CN116732146A - Fluorescent biosensor for label-free detection of miR-21 based on strand displacement amplification and primer exchange reaction - Google Patents
Fluorescent biosensor for label-free detection of miR-21 based on strand displacement amplification and primer exchange reaction Download PDFInfo
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Abstract
Disclosed herein is a method for detection of miR-21 based on Strand Displacement Amplification (SDA) and Primer Exchange Reaction (PER) cascade amplification. The biosensor comprises SDA template H1, PER template H2, miR-21, bstDNA polymerase, nb.BbvCI endonuclease and thioflavin T (ThT). MiR-21 can be combined with H1, and can be extended forwards under the action of BstDNA polymerase, nb.BbvCI endonuclease specifically recognizes a cutting site to generate a large number of single-stranded DNA, the single-stranded DNA can trigger a Primer Exchange Reaction (PER) to finally generate a large number of G-quadruplex fragments with different lengths, and under the potassium ion environment, thT and the G-quadruplex are combined to generate a strong fluorescent signal, so that the detection of miR-21 is realized. The biosensor can detect miR-21 under isothermal conditions, is simple to operate, and can realize quantitative detection of miR-21 in 90 minutes. The invention has potential application value in early diagnosis of cancer, research and development of anticancer targeted drugs and the like.
Description
Technical Field
The invention belongs to a biochemical analysis method, and particularly relates to a fluorescence biosensor for detecting miR-21 without labels based on cascade amplification of strand displacement amplification and primer exchange reaction.
MicroRNAs (miRNAs) is an endogenous small RNA molecule, and is involved in the regulation of gene expression after transcription, and abnormal expression of miRNAs is closely related to occurrence of human cancers, tumor staging and tumor treatment. Thus, miRNAs are considered as a potential biomarker, and quantitative analysis of expression of miRNAs enables better understanding of their role in cancer, thereby enabling early diagnosis of cancer and development of anticancer targeted drugs. However, due to the high sequence homology among the members of the miRNAs family, the low abundance of the miRNAs in the total RNA sample, and the easy degradation, the detection of the miRNAs is very difficult. Currently common methods for detecting miRNAs are mainly real-time polymerase chain reaction (qPCR), microarray and Northern blotting. Although these methods can effectively detect miRNAs, they have certain limitations such as complex primer design and high cost. More and more detection methods adopt isothermal amplification methods to replace the traditional detection methods, the developed isothermal amplification technology often has the defects of non-ideal sensitivity, complex nucleic acid sequence design, complex operation and the like, and the Strand Displacement Amplification (SDA) and the Primer Exchange Reaction (PER) only need one template and one primer, so the system is simple.
Disclosure of Invention
In view of the above, the invention simultaneously carries out target recognition, strand Displacement Amplification (SDA) and Primer Exchange Reaction (PER) cascade signal amplification under the constant temperature condition, and provides a fluorescent biosensor which is simple and convenient to operate and can sensitively detect miR-21. Mainly consists of 4 parts:
(1) miR-21 specifically recognizes a template H1;
(2) Extending forward under the action of BstDNA polymerase, and recognizing and cutting at specific sites by Nb.BbvCI endonuclease, and continuously polymerizing and cutting to generate a large amount of single-stranded DNA
(3) The generated single-stranded DNA is used as a primer to trigger a Primer Exchange Reaction (PER) to form a cascade amplification mode, and a large number of G-quadruplex fragments with different lengths are generated;
(4) Thioflavin T (ThT) dye intercalates into a number of G-quadruplex fragments of different length, generating a strong fluorescent signal;
the 3' end of the template H2 is modified with an inverted T to prevent nonspecific amplification; the CG base pair at the middle position of H2 is also subjected to methylolation modification, and the single-stranded DNA generated by polymerization cleavage after the H1 is combined with a target miR-2 is preferably 21 bases as a termination site.
Further, the template H1 is preferably 43 bases, and the template H2 is preferably 34 bases.
