CN113322306A - Biosensor combining exponential amplification reaction and CRISPR-Cas12a as well as detection method and application thereof - Google Patents
Biosensor combining exponential amplification reaction and CRISPR-Cas12a as well as detection method and application thereof Download PDFInfo
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Abstract
The invention discloses a biosensor combining exponential amplification reaction and CRISPR-Cas12a as well as a detection method and application thereof.A DNA template of the biosensor is formed by connecting two sections of same sequences through enzyme cutting sites of Nt.BstNI; wherein, the 5 'end of the DNA template is copied to form an antisense strand, and the adjacent 3' end of the antisense strand can be complementarily paired with the crRNA of the CRISPR-Cas12 a; when a sample contains a sequence of Let-7a, a DNA template and the sequence of Let-7a are complemented into a double strand, an amplification reaction of the whole system is started under the action of polymerase, and then under the action of Nt.BstNI, a single-stranded DNA which can be complemented with crRNA of CRISPR-Cas12a is cut out, so that the ability of the CRISPR-Cas12a for cutting the single-stranded DNA marked by a fluorophore is activated; on the other hand, if Let-7a is not present in the sample, the entire reaction will not occur.
Description
Technical Field
The invention relates to a biosensor and a detection method and application thereof, in particular to a biosensor combining exponential amplification reaction and CRISPR-Cas12a and a detection method and application thereof.
Background
microRNA, abbreviated as miRNA, is a class of endogenous, non-coding, short RNA molecules (19-23 nt). It can mediate the inhibition of transcription and the degradation of mRNA by binding with target mRNA, and plays an important role in regulating gene expression. Their stability in biological samples, resistance to adverse storage environments, etc., make them useful as a biomarker. micrornas play important roles in a wide range of biological processes, including proliferation, development, metabolism, immune response, tumorigenesis, and viral infection. In order to better understand the function of these biomolecules, they must be detected, which has great potential for early diagnosis of human diseases and for discovery of new drugs by using these molecules as targets. Northern blotting techniques are considered as standard methods for the detection of microRNAs. However, this method involves a large number of manual operations and, in addition, is time-consuming, energy-consuming and not very suitable for rapid detection in the field. Let-7a, as a microRNA, plays an important role in regulation and control in the process of generating diseases such as tumors and is closely related to tumorigenesis. However, the microRNAs themselves are present in very low amounts. Therefore, it is necessary to develop a simple and rapid detection method with high sensitivity and specificity.
Isothermal enzymatic amplification techniques utilize unique DNA and RNA polymerases, such as Phi29, Bst, Vent exo-DNA polymerase, and T7 RNA polymerase, that ultimately produce single-stranded DNA or RNA containing tens to hundreds of tandem repeats. This powerful amplification technique has become an excellent tool in biomedical research and nanotechnology. Isothermal exponential amplification (EXPAR), a technique combining single-strand cleavage and polymerase extension, is an isothermal amplification technique with high amplification efficiency. The method is characterized in that a special nicking endonuclease is used for recognizing a specific base sequence and only one strand of double-stranded DNA is cut, but the background signal of EXPAR is strong.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to amplify a detection signal by designing a biosensor combining exponential amplification reaction and CRISPR-Cas12a so as to improve the sensitivity and specificity of the whole detection method. The template designed by the invention comprises two symmetrical parts and a nickase enzyme cutting site, so that the signal is exponentially amplified. The invention also aims to provide a detection method and application of the biosensor.
The technical scheme is as follows: the invention relates to a biosensor combining exponential amplification reaction and CRISPR-Cas12 a.A DNA template of the biosensor is formed by connecting two sections of same sequences through enzyme cutting sites of Nt.BstNI; wherein, the 5 'end of the DNA template is copied to form an antisense strand, and the adjacent 3' end of the antisense strand can be complementarily paired with the crRNA of the CRISPR-Cas12 a; when a sample contains a sequence of Let-7a, a DNA template and the sequence of Let-7a are complemented into a double strand, an amplification reaction of the whole system is started under the action of polymerase, and a single-stranded DNA which can be complemented with crRNA of CRISPR-Cas12a is cut under the action of Nt.BstNI, so that the capability of the CRISPR-Cas12a for cutting the single-stranded DNA marked by a fluorophore is activated; on the other hand, if Let-7a is not present in the sample, the entire reaction does not occur.
The biosensor combining the exponential amplification reaction and the CRISPR-Cas12a has a template sequence: 5'-AACT ATAC AACC TACT ACCT CAAA CAGA CTC AAAC TATA CAAC CTAC TACC TCAA-3', respectively;
let-7a sequence: 5'-UGA GGU AGU AGG UUG UAU AGU U-3', respectively;
crRNA sequence: 5'-UAA UUU CUA CUA AGU GUA GAU AAC UAU ACA ACC UAC UAC CUC A-3' are provided.
