CN114540503B - Tumor inhibition factor kit Let-7a detection kit based on strand displacement and enzyme-assisted circulation signal amplification and application method thereof - Google Patents

Tumor inhibition factor kit Let-7a detection kit based on strand displacement and enzyme-assisted circulation signal amplification and application method thereof Download PDF

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CN114540503B
CN114540503B CN202210404541.5A CN202210404541A CN114540503B CN 114540503 B CN114540503 B CN 114540503B CN 202210404541 A CN202210404541 A CN 202210404541A CN 114540503 B CN114540503 B CN 114540503B
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CN114540503A (en
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戴嘉文
闫雪春
王倩
曾小刚
黎泓波
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Jiangxi Normal University
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Abstract

The invention belongs to the field of biological analysis, and relates to a tumor suppressor kit Let-7a detection kit based on strand displacement and enzyme-assisted circulation signal amplification. In the presence of the target gene, the target gene is assisted to participate in circulation by a strand displacement reaction, a fluorescent group in a three-chain composite structure is far away from a quenching group, so that a fluorescent signal is released, a large number of A2 single chains are displaced, then the A2 single chains are hybridized with the DNA chains modified with the fluorescent group quenching agent in a DNA double-chain track Q+F through a foot-point-mediated displacement reaction to release FAM modified DNA chains with fluorescence on, and then the DNA chains modified with the fluorescent group quenching agent are hybridized with the A2 chains under the action of an added exonuclease, so that the A2 chains can be circularly regenerated from the concave 3 end of the DNA chains, and a strong fluorescent signal is generated. The fluorescence signal intensity has a definite relation with the target gene concentration, thereby realizing the sensitive detection of the tumor suppressor Let-7 a.