The detection principle of the invention is shown in figure 1. miR-21 binds to hairpin template H1 by the principle of base complementary pairing and extends into double strand (dsDNA) with the aid of Bst polymerase and dNTPs, nb.BbvCI specifically recognizes and cleaves dsDNA exposing the 3' hydroxyl end. Under the action of the polymerase, the DNA continues to replicate forward. After multiple polymerization cleavage, a large number of single stranded DNA is obtained which hybridizes complementarily to the primer binding domain on H2, replicates forward with the aid of a polymerase and stops at a stop site. The sequence domain in hairpin H2 competes with the extension product, which is released. The released extension product has the same composition as the primer DNA bases, so that the extended DNA can in turn act as a primer to trigger the next round of Primer Exchange Reaction (PER). These ssDNA of different lengths are rich in a large number of G bases, folding into a G-quadruplex structure in a potassium environment. With the addition of ThT, the G4/ThT complex produced a significant fluorescent signal.
Preferably, the method for detecting miR-21 by using target-triggered Strand Displacement Amplification (SDA) and Primer Exchange Reaction (PER) cascade amplification comprises the following steps:
(1) Template H1 and template H2 are annealed by using 1-th TE buffer solution, the annealing reaction condition is preferably denaturation at 95 ℃ for 5min, and the constant temperature reaction is carried out for 60min after the temperature is slowly reduced to 25 ℃.
(2) Adding 4 mu L of a sample to be detected into the reaction solution I, carrying out miR-21 recognition and cascade amplification reaction to generate a large number of G-quadruplex fragments with different lengths, and then carrying out high-temperature inactivation treatment. The conditions for miR-21 recognition and cascade amplification are preferably 25-45 ℃ (preferably 37 ℃) for 60-120min (preferably 90 min), and the high-temperature inactivation temperature is preferably 80 ℃ for 10-20min (preferably 20 min).
(3) By K + Diluting the solution after the reaction in the step (2) to 100 mu L by using a buffer solution, adding the THT dye into the solution, incubating the solution in a dark place, measuring fluorescence with a fluorescence excitation wavelength of 425nm and measuring a fluorescence intensity value at an emission wavelength of 497nm, and substituting the fluorescence intensity value into the formula F=34.17 LgC miR-21 +271.02, calculating the quantity of miR-21 contained in the obtained sample; the conditions for the incubation in the dark are preferably 25-45 ℃ (preferably 37 ℃) and 10-30min (preferably 30 min) in the dark.
Template H1 and template H2 in step (1) were 75nM and 150nM, respectively.
In the step (2), the reaction solution I comprises the template H1 (75 nM), the template H2 (150 nM), the Nb.BbvCI endonuclease (0.25U/. Mu.L), the BstDNA polymerase (1U/. Mu.L), the reaction buffer and water, which are used in the step (1), so as to form a 40. Mu.L reaction system.
In the step (3), 4. Mu. LThT dye was added.
The invention has the advantages that:
(1) According to the invention, target recognition, strand Displacement Amplification (SDA) and Primer Exchange Reaction (PER) cascade amplification and signal amplification are simultaneously carried out under a constant temperature condition, the operation is simple, convenient and rapid, quantitative detection of miR-21 can be realized within 90min, and multiple complex instruments and equipment are not required.
(2) The DNA used in the invention is non-fluorescent marked DNA, and the amplified product is added with the THT dye for quantitative detection. Not only avoids the interference of background fluorescent signals, but also greatly reduces the detection cost.
(3) The miR-21 quantitative detection can be completed by only using a trace sample (4 mu L).
(4) The method is suitable for detecting miR-21 in complex samples, and comprises blood and cell extract.
Drawings
FIG. 1 is a schematic diagram of the operation of the present invention for the sensitive detection of miR-21 using a Strand Displacement Amplification (SDA) and Primer Exchange Reaction (PER) cascade amplification reaction.
FIG. 2 is a diagram of polyacrylamide gel electrophoresis for verifying the feasibility of the experimental principle.
FIG. 3 is a fluorescence spectrum for verifying the feasibility of the experimental principle.
FIG. 4 shows the optimization of template H1 concentration (A), template H2 concentration (B), bstDNA polymerase concentration (C), nb.BbvCI endonuclease concentration (D), thT concentration (E) and reaction time (F) during the reaction.
FIG. 5 is a fluorescence spectrum of miR-21 detection at different concentrations.
FIG. 6 is a graph of the linear relationship between fluorescence intensity and the logarithmic concentration of miR-21.
FIG. 7 is a specific assay of the present invention.
FIG. 8 is a reproducibility analysis of the present invention.
Fig. 9 is an analysis of an actual sample according to the present invention.
