Hairpin primer for detecting miRNA and application thereof

Abstract

This invention relates to a hairpin primer for detecting miRNA and its applications. The invention develops a reassembly ligation detection scheme for miRNA detection, designing hairpin primers and developing a two-step detection process. The first hairpin primer loop structure binds to the target miRNA, opening the stem structure. The opened stem structure attracts the remaining miRNA sequence, reassembling with the second hairpin primer to form a T-shaped double-stranded structure. The two hairpin primers, using the DNA strand as a clamp, can be ligated by DNA ligase to form a long fragment containing both the miRNA's reverse-specific and universal sequences for qPCR detection. The stem-loop design improves ligation specificity, eliminates Splint ligase dependence, reduces cost, and increases efficiency. The ligated long fragment contains both specific and universal sequences, facilitating multiplex qPCR detection.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology and relates to a hairpin primer for detecting miRNA and its application. Background Technology

[0002] MicroRNAs (miRNAs) are a class of endogenous, short, single-stranded non-coding RNAs ranging from 19 to 25 nucleotides in length. They regulate gene expression in various physiological processes by interacting with a variety of targets, including messenger RNAs and long non-coding RNAs. Current research has found that abnormal miRNA expression is associated with the occurrence and progression of various diseases. Specific miRNAs related to diseases can serve as key diagnostic and prognostic biomarkers. However, miRNAs are characterized by low abundance, with concentrations ranging from fM to pM in biological samples. Accurate and sensitive detection of miRNAs is of great significance for disease diagnosis and research into related basic mechanisms.

[0003] In recent years, various detection techniques have been developed and applied in the research field to meet the needs of quantitative analysis of miRNAs. These include Northern blotting, high-throughput RNA sequencing, microarray chips based on hybridization principles, electrochemical sensor detection systems, real-time quantitative PCR (RT-qPCR) using stem-loop reverse transcription, and clip-on ligation detection methods based on nucleic acid ligation reactions. Northern blotting, as a classic standard method in the early stages of miRNA research, has revealed significant drawbacks in practice, such as cumbersome procedures, the need for radiolabeled probes, and relatively limited overall detection sensitivity. While RNA sequencing and microarray technologies can achieve parallel detection of multiple miRNA targets, their widespread application is limited by expensive equipment, high experimental costs, and complex data analysis. Sensor detection systems typically utilize electrochemical signal conversion mechanisms to detect multiplex miRNAs; however, this technology still has limitations in terms of detection sensitivity and target specificity. Stem-loop RT-qPCR involves two key steps: reverse transcription and quantitative PCR. The reverse transcription step typically requires the design of a specific complementary sequence of approximately six bases. This design limitation restricts the potential for increasing the reverse transcription reaction temperature. Furthermore, commonly used reverse transcriptases have low catalytic efficiency for short-chain miRNA templates, making it difficult for this method to achieve the desired sensitivity when detecting low-abundance miRNAs. While splint ligation detection technology bypasses the problems associated with the reverse transcription step through improved probe design, it relies on the activity of Splint ligase and is prone to generating false-positive ligation products due to non-specific probe binding during the reaction, affecting the reliability of the detection results.

[0004] In conclusion, developing efficient, accurate, and convenient miRNA detection methods is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a hairpin primer for detecting miRNA and its application, and further develops a reassembly ligation detection method, aiming to achieve efficient, accurate and convenient miRNA detection.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a hairpin primer for detecting miRNA, the hairpin primer comprising a first hairpin primer and a second hairpin primer; The first hairpin primer includes, from 5' to 3' end, a universal primer sequence, a first stem sequence, a first target recognition region, a first dangling base sequence, and a second stem sequence, wherein the universal primer sequence is the sequence that binds to one end of the qPCR primer, the first stem sequence and the second stem sequence are reverse complementary sequences, the first target recognition region is the sequence that binds to the 3' end of the target miRNA, and the first dangling base sequence is the sequence of the next base mismatch of the target miRNA that binds to the first target recognition region; The second hairpin primer includes, from 5' to 3', a third stem sequence, a universal primer loop sequence, a fourth stem sequence, a second stem sequence recognition region, a second dangling base sequence, and a second target recognition region, wherein the third stem sequence and the fourth stem sequence are reverse complementary sequences, the universal primer loop sequence is a sequence that binds to the other end of the qPCR primer, the second stem sequence recognition region is a sequence that binds to the second stem sequence in the hairpin primer 1, the second target recognition region is a sequence that binds to the 5' end of the target miRNA, and the second dangling base sequence is a sequence with a mismatched next base of the target miRNA that binds to the second target recognition region.