Description

Tumor inhibition factor kit Let-7a detection kit based on strand displacement and enzyme-assisted circulation signal amplification and application method thereof
Technical Field
The invention belongs to the field of biological analysis, and relates to a tumor inhibition factor Let-7a detection kit based on strand displacement and enzyme-assisted circulation signal amplification and a use method thereof.
Background
MicroRNA is a non-coding, small fragment (about 20-22 nucleotides in length), single-stranded RNA molecule that plays an important role in different physiological processes. Many evidences show that micrornas can inhibit gene expression as a novel gene regulatory factor, micrornas are involved in regulating more than 60% of the activity of protein-encoding genes in mammals, and alterations in MicroRNA expression are closely related to physiological dysfunction of humans, especially tumor processes. Micrornas have therefore been selected as biomarkers in the early diagnosis of many diseases, including various cancers. There are 12 members of the Let-7 miRNA family, of which Let-7a is one of the important tumor suppressors. Abnormal expression of let-7a will lead to a variety of cancers, the low expression of which is associated with poorly differentiated normal cells and can lead to the development of aggressive cancers such as prostate, stomach and cervical cancers. Accurate detection of the concentration of certain micrornas in humans will help us to further understand the pathogenesis of certain cancers.
At present, due to strong sequence homology of micrornas, low concentration and easy degradation, it is not easy to realize high-sensitivity detection of mirnas. The most common methods for detection of mirnas are Northern blot hybridization, RT-PCR and miRNA microarrays. These methods have been successfully applied to miRNA detection in clinical applications and other basic studies, but Northern blot hybridization has limited sensitivity, complex operation, need of radioisotope labeling, and serious environmental damage. Although the RT-PCR and chip detection methods have higher detection sensitivity, the production cost is high or the operation steps are complex. In recent years, isothermal amplification of nucleic acid has attracted research interest in the introduction of the isothermal amplification into miRNA detection due to the advantages of high amplification speed, capability of effectively improving the sensitivity and accuracy of a sensor and the like. Amplification methods include hybridization chain reactions, rolling circle amplification reactions, double-strand specific nuclease amplification, strand displacement amplification, and the like. While various signal amplification techniques have been developed for highly sensitive measurement of miRNAs in combination with various signal sensors, such as Fluorescence (FL), electrochemiluminescence (ECL), and surface enhanced raman scattering. Among these methods, electrochemical methods have the general disadvantages of long time consumption, high operation difficulty, poor reproducibility and the like; while the chemiluminescent method requires additional modification of the active substance and is technically costly. Fluorescence spectroscopy is widely used because of its advantages of simple testing operation, low cost, and the like. Therefore, the development of a fluorescent signal amplified nucleic acid biosensor for sensitively detecting low-abundance microRNA is still of great significance. Patent CN105274194a describes a method for detecting a tumor marker let7a based on a multiplex amplification technique. After hybridization with the target let7a using the bead-linked deoxyribozyme, the let7a can be cut off; the cut-off product is utilized to carry out the cycle of twice shearing polymerization, thus realizing the amplification of signals; the product after shearing polymerization is utilized to carry out loop-mediated amplification reaction, fluorescent reagent is inserted to carry out fluorescence detection, the concentration of let7a can be calculated, and the detection limit can reach 10 -18 mol/L. The patent achieves a lower background signal by magnetic bead recovery and synergy between the shearing and polymerase enzymesThe signal is circularly amplified under the action, and finally, the high-sensitivity detection of the biosensor is realized, but the problems of complex operation and compatibility of a buffer solution system caused by the use of multiple enzymes still exist. The method utilizes the shear characteristics of the entropy driven strand displacement reaction and the exonuclease, avoids the simultaneous use of multiple enzymes, is simple to operate, and can realize rapid and sensitive detection of targets.
Disclosure of Invention
In order to solve the problems, the invention provides a tumor suppressor kit Let-7a detection kit based on strand displacement and enzyme-assisted circulation signal amplification and a use method thereof.
The technical scheme of the invention is realized as follows:
the detection kit of the tumor suppressor kit Let-7a based on strand displacement and enzyme-assisted circulation signal amplification comprises an A1 chain, an A2 chain, an A3 chain, a Q chain, an F chain, an auxiliary probe, a buffer system and exonuclease EXO III.
The A1 chain sequence is 5'-CAGAGACTCTCTACGCT- (FAM) -ACTAGGACTCT-3', the A2 chain sequence is 5'-AGTAGGTTGTATAGTTTCCCTGCCATGGATCGACC-3', the A3 chain is 5'-BHQ-AGCGTAGAGAGTCTCTGGGGAAACTATACAACCTACTACCTCA-3', the Q chain sequence is 5'-TCCATGGCAGGGAAACTATACAT- (BHQ) -CCTACT-3', the F chain sequence is 5'-FAM-ATGTATAGTTTCCCTGCCATGGAGTATGA-3', and the auxiliary probe sequence is 5'-AGTAGGTTGTATAGTTTCCCCAGAGACTCTCTACGCTACGCAT-3'.
The Buffer system was 1 XNEB Buffer1 (10 mM Bis-Tris-Propane-HCl, 10mM MgCl) 2 1 mM DTT, pH=7), the concentration of exonuclease EXO III is 600U/mL.
The application method of the tumor inhibition factor Let-7a detection kit comprises the following steps:
(1) Preparing an A1 chain solution, an A2 chain solution and an A3 chain solution, uniformly mixing, and incubating to obtain a multifunctional three-chain composite substrate;
(2) Preparing a Q chain solution and an F chain solution, uniformly mixing and incubating to obtain a DNA double-chain Q+F;
(3) Mixing the multifunctional three-chain composite substrate in the step (1) with a liquid to be detected, sequentially adding an auxiliary probe and a buffer system, incubating, adding the DNA double-strand Q-F and the exonuclease EXO III in the step (2), incubating, heating to inactivate the exonuclease EXO III, and collecting fluorescent signals by using a fluorescence photometer;
(4) And (3) bringing the fluorescent signal in the step (3) into a standard curve to calculate the content of the tumor suppressor Let-7a in the solution to be detected.
In the step (1), the concentration of the A1 chain solution, the concentration of the A2 chain solution and the concentration of the A3 chain solution are all 10 mu M, and the volume ratio of the A1 chain solution to the A2 chain solution to the A3 chain solution is 1:1:1.
In the step (2), the concentrations of the Q chain solution and the F chain solution are 10 mu M, and the volume ratio of the Q chain solution to the F chain solution is 1:1.
The hatching conditions in the steps (1) and (2) are that hatching is 2 h in a metal bath at 37 ℃.
The concentration of the auxiliary probe in the step (3) is 10 mu M, DNA, the concentration of double-strand Q-F is 5 mu M, and the concentration of exonuclease EXO III is 600U/mL.
The excitation wavelength adopted in the fluorescent signal collection is 492-nm, the detection range of the emission wavelength is 490-650 nm, and the scanning speed is 1200nm/min; the specific conditions of incubation and heating are that incubation is carried out for 90min at 37 ℃, and then heating is carried out for 20 min at 75 ℃.
The standard curve in the step (4) is y=19463.8396x+109619.3636; r is R 2 =0.99041。
The invention has the following beneficial effects:
1. the technical principle of the detection method of the application is as follows: in the presence of the target gene, the target gene is assisted to participate in circulation by a strand displacement reaction, a fluorescent group in a three-chain composite structure is far away from a quenching group, so that a fluorescent signal is released, a large number of A2 single chains are displaced, then the A2 single chains are hybridized with the DNA chains modified with a fluorescent group quencher in a DNA double-chain track Q+F through a foot-point-mediated displacement reaction to release FAM modified DNA chains with fluorescence on, and then the DNA chains modified with the fluorescent group quencher are hybridized with the A2 chains under the action of added EXO III exonuclease, so that the recessed 3 end of the DNA chains can be hydrolyzed to circularly regenerate the A2 chains, and a strong fluorescent signal is generated. In the absence of the target gene, the strand displacement reaction cannot occur, and the fluorescence signal is low. The fluorescence signal intensity has a definite relation with the target gene concentration, thereby realizing the sensitive detection of the tumor suppressor Let-7 a.
2. The multiplex composite structure is used for replacing the DNA hairpin structure, so that the reversibility of each step of the signal amplification system is reduced, the simultaneous use of multiple enzymes is avoided by utilizing the shear characteristics of entropy-driven strand displacement reaction and exonuclease, the operation is simple, and the rapid and sensitive detection of a target can be realized.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a detection principle of the present application.
FIG. 2 is a fluorescence spectrum of detection Let-7a measured in example 1.
FIG. 3 shows the results of the standard sample test in example 1. A: example 1 response curve of fluorescence intensity change with detection target concentration, B: a log linear plot of fluorescence intensity change versus detection target concentration.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
Examples
The tumor suppressor kit Let-7a detection kit based on strand displacement and enzyme-assisted circulation signal amplification comprises an A1 chain, an A2 chain, an A3 chain, a Q chain, an F chain, an auxiliary probe, a buffer system and exonuclease EXO III.
The DNA sequences used were as follows:
Figure 4772DEST_PATH_IMAGE001
the Buffer system was 1 XNEB Buffer1 (10 mM Bis-Tris-Propane-HCl, 10mM MgCl) 2 1 mM DTT, pH=7), the concentration of exonuclease EXO III is 600U/mL.
The kit can construct a high-sensitivity biosensor:
(1) Before the experiment, 10 mu M DNA single-stranded A1, A2 and A3 chains with the concentration of 10 mu M and the same volume are incubated for 2 h in a 37 ℃ metal bath, so that the multifunctional three-chain composite substrate is obtained. A DNA double strand Q+F was obtained in the same manner as described above. The method comprises the steps of carrying out a first treatment on the surface of the
(2) mu.L of the multifunctional three-chain composite substrate in (1) was taken, and 2. Mu.L of 10 XNEB buffer1 (100 mM MgCl) 2 500 mM NaCl,10mM dithiothreitol, 100 mM Tris-HCl, pH=7.9) and 11.5. Mu.L of ultrapure water were mixed, and then 1. Mu.L of 10. Mu.M auxiliary probe and 1. Mu.L of a standard aqueous solution of let-7a at a concentration of 125 nM,50 nM,25 nM,10 nM,5 nM,1 nM,500 pM,250 pM,100 pM,50 pM were added, followed by incubation at 37℃for 1 h. Subsequently, 2. Mu.L of 5. Mu.M DNA double strand Q+F and 1. Mu.L of 600U/mL EXO III were added to the resulting reaction sample, and incubated at 37℃for 90min, followed by heating at 75℃for 20 min to inactivate the enzyme.
Next, the tumor suppressor Let-7a was detected:
(3) 180. Mu.L of ultrapure water was added to the reaction mixture obtained in the step (2), followed by fluorescence measurement; when the change of the fluorescent signal is detected, the excitation wavelength is 492 nm, the detection range of the emission wavelength is 490nm-650nm, and the scanning speed is 1200nm/min. The fluorescence intensity has a definite relation with the concentration of the target gene, thereby realizing the sensitive detection of the tumor suppressor Let-7 a;
(5) Drawing a standard curve according to the detected change value of the fluorescent signal and the concentration of the standard solution of the corresponding Let-7 a;
(6) The ultrapure water in (2) is replaced by 1% of human serum, four types of Let-7a to-be-detected liquids with different concentrations are prepared, the to-be-detected liquids are used for replacing standard solutions for detection, and the feasibility of the method in detecting target DNA in complex biological matrixes is examined according to the comparison of the detected fluorescent signals and standard curve signals.
FIG. 1 is a schematic diagram showing the detection process of a biological tumor suppressor gene Let-7a with strand displacement and enzyme-assisted double-order cycle signal amplification according to the present invention. In the presence of the target gene, the target gene is assisted to participate in circulation by a strand displacement reaction, a fluorescent group in a three-chain composite structure is far away from a quenching group, so that a fluorescent signal is released, a large number of A2 single chains are displaced, then the A2 single chains are hybridized with the DNA chains modified with a fluorescent group quencher in a DNA double-chain track Q+F through a foot-point-mediated displacement reaction to release FAM modified DNA chains with fluorescence on, and then the DNA chains modified with the fluorescent group quencher are hybridized with the A2 chains under the action of added EXO III exonuclease, so that the recessed 3 end of the DNA chains can be hydrolyzed to circularly regenerate the A2 chains, and a strong fluorescent signal is generated. In the absence of the target gene, the strand displacement reaction cannot occur, and the fluorescence signal is low. The fluorescence signal intensity has a definite relation with the target gene concentration, thereby realizing the sensitive detection of the tumor suppressor Let-7 a.
FIG. 2 is a fluorescence spectrum of the detection target gene Let-7 a. As can be seen from fig. 2, in the absence of the target gene, the fluorescence intensity is weak (curve a); when the target gene exists, a stronger fluorescent signal appears at 510nm (curve b), the fluorescence intensity is further enhanced after exoenzyme is added (curve c), and the peak height of the fluorescent signal is related to the concentration of the target gene ret-7 a.
FIG. 3A is a response curve of the fluorescence intensity change obtained by the standard sample detection according to example 1, corresponding to the concentration of the detection target. y=19463.8396x+109619.3636; r is R 2 = 0.99041. In the target gene concentration range of 50 pM to 125 nM, there is a good linear relationship between fluorescence intensity and the logarithm of target gene concentration, and the detection limit is as low as 16.7 pM (fig. 3B).
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
<110> university of Jiangxi teachers
<120> tumor suppressor kit Let-7a detection kit based on strand displacement and enzyme-assisted circulation signal amplification and use thereof
Method of use
<130> 202204-0471
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 28
<212> DNA
<213> Unknown
<220>
<223> Artificial sequence
<400> 1
cagagactct ctacgctact aggactct 28
<210> 2
<211> 35
<212> DNA
<213> Unknown
<220>
<223> Artificial sequence
<400> 2
agtaggttgt atagtttccc tgccatggat cgacc 35
<210> 3
<211> 43
<212> DNA
<213> Unknown
<220>
<223> Artificial sequence
<400> 3
agcgtagaga gtctctgggg aaactataca acctactacc tca 43
<210> 4
<211> 22
<212> DNA
<213> humo spaiens
<400> 4
tgaggtagta ggttgtatag tt 22
<210> 5
<211> 29
<212> DNA
<213> Unknown
<220>
<223> Artificial sequence
<400> 5
tccatggcag ggaaactata catcctact 29
<210> 6
<211> 29
<212> DNA
<213> Unknown
<220>
<223> Artificial sequence
<400> 6
atgtatagtt tccctgccat ggagtatga 29
<210> 7
<211> 43
<212> DNA
<213> Unknown
<220>
<223> Artificial sequence
<400> 7
agtaggttgt atagtttccc cagagactct ctacgctacg cat 43

