MiRNA-SNPs fluorescence sensor based on programmable nuclease activity of PfAgo protein and sensing method and application of miRNA-SNPs fluorescence sensor

Abstract

The invention discloses a miRNA-SNPs fluorescent sensor based on PfAgo protein programmable nuclease activity and a sensing method and application of the miRNA-SNPs fluorescent sensor. The fluorescent sensor comprises a chain A, a chain B, SplintR ligase, FQ and PfAgo nuclease. The chain A and the chain B generate a chain AB complementary to the miRNA to be detected through a base complementary pairing principle and the action of SplintR ligase; the AB chain excites PfAgo nuclease to perform targeted cleavage on an FQ single chain, so that a fluorophore FAM on the FQ single chain is separated from a quenching group BHQ1. According to the sensor and the sensing method thereof disclosed by the invention, zero background and accurate quantification of miRNA with mutated single basic group can be realized. The invention provides a simple, rapid and ultra-sensitive miRNA-SNPs accurate quantification strategy, and provides a powerful and universal detection platform for prognosis of human cancers related to miRNA related single nucleotide polymorphism.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection and biosensor, and specifically relates to a miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of a PfAgo protein, and a sensing method and application thereof. Background Art

[0002] Single nucleotide polymorphisms (SNPs) refer to polymorphisms in nucleic acid sequences caused by changes in a single nucleotide base. MicroRNA (miRNA) is a type of non-coding single-stranded RNA with a length of about 22 nucleotides. It participates in post-transcriptional gene expression regulation and is highly related to the etiology, development and prognosis of cancer. Almost all miRNAs are differentially expressed in different types of cancer. Single nucleotide polymorphisms in miRNA genes (miR-SNPs) mainly refer to nucleic acid sequence polymorphisms caused by single nucleotide variations at the genomic level corresponding to miRNAs, which have an important impact on the expression of miRNAs and the interaction between miRNAs and messenger RNAs. Since miRNAs are involved in regulating more than 30% of protein-coding genes in the human genome, minor variations in miRNAs may have an immeasurable impact on thousands of cell pathways, leading to disordered gene expression, multiple functional disorders, and then inducing cancer. However, the expression of miRNAs is regulated by genes, enzymes, and transcription factors, resulting in differences in their information and the DNA information stored in the genome. This means that detecting SNPs at the gene level cannot intuitively reflect the SNP level on miRNAs. Therefore, directly detecting SNPs in miRNA is of great significance and can more intuitively reflect its relationship with cancer.

[0003] However, the difficulty in identifying single nucleotide differences, the sequence homology and small size of miRNAs, and the low abundance of miRNA-SNPs in the human body pose challenges to the simple, sensitive, and specific quantification of miRNA-SNPs. Traditional RNA detection methods, such as quantitative reverse transcription polymerase chain reaction (RT-qPCR), are usually insensitive to the detection of miR-SNPs due to the short length of miRNAs, which hinders the stable binding of SNP-sensitive probes to the mutation site during complementary DNA (cDNA) amplification. At present, some novel detection methods have emerged, such as allele-specific ligation-driven exponential replication and reduced graphene oxide-assisted rolling circle amplification, which show high sensitivity in miRNA sensing. However, these methods require multiple steps, are cumbersome to operate, and take more than 2 hours. Opening the lid or transferring the solution in the middle also makes them susceptible to contamination and leads to RNA degradation. Therefore, it is urgent to develop a new biosensing strategy for accurate quantification of miRNA-SNPs that is fast, simple, and sensitive.

[0004] SplintR ligase can effectively catalyze the ligation of adjacent single-stranded DNA with complementary RNA strands as splints. It promotes the formation of phosphodiester bonds between 3'-hydroxyl and 5'-phosphate in nucleic acid residues, in which the 5'-P of the phosphate donor strand is required. Notably, the ligation process of SplintR is significantly inhibited when there is a single nucleotide mismatch at the junction, making SplintR ligase an ideal choice for ligation-dependent recognition of RNA SNPs.