Detailed Description
A fluorescent biosensor for label-free detection of miR-21 based on Strand Displacement Amplification (SDA) and Primer Exchange Reaction (PER) cascade amplification is shown in FIG. 1, in which miR-21 binds to hairpin template H1 by the principle of base complementary pairing and extends into double strand (dsDNA) with the aid of Bst polymerase and dNTPs, nb.BbvCI specifically recognizes and cleaves dsDNA exposing the 3' hydroxyl end. Under the action of the polymerase, the DNA continues to replicate forward. After multiple polymerization cleavage, a large number of single stranded DNA is obtained which hybridizes complementarily to the primer binding domain on H2, replicates forward with the aid of a polymerase and stops at a stop site. The sequence domain in hairpin H2 competes with the extension product, which is released. The released extension product has the same composition as the primer DNA bases, so that the extended DNA can in turn act as a primer to trigger the next round of Primer Exchange Reaction (PER). These ssDNA of different lengths are rich in a large number of G bases, folding into a G-quadruplex structure in a potassium environment. With the addition of ThT, the G4/ThT complex produced a significant fluorescent signal at 497nm under excitation light at 425nm wavelength. Along with the increase of the concentration of miR-21 in the sample, the fluorescence signal is continuously enhanced, so that the quantitative detection of miR-21 is realized.
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
EXAMPLE 1 pretreatment of probe DNA
First, powdered primer DNA ordered from Shanghai, was centrifuged at 4000rpm/min for 1min before use, and dissolved in 1X TE buffer as required to form a 100. Mu. MDNA solution. For use, diluted with 1 x te buffer. The 1 x te buffer composition was 10mm tris,1mm fidta, ph=8.
Template H1 has the sequence of
5'-CCCTAACCATCAGACCTCAGCTCAACATCAGTCTGATAAGCTA-3'
Wherein the underlined sequence is the nb.bvci endonuclease recognition sequence.
The sequence of the template H2 is 5 '-TTAGGGmCmCCGGTTTTCCGmGCCCTAACCCTAACCdT-3'.
Wherein m is a methylolation modification; dT is an inverted T modification.
Example 2 validation of protocol feasibility by electrophoresis on a 12% polyacrylamide gel
In FIG. 2, lane one is miR-21, lane two is H1, lane four is H2, and the band above lane three, compared with lanes one and two, shows that most of the target and H1 are successfully bound together, lane five is a negative control, and it can be seen that in the absence of target, only H1 and H2 bands are present, no new bands are added, and no amplification is demonstrated. In the presence of the target in lane six, a bright band is seen at the bottom of the lane, which is the single-stranded product (i.e., the primer for PER amplification) successfully cut by Strand Displacement Amplification (SDA), while a continuous band of different molecular weights is observed above the lane, demonstrating that the reaction was successful and that the amplification produced a large number of tandem repeat G-quadruplex fragments of different lengths. The results of polyacrylamide gel electrophoresis validated the feasibility of our protocol.
Example 3 fluorescence analysis verifies the feasibility of the protocol.
In FIG. 3, the fluorescence signal was significantly increased by detecting miR-21 (100 nM) diluted with ultrapure water using this method; whereas the fluorescence signal was maintained at a lower level when miR-21 was absent. The method can realize the detection of miR-21.
Example 4 experimental conditions of the present method were optimized.
Firstly, the concentrations of the template H1 and the template H2 are optimized, as shown in FIG. 4, when the added concentrations of the H1 and the H2 are 75nM and 150nM respectively, the fluorescence intensity F/F0 is not increased any more and is stable, so that the concentrations of the H1 are taken to be 75nM and the concentrations of the H2 are taken to be 150nM and are optimal; next, the concentration of BstDNA polymerase was optimized, and as shown in FIG. 4 (C), 1U/. Mu.LBstDNA polymerase reaction was taken as optimum; optimization of the concentration of Nb.BbvCI endonuclease as shown in FIG. 4 (D), F/F0 was highest when 0.25U/. Mu.L was added, so 0.25U/. Mu.L was taken as the optimal concentration of Nb.BbvCI endonuclease; thT concentration was also optimized, as shown in fig. 4 (E), taking 4uM as the optimal concentration; finally, the reaction time of the method is optimized, as shown in fig. 4 (F), and 90min is selected as the optimal reaction time.
Wherein, miR-21 with the final concentration of 100nM is used in all optimization processes.