The detection method of the biosensor combining the exponential amplification reaction and the CRISPR-Cas12a comprises the following steps:
(1) adding a sample; under the condition that the target exists, the whole amplification reaction is started under the action of DNA polymerase and Nt.BstNBI nicking endonuclease so as to achieve the purpose of amplifying signals;
(2) adding an aptamer complementary to the template, wherein the aptamer and the vicinity of the 5' end of the template are combined through base complementary pairing;
(3) adding DNA polymerase and Nt.BstNBI nicking endonuclease, and amplifying the aptamer along the template to obtain a DNA fragment capable of activating CRISPR-Cas12 a; the newly amplified DNA fragment is separated from the template due to the cutting of the Nt.BstNBI nicking endonuclease, so that the amplification result shows exponential amplification;
(4) adding CRISPR-Cas12a, crRNA, fluorescence-labeled single-stranded DNA and reaction buffer solution 3 into the product obtained in the step (3), and measuring the fluorescence intensity of the product by using a microplate reader;
(5) and (3) detecting the sample in the step (1) by using a Real-Time PCR method, and verifying the accuracy of the method.
The detection method of the biosensor combining the exponential amplification reaction and the CRISPR-Cas12a comprises the step (3) that the DNA polymerase is in a reaction buffer solution 1, wherein the reaction buffer solution contains Tris-HCl, (NH)4)2SO4、KCL、MgSO4、
X-100; BstNBI nicking endonuclease in the step (3) is in reaction buffer solution 2, wherein the nicking endonuclease contains NaCl, Tris-HCl and MgCl2BSA; the reaction buffer 3 in the step (4) contains NaCl, Tris-HCl and MgCl2、BSA。
The detection method of the biosensor combining the exponential amplification reaction and the CRISPR-Cas12a comprises the step (3) of reacting the reaction mixed solution of the Nt.BstNBI nicking endonuclease, the template and the solution containing the target substance for 30-60min at the temperature of 50-65 ℃.
According to the detection method of the biosensor combining the exponential amplification reaction and the CRISPR-Cas12a, the concentration of the Let-7 a-containing sample in the step (3) is 0-100 nM.
The index amplification reaction and the application of the biosensor combined with the CRISPR-Cas12a in the preparation of Let-7a diagnostic reagents.
According to the invention, through specific design, the 3' end of the template is modified by a phosphate group, so that the occurrence of non-specific amplification can be effectively avoided. When containing Let-7a, the template and Let-7a are complementarily paired, and a plurality of fragments of Let-7a are continuously amplified under the action of Vet DNA polymerase. The amplified template is continuously cut out under the action of nickase-Nt.BstNBI, so that the single-stranded DNA activating the CRISPR/Cas12a is exponentially increased. The resulting fluorescence will increase significantly. In the case of no target, the amplification reaction cannot be carried out due to the absence of the amplified primer, and finally lower fluorescence intensity is obtained.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: (1) the method greatly increases detection signals and reduces the influence of non-specific signals by utilizing the programmability of nucleic acid and reasonably designing the sequence of an amplification template. (2) The method relies on an exponential amplification technology as a means of signal amplification, avoids the complexity of the traditional amplification method, and ensures that the whole reaction is carried out at constant temperature. Has great significance for detecting samples at fixed points in real time. (3) The invention adopts a sensing method for detecting trace microRNA by combining a symmetrical amplification template with a constant-temperature amplification technology. The kit can be used for detecting microRNA in a human body, has the advantages of no need of additional labeling, temperature-changing treatment, high sensitivity, low cost and high-flux detection, and has a good application prospect.
Drawings
FIG. 1 is a graph showing the trend of the fluorescence intensity of the method and Real-Time PCR in samples containing different concentrations of Let-7 a; wherein series 1 is the fluorescence value of the method and series 2 is the fluorescence value of RT-PCR;
FIG. 2 is a specific detection assay for this method. And detecting the fluorescence values of other microRNAs and Let-7a under the same conditions.
Detailed Description
Drugs and reagents: all DNA used in the experiment was synthesized by biological engineering (shanghai, china) and purified by HPLC.
Example 1
(1) A reaction mixture solution of 1. mu.M of the DNA template solution and 10nM Let-7a, and the enzyme, respectively, was taken, and the mixture solution contained 10 XVent DNA polymerase Buffer, 10 XNt. BstNBI Buffer, 10nM dNTPs, DNA polymerase and Nt. BstNBI. The reaction was carried out at 55 ℃ for 30min, and then at 80 ℃ for 20 min. Incubate for 30 min.
(2) 10 μ L of each of the above products was added to 500nM CRISPR/Cas12a, 100nM crRNA, deionized water containing RNA inhibitory enzyme, and single stranded DNA of fluorophore and quencher in a reaction buffer of 10 × CRISPR/Cas12 a. The whole reaction was incubated at 37 ℃ for 60min and at 65 ℃ for 10min to inactivate the enzyme.
(3) 80. mu.L of each of the above-obtained products was placed on an ELISA plate, and the fluorescence results were measured.