[0005] Pyrococcus furiosus Argonaute (PfAgo) is a prokaryotic Ago protein derived from Archaea, which consists of four domains: amino terminus (N), PIWI, middle domain (MID) and PIWI-Argonaute-Zwille (PAZ). Studies on the cleavage activity of PfAgo have shown that it uses 5' phosphorylated single strand DNA (ssDNA) with a length of at least 15 nt as a guide DNA (gDNA), and targets and cleaves its complementary ssDNA at high temperature. The cleavage site is the target ssDNA that is complementary to the 10th and 11th bases from the 5' to 3' end of the gDNA. Unfortunately, there is no research on the application of PfAgo in the detection of miRNA-SNPs. Therefore, the use of PfAgo to construct a sensing strategy for miRNA-SNPs has great application prospects in in vitro diagnosis and is expected to provide a new solution for disease monitoring in resource-poor areas. Summary of the invention

[0006] Purpose of the invention: In view of the problems of existing miRNA-SNPs detection technology, such as cumbersome operation, long time consumption, easy contamination of the system, which leads to degradation of the target miRNA, the present invention provides a miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of PfAgo protein and a sensing method thereof. The fluorescent sensor and its sensing method adopt a one-pot detection method. After mixing the target miRNA with the reaction solution, the detection can be quickly completed within 50 minutes, thereby avoiding degradation of miRNA due to solution contamination.

[0007] The present invention also provides the application of the miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of the PfAgo protein.

[0008] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses a miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of PfAgo protein, wherein the fluorescent sensor mainly comprises a reaction solution, wherein the reaction solution comprises an A chain, a B chain, a SplintR ligase, FQ, and a PfAgo nuclease; the A chain and the B chain generate an AB chain complementary to the miRNA to be tested through the principle of base complementary pairing and the action of the SplintR ligase; the AB chain stimulates the PfAgo nuclease to perform targeted cutting of the FQ single chain, resulting in the separation of the fluorescent group FAM and the quenching group BHQ1 on the FQ single chain, thereby generating a fluorescent signal.

[0009] The reaction solution includes A chain, B chain, SplintR ligase, ligation reaction buffer, FQ, MnCl2, and PfAgo nuclease; and the ligation reaction buffer components include Tris-HCl, MgCl2, ATP, and DTT.

[0010] Wherein, the A chain, B chain, AB chain, and FQ sequences are:

[0011] PHO-GTAACTCAG

[0012] PHO-ACAGTTTC

[0013] PHO-GTAACTCAGACAGTTTC

[0014] FAM-CGCACCACAAGAAACTGTCTGAGTTACGGGTGCG-BHQ1.

[0015] Preferably, the reaction solution consists of 10μM A chain, 10μM B chain, 0.5U / μL SplintR ligase, 1× ligation reaction buffer, 10μM FQ, 40μM MnCl2, and 200U / μL PfAgo nuclease. Among them, the components of 1× ligation reaction buffer are 50mM Tris-HCl, 10mM MgCl2, 1mMATP, 10mM DTT, pH7.5. The reaction program set for qPCR is 37.0℃, 20min+95.0℃, 30min. Among them, 37.0℃, 20min belongs to program 1 ligation reaction, and 95.0℃, 30min belongs to program 2 nuclease cleavage reaction.

[0016] Preferably, the concentrations of the A chain, B chain and FQ in the reaction solution are 10 μM, the concentration of MnCl2 is 40 μM, the concentration of SplintR ligase is 0.5 U / μL, and the concentration of PfAgo nuclease is 200 U / μL.

[0017] The present invention provides a fluorescence sensing method of a miRNA-SNPs fluorescence sensor based on the programmable nuclease activity of the PfAgo protein. The fluorescence sensing method is a one-pot method, specifically comprising the following steps:

[0018] The miRNA sample to be tested is mixed with the fluorescent sensor, placed in a qPCR instrument, and a reaction program is set and reacted, the program including a ligation reaction and a nuclease cleavage reaction.

[0019] Wherein, the reaction procedure is 25-40°C, 5-30min+90-95°C, 30-60min.

[0020] Preferably, the reaction procedure is 37.0°C, 20 min+95.0°C, 30 min.