Example 5 fluorescence detection of different concentrations of miR-21.
FIG. 5 is a graph of fluorescence spectra of miR-21 at different concentrations, wherein the fluorescence intensity at 497nM is gradually increased along with the gradual increase of the miR-21 concentration of 10fM-200 nM.
Example 6 the relationship between fluorescence detection results and the logarithmic value of miR-21 concentration was calculated.
FIG. 6 shows that there is a good linear relationship between fluorescence intensity value at 497nm and the logarithmic value of miR-21 concentration measured by the method in the concentration interval of 10fM-1 nMIR-21. The linear relationship is formulated as 34.17LgC miR-21 +271.02(R 2 =0.9945), the lowest detection limit is 1.25fM.
Example 7 the specificity of the present method was analyzed.
Let-7a, miR-155, miR-144, miR-126, single-base mismatch miR-21 (M1), double-base mismatch miR-21 (M2) and complete non-complementary sequences (NCM) with the concentration of 100nM are detected by the method, and fluorescence intensity at 497nM is obtained; as shown in FIG. 7, the method has good specificity.
Example 8 the reproducibility of the method was analyzed.
Example 9 actual samples were analyzed by the present method.
Total RNA in the cells MCF-7, hela and MCF-10A is extracted respectively, and then used as a sample to be detected by the method. As shown in FIG. 9, compared with the normal cell MCF-10A, the cancer cells MCF-7 and Hela have higher content, and the results are basically consistent with RT-qPCR, thus proving that the method can distinguish different cells in vitro.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (8)
1. A fluorescent biosensor for label-free detection of miR-21 based on strand displacement amplification and primer exchange reactions, wherein the biosensor comprises a Strand Displacement Amplification (SDA) template H1, a Primer Exchange Reaction (PER) template H2, miR-21, bst DNA polymerase, nb.bvci endonuclease, and thioflavin T (ThT).
2. The biosensor of claim 1, wherein the 3' end of the template H2 is modified with an inverted T to prevent non-specific amplification; CG base pairs in the H2 intermediate position have also been hydroxymethylated as termination sites.
3. The biosensor of claim 1, further comprising dNTPs consisting of four deoxynucleotide triphosphates, dATP, dCTP, dGTP and dTTP, mixed in equal proportions.
4. A method of detecting miR-21, comprising detecting using the biosensor of any one of claims 1-3.
5. The method of claim 4, wherein the method comprises:
s1, using Mg-containing 2+ Annealing SDA template H1 and PER template H2 to form a hairpin;
s2, adding a sample to be detected into the reaction solution I, performing miR-21 recognition, strand Displacement Amplification (SDA) and Primer Exchange Reaction (PER) cascade amplification reaction to generate a large number of G-quadruplex fragments, and performing high-temperature inactivation treatment;
s3, adding the ThT dye into the solution after the reaction in the step S2 to obtain a large number of G-quadruplex fragments embedded in the ThT;
preferably, in the step S1, the composition of the 1 x te buffer is 10mM Tris,1mM EDTA,pH =8;
preferably, in the step S2, the reaction solution I includes at least SDA template H1, PER template H2, nb.bvci endonuclease and Bst DNA polymerase;
further preferably, the reaction solution I may further include dNTPs, reaction buffer, ultrapure water, etc., so as to facilitate the smooth progress of the reaction;
preferably, in the step S2, the specific reaction conditions of miR-21 recognition and Strand Displacement Amplification (SDA) and Primer Exchange Reaction (PER) cascade amplification reactions are as follows: reacting for 90min at 37 ℃; the high-temperature inactivation temperature is preferably 80 ℃, the reaction is carried out for 10-20min (preferably 20 min), and the high detection sensitivity is achieved in the preferred time;
preferably, in the step S3, specific reaction conditions are as follows: the reaction is carried out at 25-45deg.C (preferably 37deg.C) for 10-30min (preferably 30 min).
6. The method of claim 5, further comprising performing a detection assay on the reaction product obtained in step S3; preferably, the detection assay is a fluorescent detection assay.
7. The biosensor of claim 5, wherein the concentration of miR-21 of step S2 is in the range of 10fM "1 nM.
8. Use of the biosensor of any one of claims 1-3 and/or the detection method of any one of claims 5-7 in a biological sample miR-21 detection assay.
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