3SD/k was the lowest detection limit according to the standard curve formula y obtained above of 0.0125X + 1.0135. The standard deviation SD of the blank group is 0.047, and the standard curve is substituted to obtain the minimum detection limit of the built double detector to the chloramphenicol as 11.28 ng/ml.
The standard curve of the kanamycin concentration and the 2^ -delta Delta Ct value is that y is 0.0241X +0.9724, and the correlation coefficient R2Linear detection ranged from 0ng/ml to 20ng/ml 0.9812. The standard deviation SD of the fluorescence values of 10 groups was calculated from 10 groups of blank groups without chloramphenicol, and the value calculated from 3SD/k was the lowest detection limit according to the standard curve formula y obtained above of 0.0241X + 0.9724. The standard deviation SD of the blank group is 0.154, and the standard curve is substituted to obtain the minimum detection limit of the built double detector to the chloramphenicol, which is 19.17 ng/mL.
The invention has the advantages that:
(1) an aptamer sensor for simultaneously detecting two antibiotics is established
(2) When two antibiotics exist simultaneously, the common detection system can specifically identify the two antibiotics
(3) Micro detection of antibiotics by real-time fluorescent quantitative PCR method
Drawings
FIG. 1: secondary structure prediction map of aptamer strand and complementary strand for experiment
FIG. 2: experimental results of primer interference
FIG. 3: standard curve for chloramphenicol and kanamycin 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 aptamer and kanamycin aptamer
CAP-aptamer:
chloramphenicol aptamer (Apt-CAP):
5’-AGCAGCACAGAGGTCAGATGACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAGCCTATGCGTGCTACCGTGAA-3’
kanamycin aptamer (Apt-KANA):
5’-CACCTAATACGACTCACTATAGCGGATCCGTGTCCAAGTGGTCTTGAGGTTCTGGCTCGAACAAGCTTGC-3’
Apt-CAP corresponds to the up-lead (C1): 5'-AGCAGCACAGAGGTCAGATG-3'
Apt-CAP corresponds to the drop down (C2): 5'-CCTATGCGTGCTACCGTGAA-3'
Apt-Kana corresponds to the up-lead (K1): 5'-CACCTAATACGACTCACTATA-3'
Apt-Kana corresponds to the drop down (K2): 5'-CTGGCTCGAACAAGCTTGC-3'
Example 2: establishment of antibiotic double detection system
Buffers used for the experiments:
f1 buffer: 10mM of Tris (hydroxymethyl) aminomethane (Tris), 100mM of sodium chloride (NaCl), 1mM of disodium ethylenediaminetetraacetate (EDTA Na2), and pH 8.0.
F2 buffer: 20mM of Tris (hydroxymethyl) aminomethane (Tris), 200mM of sodium chloride (NaCl), 1mM of disodium ethylenediaminetetraacetate (EDTA Na2), 0.02% of Triton X-100 and pH 7.8.
1 XB buffer: tris 50mM, magnesium chloride (MgCl2)1mM, sodium chloride (NaCl)200mM, potassium chloride (KCl)5mM, Triton X-100 0.02% and pH 7.4.
2 × B buffer: tris 100mM, MgCl2 2mM, NaCl 400mM, KCl 10mM, Triton X-100 0.02% and pH 7.4.
2.1 double detector feasibility verification experiment
2.1.1 respectively simulating and predicting the secondary structures of two aptamers through mfold, determining the complementary region sequences of two aptamer chains Apt-CAP, Apt-KANA and a complementary chain C-DNA by consulting data, and determining the complementary chain C-DNA according to the complementary region sequences;
2.1.2 sequence alignment is carried out on the pair primers (C1/C2 and K1/K2) of the two aptamer chains respectively, and in order to avoid non-specific binding between the two groups, the specificity of the two groups of primers to the aptamer sequences in the mixed system is verified through design experiments.
2.1.3 preparing Apt-CAP, Apt-KANA and C-DNA nucleic acid chain solutions with the same concentration of 1nM as templates, adding two pairs of primers respectively, and performing real-time fluorescence quantitative PCR experiment, wherein the q-PCR experiment comprises 2 × SYBRMixture 10. mu.L, Forward Primer (10. mu.M) 0.5. mu.L, Reverse Primer (10. mu.M) 0.5. mu.L, Template 2. mu.L, ddH2O7. mu.L, wherein the qPCR program was set to 1) preheat at 95 ℃ for 1min, 2) cycle 30 × at 95 ℃ for 15s, 57 ℃ for 15s, 72 ℃ for 3)30s, dissolution curve Ct values obtained by exchanging primers for two sets of aptamers, with a difference of about 220Double, i.e. about 6 orders of magnitude, it can be seen that the effect of non-specific amplification in the two aptamer mixture systems is negligible.