[0021] The ligation reaction is as follows: the miRNA sample to be tested and the A chain and the B chain are subjected to a ligation reaction at 25-40° C. for 5-30 minutes through the principle of complementary base pairing and the action of SplintR ligase to generate an AB chain complementary to the miRNA, and the AB chain and the miRNA are in a hybridized state under the action of SplintR ligase.

[0022] The AB chain is 17 nt in length and has a 5' end phosphorylated.

[0023] Preferably, the ligation reaction is the generation of gDNA (AB chain): the miRNA and A chain and B chain in the mixed solution are subjected to ligation reaction at 37°C for 20 minutes through the principle of base complementary pairing and the action of SplintR ligase, so that A chain and B chain are connected into a complete AB chain with a length of 17nt and a phosphorylation modification at the 5' end. The AB chain and miRNA are in a hybridized state under the action of SplintR ligase.

[0024] Wherein, the nuclease cleavage reaction is: the AB chain and the miRNA are complementary to form a double chain, reacting at a temperature of 90-95°C for 30-40 minutes, the double chain is unzipped, the AB chain is released, and the nuclease activity of the PfAgo protein is stimulated, so that it performs targeted cleavage on the FQ single chain, resulting in the separation of the fluorescent group FAM and the quenching group BHQ1, thereby increasing the fluorescence intensity. The SNP in the present invention is mainly aimed at the polymorphism in miR-196a2. When the miRNA undergoes a SNP mutation (i.e., miR-196a2T), SplintR can play a connecting role, generate an AB chain, trigger the cleavage activity of PfAgo, and produce increased fluorescence, while the wild type (i.e., miR-196a2C) will not have an increase in fluorescence, thereby being able to distinguish the polymorphism of the miRNA.

[0025] Preferably, the nuclease cleavage reaction is an AB chain activation of PfAgo nuclease activity: the AB chain and the miRNA are complementary to form a double strand, reacting at a temperature of 95°C for 30 minutes, the double strand is melted, the AB chain is released, and the nuclease activity of the PfAgo protein is stimulated to perform targeted cleavage on the FQ single strand in the environment, resulting in the separation of the fluorescent group FAM and the quenching group BHQ1, thereby increasing the fluorescence intensity.

[0026] The invention relates to the application of the miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of the PfAgo protein in the detection of miRNA-related single nucleotide polymorphisms.

[0027] The invention provides an application of the miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of the PfAgo protein in the preparation of a detection reagent or tool for miR-196a2T, a potential biomarker for non-small cell lung cancer.

[0028] The present invention selects miR-196a2T, a potential biomarker of non-small cell lung cancer, as the detection target, and develops a method based on the programmable nuclease activity of PfAgo protein to construct a fluorescent biosensor for miRNA-SNPs, which has high sensitivity, simple operation and fast reaction speed.

[0029] The present invention uses miR-196a2T, a potential biomarker of non-small cell lung cancer, as the detection target, generates an AB chain that can stimulate the subsequent nuclease cleavage reaction through a ligation reaction, and the AB chain is complementary to the miRNA to form a double chain. At a temperature of 95°C, the double chain is unwound, the AB chain is released, and the nuclease activity of the PfAgo protein is stimulated, so that it performs targeted cleavage on the FQ single chain in the environment, resulting in the separation of the fluorescent group FAM and the quenching group BHQ1, thereby increasing the fluorescence intensity, and achieving zero background and accurate quantification of miR-196a2T with a single base mutation. The present invention provides a simple, rapid, ultra-sensitive, low-cost miRNA-SNPs accurate quantification strategy, which is expected to provide a powerful and universal detection platform for the prognosis of human cancers related to miRNA-related single nucleotide polymorphisms.