Claims (2)

1. The tumor suppressor kit Let-7a detection kit based on strand displacement and enzyme-assisted circulation signal amplification is characterized in that: comprises an A1 chain, an A2 chain, an A3 chain, a Q chain, an F chain, an auxiliary probe, a buffer system and an exonuclease EXO III;
the A1 chain sequence is 5'-CAGAGACTCTCTACGCT- (FAM) -ACTAGGACTCT-3', the A2 chain sequence is 5'-AGTAGGTTGTATAGTTTCCCTGCCATGGATCGACC-3', the A3 chain is 5'-BHQ-AGCGTAGAGAGTCTCTGGGGAAACTATACAACCTACTACCTCA-3', the Q chain sequence is 5'-TCCATGGCAGGGAAACTATACAT- (BHQ) -CCTACT-3', the F chain sequence is 5'-FAM-ATGTATAGTTTCCCTGCCATGGAGTATGA-3', and the auxiliary probe sequence is 5'-AGTAGGTTGTATAGTTTCCCCAGAGACTCTCTACGCTACGCAT-3'.
2. The tumor suppressor kit of claim 1, wherein: the Buffer system is 1 XNEB Buffer1 with pH=7, which contains 10mM Bis-Tris-Propane-HCl and 10mM MgCl 2 And 1 mM DTT.
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US8206904B2 (en) * 2002-12-18 2012-06-26 Third Wave Technologies, Inc. Detection of nucleic acids
CN105274194A (en) * 2014-12-10 2016-01-27 临沂大学 Detection method for tumor marker let7a based on multiple amplification technology
CN104894260A (en) * 2015-06-01 2015-09-09 清华大学 DNA (deoxyribonucleic acid) nano machine for determining miRNA (microribonucleic acid), and establishment and determination method thereof
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