2.1.4 measurement of magnetic nanoparticle Properties, i.e., measurement of the amount of aptamer that can be loaded on magnetic beads, 80. mu.L of magnetic beads were placed in a 200. mu.L vial and washed 3 times with F2 buffer for use. Respectively taking 10 mu M of complementary strand C-DNA, Apt-CAP and Apt-KANA, respectively, placing 10 mu L into 200 mu L small tubes, adding 50 mu L F2 buffer solution into each tube, mixing uniformly, heating at 95 ℃ for 5min, then carrying out ice bath for 15min, and respectively measuring the ssDNA content by using an ultra-micro ultraviolet-visible spectrophotometer.
2.1.5 the prepared magnetic beads are mixed with C-DNA, made up to 200. mu.L with F2 buffer, shaken for 30min at 150rpm in a shaker at 27 ℃. Placing the suspension on a magnetic separation frame, removing the supernatant, and measuring the content of unbound C-DNA by using an ultramicro ultraviolet-visible spectrophotometer. The effective binding amount of C-DNA under the experimental conditions was 13.68 ng/. mu.L of magnetic beads.
2.1.6 the magnetic beads from the previous step were washed 3 times with F2 buffer, resuspended in F2 buffer, and added with prepared Apt-CAP solution, made up to 200. mu.L with F2 buffer, and shaken for 50min at 150rpm in a shaker at 27 ℃. And placing the suspension on a magnetic separation frame, removing the supernatant, and measuring the content of the remaining Apt-CAP by using an ultramicro ultraviolet-visible spectrophotometer. The effective binding capacity of Apt-CAP under the experimental conditions was 3.8 ng/. mu.L of magnetic beads.
2.1.7 the same procedure as the previous step is used to replace Apt-CAP with Apt-KANA. The effective binding capacity of Apt-KANA was found to be 6.475 ng/. mu.L of magnetic beads under the experimental conditions.
2.2 method for establishing detection System
2.2.1 time and salinity are important factors influencing a detection system, and are optimized in turn. And (3) optimizing the reaction time after the antibiotics are added at three time points of 30min, 45min and 60 min. The results of the experiments show that the Δ Ct value obtained at 30min is better. The salt concentration in 1 XB buffer solution with different NaCl concentration of 100mM, 200mM and 300mM is respectively prepared for experiment, and 200mM NaCl is obtained as the best experimental condition.
2.2.2 respectively putting 2 μ L of Apt-CAP, Apt-KANA and complementary strand C-DNA with the concentration of 1 μ M into a 200 μ L tubule, adding 34 μ L of F2 buffer solution, mixing, heating at 95 ℃ for 5min, putting on ice for 15min, finally putting in a 20 ℃ shaking table, and shaking at 150rpm for 1 h;
2.2.3 vortex and mix the magnetic beads modified with streptavidin for 20s, take 75 μ L of magnetic beads into a centrifuge tube, separate the magnetic beads on a magnetic separation rack, and remove the supernatant. Adding F2 buffer solution, mixing, magnetically separating again, removing supernatant, and repeating the washing operation for 3 times. Finally, resuspending the suspension in F2 buffer solution for later use;
2.2.4 Add the solution of the complex strand from step 2.1.1 to the suspension of magnetic beads from the previous step and add F2 buffer to the final incubation system at 625. mu.L. Placing the mixture in a shaking table at 20 ℃ and shaking the mixture for 1h at 150 rpm. Then placing the mixture in a refrigerator at 4 ℃ for overnight;
2.2.5 Place the tubules from the previous step on a magnetic separation rack and remove the supernatant. Adding 1 XB buffer solution, blowing, beating and mixing uniformly, then carrying out magnetic separation again, removing supernatant, and repeating the operation and washing for 3 times. Then 2 XB buffer is added for resuspension.
2.2.6 put 10. mu.L of sterilized water, chloramphenicol and kanamycin solution into three 200. mu.L vials, and add 100. mu.L of the magnetic bead suspension from the above step and mix them well. Triplicate were made for each sample.
2.2.7 the sample is then placed in a 20 ℃ shaker and shaken for 1h at 150 rpm. After the reaction, the suspension was placed on a magnetic separation rack and the supernatant carefully pipetted into small tubes for use.
2.2.8 taking the supernatant to do real-time fluorescence quantitative PCR, analyzing the experimental result, wherein the q-PCR experiment comprises the following components of 2 × SYBR Mixture 10 mu L, Forward Primer (10 mu M)0.5 mu L, Reverse Primer (10 mu M)0.5 mu L, Template 2 mu L, ddH2O7. mu.L, wherein the qPCR program was set up 1) preheating at 95 ℃ for 1min, 2) cycling 30 × at 95 ℃ for 15s, 57 ℃ for 15s, 72 ℃ and 3)30s, dissolution curve.