[0030] The principle diagram of the present invention is shown in the attached Figure 1. Taking miR-196a2 as the detection target, when a single base site mutation occurs, the C base at the specific site mutates to a U base. The designed A and B chains are both 5' phosphorylated and complementary paired with the mutant miR-196a2. When the A chain, B chain and miRNA complementarily pair to form an AB-miR complex, the SplintR ligase connects the A chain and the B chain to form a complete DNA chain, namely the AB chain. When the miRNA has not mutated, the first base A at the 5' end of the B chain cannot complementarily pair with the mutable site C of miR-196a2, and the AB-miR complex cannot be formed. The SplintR ligase cannot catalyze the ligation reaction, and the AB chain cannot be formed. The subsequent stimulation of PfAgo nuclease activity requires a 5' phosphorylated single-stranded DNA with a length of 15-30nt as a guide chain (gDNA). After activation, PfAgo cuts the single strands in the environment that complementarily pair with the gDNA. It is known that the length of chain A is 9nt and the length of chain B is 8nt, and neither can stimulate the cleavage activity of PfAgo, so that the FQ hairpin structure modified with the fluorescent group at the 5' end and the quenching group at the 3' end remains intact, and there is no fluorescence increase. When the miRNA mutates, the first base A at the 5' end of the B chain complements the mutation site U of miR-196a2T, and the SplintR ligase plays a role and connects the adjacent linkers of the A chain and the B chain, and the AB chain is generated. At a high temperature of 95°C, the AB chain unwinds from the miRNA and binds to PfAgo, becoming the gDNA of PfAgo, stimulating the nuclease activity of PfAgo, and cutting the single-stranded FQ complementary to the gDNA in the environment, thereby separating the fluorescent group FAM from the quenching group BHQ1, and the fluorescence increases.

[0031] The present invention establishes a PfAgo protein-based programmable nuclease activity biosensor for detecting miRNA-related nucleic acid polymorphisms. The combination of SplintR ligase and PfAgo nuclease makes the identification and detection of single gene mutations more accurate. The one-pot method makes the experimental steps simple and easy to operate, reduces human experimental errors, and has ultra-high sensitivity.

[0032] Design principle: Figure 1 As shown, the fluorescent sensor and detection method of the present invention is a single-tube detection strategy, including two steps: a ligation reaction of SplintR ligase and a cleavage reaction of PfAgo nuclease. Figure 1As shown in a, when wild-type miR-196a2C is present, the single nucleotide mismatch at the RNA-DNA junction prevents the SplintR ligase from functioning normally, and the 3'-OH of the A chain and the 5'-P of the B chain cannot be effectively connected, resulting in the ineffective formation of the AB chain. Since the minimum gDNA length required by PfAgo is 15nt, while the length of the B chain is only 8nt and the A chain is only 9nt, although both have 5'-P, neither can independently activate PfAgo as gDNA. Therefore, in the presence of wild-type miR-196a2C, due to the lack of suitable gDNA, PfAgo cannot cut FQ and thus cannot generate a fluorescent signal. In contrast, in the presence of miR-196a2T, the perfect match between the B chain and the miR-196a2T mutation site enables the SplintR ligase to catalyze the formation of a phosphodiester bond between the 3'-OH of the A chain and the 5'-P of the B chain, thereby generating an AB chain with a length of 17nt. At 95°C, the AB chain is released from the DNA-RNA hybrid chain and binds to PfAgo as gDNA, guiding PfAgo to recognize and cut the FQ that is complementary to the AB chain, causing FAM and BHQ1 to separate, resulting in enhanced fluorescence signal ( Figure 1 b).

[0033] The present invention integrates the specific recognition of RNA single-base mutations by SplintR ligase, the programmable nuclease activity of PfAgo and the fluorescent signal sensor based on this, and sensitively distinguishes miRNA with single nucleotide mutations from wild-type miRNA. This method is a one-pot method, which avoids contamination caused by opening the lid and pipetting in the middle; it can also directly detect the level of miR-SNP without pre-amplification, which is time-consuming and can complete all detections in 50 minutes.

[0034] The detection method of the present invention is a one-pot method without pollution and is simple to operate; it is short in time, and the current miR-SNP detection methods almost all take more than 2 hours, while the sensor and detection method of the present invention can produce results in 50 minutes; it has good specificity and can distinguish mutation levels of 0.005%.

[0035] The present invention cascades SplintR and PfAgo to detect miR-SNP in a one-pot method, and uses PfAgo for the first time in the detection application on miRNA.