TABLE 1 template sequences used
Example 2
Sensitivity test
Sample solutions with Let-7a concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100pM, respectively, were selected for validation and the results are shown in fig. 1. The linear range of detection is determined to be 0-100pM, and the detection limit reaches 0.04 pM.
Example 3
Selectivity test
The steps in the example 1 are repeatedly implemented, other conditions are unchanged, and the invention selects Let-7b, Let-7c, Let-7d, Let-7e, Let-7f and Let-7g as interference elements to detect fluorescence, thereby obtaining a selectivity result chart of the method. Under the same conditions, fluorescence values of other microRNAs and Let-7a are detected as shown in FIG. 2, and it can be seen that the method has good selectivity on target molecules.
Example 4
Recovery rate test of the detection method of the present invention
The sample is changed into deionized water, and equal amount of 20, 40 and 60pM of Let-7a is respectively added into the sample, so that the recovery rate of the method in the detection of target molecules of the actual sample is obtained, and the result is shown in Table 2.
TABLE 2 recovery rate experiment
Example 5
The invention is compared with the detection result based on Real-Time PCR
Since the method still detects the final target concentration by taking the final fluorescence intensity as a judgment, the invention uses a gold standard method Real-Time PCR for detecting the fluorescence intensity to carry out a verification test. The results are shown in FIG. 1. The method is more sensitive and Time-saving than the Real-Time PCR method.
Claims (7)
1. A biosensor combining exponential amplification reaction and CRISPR-Cas12a is characterized in that a DNA template of the biosensor is formed by connecting two sections of identical sequences through enzyme cutting sites of Nt.BstNI; wherein, the 5 'end of the DNA template is copied to form an antisense strand, and the adjacent 3' end of the antisense strand can be complementarily paired with the crRNA of the CRISPR-Cas12 a; when a sample contains a sequence of Let-7a, a DNA template and the sequence of Let-7a are complemented into a double strand, an amplification reaction of the whole system is started under the action of polymerase, and a single-stranded DNA which can be complemented with crRNA of CRISPR-Cas12a is cut under the action of Nt.BstNI, so that the capability of the CRISPR-Cas12a for cutting the single-stranded DNA marked by a fluorophore is activated; on the other hand, if Let-7a is not present in the sample, the entire reaction does not occur.
2. The biosensor in combination with the exponential amplification reaction and CRISPR-Cas12a according to claim 1, wherein the template sequence: 5'-AACT ATAC AACC TACT ACCT CAAA CAGA CTC AAAC TATA CAAC CTAC TACC TCAA-3', respectively;
let-7a sequence: 5'-UGA GGU AGU AGG UUG UAU AGU U-3', respectively;
crRNA sequence: 5'-UAA UUU CUA CUA AGU GUA GAU AAC UAU ACA ACC UAC UAC CUC A-3' are provided.
3. A method for detecting a CRISPR-Cas12 a-bound biosensor in accordance with the exponential amplification reaction of claim 1, comprising the steps of:
(1) adding a sample; under the condition that the target exists, the whole amplification reaction is started under the action of DNA polymerase and Nt.BstNBI nicking endonuclease so as to achieve the purpose of amplifying signals;
(2) adding an aptamer complementary to the template, wherein the aptamer and the vicinity of the 5' end of the template are combined through base complementary pairing;
(3) adding DNA polymerase and Nt.BstNBI nicking endonuclease, and amplifying the aptamer along the template to obtain a DNA fragment capable of activating CRISPR-Cas12 a; the newly amplified DNA fragment is separated from the template due to the cutting of the Nt.BstNBI nicking endonuclease, so that the amplification result shows exponential amplification;
(4) adding CRISPR-Cas12a, crRNA, fluorescence-labeled single-stranded DNA and reaction buffer solution 3 into the product obtained in the step (3), and measuring the fluorescence intensity of the product by using a microplate reader;
(5) and (3) detecting the sample in the step (1) by using a Real-Time PCR method, and verifying the accuracy of the method.
4. The method for detecting a CRISPR-Cas12 a-associated biosensor in accordance with claim 3, wherein the DNA polymerase of step (3) is Tris-HCl, (NH) in reaction buffer 14)2SO4、KCL、MgSO4、
X-100; BstNBI nicking endonuclease in the step (3) is in reaction buffer solution 2, wherein the nicking endonuclease contains NaCl, Tris-HCl and MgCl2BSA; the reaction buffer 3 in the step (4) contains NaCl, Tris-HCl and MgCl2、BSA。
5. The method for detecting a biosensor combined with an exponential amplification reaction and CRISPR-Cas12a according to claim 3, wherein the reaction mixture of Nt.BstNBI nicking endonuclease, the template and the solution containing the target in the step (3) is reacted for 30-60min at 50-65 ℃.
6. The method for detecting the biosensor combined with the exponential amplification reaction and the CRISPR-Cas12a according to claim 3, wherein the concentration of the Let-7 a-containing sample in the step (3) is 0-100 nM.
7. Use of the exponential amplification reaction of claim 1 in combination with a CRISPR-Cas12a biosensor in the preparation of Let-7a diagnostic reagents.
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