EXAMPLE 3 determination of the Linear Range and detection Limit of the Detector
Chloramphenicol and antibiotics at final concentration were added to the reaction system as an experimental group, and sterile water was added as a blank group. And (3) taking the antibiotic concentration as an abscissa and the 2^ -delta Ct values of the experimental group and the blank group as an ordinate to draw a standard curve. The standard curve of the chloramphenicol concentration and the 2^ -delta Delta Ct value is that y is 0.0125X +1.0135, and the correlation coefficient R2Linear detection ranged from 0ng/ml to 20ng/ml 0.9867. The standard deviation SD of the fluorescence values of 10 groups was calculated from 10 blank groups without chloramphenicol, and the value calculated from 3SD/k was the lowest detection limit according to the standard curve formula y obtained above of 0.0125X + 1.0135. The standard deviation SD of the blank group is 0.047, and the standard curve is substituted to obtain the minimum detection limit of the built double detector to the chloramphenicol as 11.28 ng/ml.
The standard curve of the kanamycin concentration and the 2^ -delta Delta Ct value is that y is 0.0241X +0.9724, and the correlation coefficient R2Linear detection ranged from 0ng/ml to 20ng/ml 0.9812. The standard deviation SD of the fluorescence values of 10 groups was calculated from 10 groups of blank groups without chloramphenicol, and the value calculated from 3SD/k was the lowest detection limit according to the standard curve formula y obtained above of 0.0241X + 0.9724. The standard deviation SD of the blank group is 0.154, and the blank group is substituted into a standard curve to obtain the built double detector pairThe minimum detection limit of chloramphenicol was 19.17 ng/mL.
Sequence listing
<110> Beijing university of chemical industry
<120> establishment method of antibiotic double detection sensor based on aptamer
<141>2020-08-17
<150>2019110580881
<151>2019-11-01
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>80
<212>DNA
<213> Chloramphenicol aptamer Apt-CAP (2 Ambystoma laterale x Ambystomajeffersonanium)
<400>1
agcagcacag aggtcagatg acttcagtga gttgtcccac ggtcggcgag tcggtggtag 60
cctatgcgtg ctaccgtgaa 80
<210>2
<211>70
<212>DNA
<213> kanamycin aptamer Apt-KANA (2 Ambystoma laterale x Ambystomajeffersonanium)
<400>2
cacctaatac gactcactat agcggatccg tgtccaagtg gtcttgaggt tctggctcga 60
Claims (1)
1. An establishment method of an antibiotic double detection sensor based on an aptamer is characterized in that:
1) the following sequences, i.e. chloramphenicol and kanamycin aptamers, were synthesized:
chloramphenicol aptamer Apt-CAP:
5’-AGCAGCACAGAGGTCAGATGACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAGCCTATGCGTGCTACCGTGAA-3’
kanamycin aptamer Apt-KANA:
5’-CACCTAATACGACTCACTATAGCGGATCCGTGTCCAAGTGGTCTTGAGGTTCTGGCTCGAACAAGCTTGC-3’
2) establishment of Dual detection System
Buffer solution for experiments
F2 buffer: tris (hydroxymethyl) aminomethane (Tris 20 mM), NaCl (200 mM), disodium ethylenediamine tetraacetate (EDTANa)21mM, 0.02 percent of Triton X-100 by mass and pH 7.8;
1 × B buffer Tris 50mM Tris hydroxymethyl aminomethane, MgCl21mM, sodium chloride NaCl 200mM, potassium chloride KCl 5mM, Triton X-100 mass percent 0.02%, pH 7.4;
2 × B buffer solution of Tris 100mM, MgCl22mM, 400mM of sodium chloride NaCl, 10mM of potassium chloride KCl, 0.02 percent of Triton X-100 by mass and 7.4 of pH;
2.1 respectively taking 2 mu L of Apt-CAP, Apt-KANA and complementary strand C-DNA with the concentration of 1 mu M into a 200 mu L tubule, adding 34 mu L of F2 buffer solution, uniformly mixing, heating at 95 ℃ for 5min, placing on ice for 15min, finally placing in a 20 ℃ shaking table, shaking at the rotating speed of 150rpm for 1h, and preparing a composite strand solution;
2.2 uniformly mixing the magnetic beads of Purimag streptavidin on a Vortex mixer for 20s in a Vortex manner, taking 75 mu L of the magnetic beads in a centrifugal tube, separating the magnetic beads on a magnetic separation frame, and removing the supernatant; adding F2 buffer solution, blowing, beating and mixing uniformly, then carrying out magnetic separation again, removing supernatant, and repeating the operation and cleaning for 3 times; finally, resuspending the suspension in F2 buffer solution for later use;
2.3 adding the solution of the complex strand in the step 2.1 into the suspension of the magnetic beads in the above step, and adding F2 buffer solution until the final incubation system is 625 μ L; placing the mixture in a shaking table at 20 ℃, and shaking for 1h at 150 rpm; then placing the mixture in a refrigerator at 4 ℃ for overnight;
2.4 placing the centrifuge tube in the previous step on a magnetic separation frame, and removing the supernatant; adding 1 XB buffer solution, blowing, beating and uniformly mixing, then carrying out magnetic separation again, removing supernatant, and repeating the operation and cleaning for 3 times; then 2 XB buffer is added for resuspension.
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