[0036] The present invention uses SplintR ligase and PfAgo nuclease in combination, SplintR plays a role in identifying the SNP of miRNA, and PfAgo plays a role in signal output. The system completes the one-pot detection of miR-SNP within 50min, with fast reaction speed, no pre-amplification, no midway opening, and avoids contamination; the detection limit is 20.40pM, with high sensitivity; it also performs well in specific experiments, and miR-196a2C has almost no interference with the detection of miR-196a2T. The current prior art has a long reaction time (all above 2h), and all require step-by-step reactions, which are easy to cause contamination midway, leading to RNA degradation. The one-pot method of the present invention not only has a short reaction time, but also requires only one-step reaction to avoid contamination. If one of the enzymes SplintR ligase or PfAgo nuclease is used in the present invention, it is ineffective, and at the same time, under the current system of the present invention, replacing it with other enzymes will not produce any effect. The combination of SplintR and Cas12a protein requires the special design of crRNA in Cas12a protein to stimulate its activity. In the system of the present invention, there is no need to stimulate the components of Cas12a.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0038] (1) The fluorescent sensor and fluorescent sensing method of miRNA-related single nucleotide polymorphism based on the programmable nuclease activity of PfAgo protein of the present invention have high sensitivity, simple operation and fast reaction speed.

[0039] (2) The PfAgo protein nucleic acid guide of the present invention is shorter. Compared with CRISPR / Cas technology, the targeting region is not restricted by a specific sequence and does not require a PAM sequence.

[0040] (3) The programmable nuclease activity of PfAgo protein combined with fluorescence sensing can significantly amplify the signal. In addition, the qPCR program setting effectively reduces the occurrence of false positives, and the detection results are accurate.

[0041] (4) The fluorescence sensor and fluorescence sensing method of the present invention adopt a one-pot method, which is easy to operate and does not require high technical personnel. In addition, there is no midway opening step, which avoids sample contamination and RNA degradation.

[0042] (5) The miRNA-related single nucleotide polymorphism fluorescent sensor and fluorescent sensing method based on the programmable nuclease activity of PfAgo protein of the present invention have rapid response, high sensitivity and good specificity, and provide a potential and reliable tool for the detection of miR-196a2T, a potential biomarker for non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1It is a schematic diagram of the miRNA-associated single nucleotide polymorphism fluorescence sensing method based on the programmable nuclease activity of the PfAgo protein;

[0044] Figure 2 This is the result of 20% non-denaturing polyacrylamide gel electrophoresis;

[0045] Figure 3 is the fluorescence result of miR-T and miR-C;

[0046] Figure 4 is the fluorescence detection result of different concentrations of miR-196a2T;

[0047] Figure 5 It is a specific test result. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0049] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0050] Drugs and reagents: DNA, RNA, and FQ used in the experiment were synthesized by Sangon Biotech (China) and purified by HPLC. SplintR ligase (Cat. No. M0375) and 10× ligation reaction buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT, pH 7.5@25°C) were purchased from New England Biolabs (NEB) Co., Ltd. (USA). MnCl2 and PfAgo nuclease (Cat. No. JY0316) were purchased from Wobo Biotech Co., Ltd. (China). Fluorescence data were obtained from QuantStudio 3 Real-Time PCR instrument, which was produced by Thermo Fisher (USA).

[0051] The nucleic acid sequences used in the examples are shown in Table 1.

[0052] Table 1 Nucleic acid sequences used in the experiment

[0053]

[0054] Note: FAM is the labeled fluorescent group, BHQ1 is the quenching group, and PHO is the phosphate group.

[0055] Example 1

[0056] Non-denaturing polyacrylamide gel electrophoresis to verify the feasibility of the scheme

[0057] In order to facilitate sample retention and gel running, a two-step method was used to complete the experiment, namely, a ligation reaction was performed first, and then a PfAgo nuclease cleavage reaction was performed.

[0058] (1) Prepare the ligation reaction system: according to the final concentration, mix 10 μM A chain, 10 μM B chain, 10 nM miR-196a2T / miR-196a2C and 25 U / μL SplintR ligase in 1× ligation reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5) and incubate at 37°C for 20 min. Keep the sample for gel running.

[0059] (2) Prepare PfAgo nuclease cleavage reaction: Add 1 μM FQ, 4 μM MnCl2, and 5 U / μL PfAgo to the reaction solution (1) according to the final concentration, and incubate at 95°C for 30 min. Keep the sample for gel running.

[0060] (3) The reaction solution samples from (1) and (2) were subjected to 20% non-denaturing polyacrylamide gel electrophoresis. The results were as follows: Figure 2As shown. Lane 12 and lane 13 are the ligation products of miR-196a2C and miR-196a2T, respectively. A bright band appeared in the miR-196a2T group (lane 13), which is the miR-196a2T-AB chain complex, indicating that the ligation reaction was successful; while the band did not appear in the miR-196a2C group (lane 12), indicating that the ligation reaction did not occur. Lane 9 and lane 10 are the PfAgo nuclease cleavage reaction products of miR-196a2C and miR-196a2T, respectively. Lane 5 is a control group with only FQ added, and lane 7 is a reaction control group of gDNA (AB chain) and PfAgo. A band appeared in the miR-196a2C group (lane 9) and the position of the band was the same as that of FQ (lane 5), indicating that FQ was not cleaved, that is, the PfAgo nuclease cleavage reaction did not occur; no band appeared in the miR-196a2T group (lane 10), which was consistent with the gDNA reaction group (lane 7), indicating that FQ was completely cleaved, further verifying the feasibility of the present invention. Lane 4 is the control group with only gDNA added. Neither the gDNA reaction group (lane 7) nor the miR-196a2T group (lane 10) has a corresponding band. The reason is that when PfAgo is present, gDNA (AB chain) will bind to PfAgo to form a gDNA-PfAgo complex. When running the gel, gDNA cannot be released, so no corresponding band appears. This gel image proves that only when miR-196a2T is present can the AB chain be generated, and only when the AB chain is generated can FQ be cut by PfAgo, proving the feasibility of the entire system, and also proving that the one-pot method of SplintR ligation reaction and PfAgo cutting reaction is feasible.

[0061] Example 2

[0062] The present invention uses a one-pot method to detect the fluorescence results of miR-196a2T and miR-196a2C respectively

[0063] (1) Prepare the reaction solution. According to the final concentration, mix 10 μM A chain, 10 μM B chain, 0.5 U / μL SplintR ligase, 1× ligation reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5), 1 μM FQ, 4 μM MnCl2, and 5 U / μL PfAgo nuclease, and use it as the reaction solution for later use.

[0064] (2) Preparation of miR-196a2T / miR-196a2C solution: The dry powders of miR-196a2T and miR-196a2C were centrifuged at 13,000 rpm for 5 min, dissolved in DEPC water, and mixed well to prepare a 10 nM solution.

[0065] (3) Take 2 μL of 10 nM miR-196a2T and miR-196a2C solutions and 18 μL of reaction solution in eight tubes (the control group replaced miRNA with 2 μL DEPC water), place the eight tubes in the Thermo Fisher qPCR instrument, set the program to 37.0℃, 20min+95.0℃, 30min, and the reporter channel to FAM. Start the qPCR instrument. Record the fluorescence values ​​of the last cycle and make a graph, as shown in Figure 2. Figure 3 As shown. It shows that the fluorescence response of the miR-196a2T group is very high, and the fluorescence intensity of the miR-196a2C group is almost the same as the DEPC water of the Control group, which proves that the selectivity of this system is excellent and miR-196a2T can be effectively detected.

[0066] Example 3

[0067] Fluorescence results of the present invention for detecting different concentrations of miR-196a2T

[0068] The reaction system was prepared as in (1)(2)(3) of Example 2 above and the same reaction procedure was set in qPCR, wherein the concentration of miR-196a2T in (2) of Example 2 was changed to: 100pM, 500pM, 700pM, 1000pM, 2000pM, 3000pM, 4000pM, 5000pM. The detection results are as follows Figure 4 As shown. This indicates that the concentration of miR-196a2T in the range of 100pM-5000pM is linearly related to the fluorescence intensity: y=11.002x+239.712, R 2 =0.9994. In the formula, x is the concentration of miR-196a2T, and y is the fluorescence intensity value at the corresponding concentration. The detection limit of the present invention is 20.40 pM, and the detection range is a linear range of 100 pM-5000 pM, or even higher.

[0069] Example 4

[0070] Specific detection results of the present invention

[0071] The reaction system was prepared as in (1)(2)(3) of Example 2 above and the same reaction procedure was set in qPCR, wherein miR-196a2T in (2) of Example 2 was replaced with miR-196a2C as an interfering substance to investigate the specificity of the method. miR-196a2T and miR-196a2C were mixed in different proportions to prepare a series of mixtures. The total concentration of the mixture was 10 nM, wherein the mutation levels were 0.005% (molar content), 0.01%, 1%, 10%, 50% and 100%. The measured mutation level was calculated according to equation (1).

[0072] Mutation level(%)=M / (M+W)×100% (1)

[0073] In this equation, M is the content of miR-196a2T, and W is the content of miR-196a2C.

[0074] like Figure 5 As shown, as the input mutation level increases, the fluorescence intensity also increases significantly, indicating that the wild-type miR-196a2C has minimal interference with the sensor and that the sensor can recognize mutations as low as 0.005% of miR-196a2T. Figure 3 , Figure 4 and Figure 5 The experimental results were all carried out in three groups in parallel, with low repeatability and good repeatability.

Claims

1. A miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of PfAgo protein, characterized in that: The fluorescent sensor mainly includes a reaction solution, which includes an A chain, a B chain, a SplintR ligase, FQ, and a PfAgo nuclease; the A chain and the B chain generate an AB chain complementary to the miRNA to be detected through the principle of base complementary pairing and the action of the SplintR ligase; the AB chain stimulates the PfAgo nuclease to perform targeted cutting of the FQ single chain, resulting in the separation of the fluorescent group FAM and the quenching group BHQ1 on the FQ single chain.

2. The miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of PfAgo protein according to claim 1, characterized in that: The reaction solution comprises A chain, B chain, SplintR ligase, ligation reaction buffer, FQ, MnCl2, and PfAgo nuclease; and the ligation reaction buffer components are Tris-HCl, MgCl2, ATP, and DTT.

3. The miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of PfAgo protein according to claim 1, characterized in that: The A chain, B chain, AB chain, and FQ sequences are: PHO-GTAACTCAG PHO-ACAGTTTC PHO-GTAACTCAGACAGTTTC FAM-CGCACCACAAGAAACTGTCTGAGTTACGGGTGCG-BHQ1.

4. A fluorescence sensing method of a miRNA-SNPs fluorescence sensor based on the programmable nuclease activity of PfAgo protein, characterized in that: The fluorescence sensing method is a one-pot method, which specifically comprises the following steps: The miRNA sample to be tested is mixed with the fluorescent sensor, placed in a qPCR instrument, and a reaction program is set and the reaction is carried out, the program including a ligation reaction and a nuclease cleavage reaction.

5. The fluorescence sensing method according to claim 4, characterized in that: The reaction procedure is 25-40°C, 5-30 min + 90-95°C, 30-60 min.

6. The fluorescence sensing method according to claim 4, characterized in that: The ligation reaction is as follows: the miRNA sample to be tested and the A chain and the B chain are subjected to a ligation reaction at 25-40° C. for 5-30 minutes through the principle of complementary base pairing and the action of SplintR ligase to generate an AB chain complementary to the miRNA, and the AB chain and the miRNA maintain a double-stranded hybridization state.

7. The fluorescence sensing method according to claim 6, characterized in that: The AB chain is preferably 17 nt in length and the 5' end is phosphorylated.

8. The fluorescence sensing method according to claim 4, characterized in that: The nuclease cleavage reaction is as follows: reacting at a temperature of 90-95°C for 30-60 minutes, the AB-miRNA hybrid chain is unzipped, the AB chain is released, and the PfAgo protein is guided to exert nuclease activity, so that it performs targeted cleavage on the FQ single chain, resulting in the separation of the fluorescent group FAM and the quenching group BHQ1, thereby increasing the fluorescence intensity.

9. Use of the miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of PfAgo protein according to claim 1 in the detection of miRNA-related single nucleotide polymorphisms.

10. Use of the miRNA-SNPs fluorescent sensor based on the programmable nuclease activity of PfAgo protein according to claim 1 in the preparation of a detection reagent or tool for the potential biomarker miR-196a2T of non-small cell lung cancer.

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