[0007] In this invention, hairpin primers with specific structures are designed and can be effectively used for miRNA detection. The loop structure of the first hairpin primer can specifically bind to the target miRNA and open the stem structure. After opening, the stem structure and the remaining miRNA sequence jointly and specifically attract the second hairpin primer for reassembly, forming a T-shaped double-stranded structure. The two hairpin primers are then linked by DNA ligase using the DNA strand as a clamp, forming long fragments containing miRNA reverse specific sequences and universal sequences for subsequent qPCR detection.

[0008] Optionally, the first stem sequence and the second stem sequence in the first hairpin primer are reverse complementary to form a hairpin structure, and the length of the hairpin structure is at least 6 bp.

[0009] Optionally, the length of the second stem sequence recognition region in the second hairpin primer that is complementary to the second stem sequence in the first hairpin primer is at least 5 bp.

[0010] Optionally, the 5' and 3' ends of the second hairpin primer are modified with phosphate groups.

[0011] Optionally, the primer at one end of the qPCR and the primer at the other end of the qPCR are primer pairs used for performing qPCR.

[0012] Optionally, the length of the first target recognition region complementary to the 3' end of the target miRNA is at least 12-18 bp.

[0013] Optionally, the length of the first suspended base sequence is 1-3 nt.

[0014] Optionally, the second target recognition region is complementary to the 5' end of the target miRNA in length of at least 3-7 bp.

[0015] Optionally, the length of the second dangling base sequence is 1-3 nt.

[0016] It is understood that the specific base types in the hairpin primers of this invention can be designed according to the actual situation (target miRNA, etc.) to meet the corresponding complementarity and mismatch requirements.

[0017] In a second aspect, the present invention provides the application of the hairpin primers described in the first aspect for detecting miRNA in the detection of miRNA.

[0018] Thirdly, the present invention provides a kit for detecting miRNA, the kit comprising the hairpin primers and qPCR primers and probes for detecting miRNA as described in the first aspect; The qPCR primer at one end is a sequence that binds to the universal primer sequence in the first hairpin primer, and the primer at the other end is a sequence that binds to the universal primer loop sequence in the second hairpin primer; The probe sequence includes the sequence of the first target recognition region in the first hairpin primer, and one end of the probe is modified with a fluorescent group and the other end is modified with a quenching group.

[0019] Fourthly, the present invention provides a method for detecting miRNA, the method comprising: mixing the hairpin primers for detecting miRNA described in the first aspect with the sample to be tested and DNA ligase to perform a ligation reaction; taking the ligation reaction product and performing qPCR detection using qPCR primers and probes; and determining the miRNA content in the sample to be tested by fluorescence amplification curve.

[0020] In this invention, a reassembly ligation detection method based on the developed hairpin primer design is proposed. The reassembly ligation method improves ligation specificity through stem-loop design, while eliminating Splint ligase dependence, replacing DNA ligase to reduce reaction costs and improve ligation efficiency. In addition, the long fragment after ligation contains both specific sequences and introduces longer universal sequences, which is helpful for multiplex qPCR detection.

[0021] It is understood that the method for detecting miRNAs in this invention has multiple applications. It can be used to detect disease-related miRNAs, as well as for non-disease diagnostic purposes. For example, in drug development, miRNAs serve as potential drug targets, and this detection method helps researchers gain a deeper understanding of the impact of drugs on miRNA expression. By detecting changes in miRNA expression before and after drug treatment, researchers can assess the efficacy and safety of drugs, providing important reference data for new drug development. In biological research, this method can help researchers explore the mechanisms of action of miRNAs in biological development, cell differentiation, and other processes. By accurately detecting miRNA expression levels, researchers can reveal the relationship between miRNAs and gene regulation and signaling pathways, providing strong technical support for basic biological research.

[0022] Optionally, the DNA ligase includes T4 DNA ligase or E. coli DNA ligase.

[0023] Optionally, the conditions for the connection reaction are: 15~30℃, 25~65 min; 93~98℃, 3~10 min.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects: This invention develops a reassembly-ligation detection scheme for miRNA detection, employing hairpin primers and a two-step detection process. The first hairpin primer loop binds to the target miRNA, opening the stem structure. The opened stem structure then attracts the remaining miRNA sequence, leading to reassembly with the second hairpin primer, forming a T-shaped double-stranded structure. The two hairpin primers, using the DNA strand as a clamp, can be ligated with DNA ligase to form a long fragment containing both the miRNA's reverse-specific and universal sequences for qPCR detection. The reassembly-ligation method improves ligation specificity through stem-loop design, eliminates Splint ligase dependence, and uses DNA ligase to reduce costs and increase efficiency. The ligated long fragment contains both specific and universal sequences, facilitating multiplex qPCR detection. This method can effectively detect miRNAs as low as 0.1 fM, with potential for further reduction in the minimum detection concentration. Attached Figure Description

[0025] Figure 1This is a schematic diagram of the hairpin primer structure and detection principle.

[0026] Figure 2 This is an image showing the electrophoresis results of the ligation products in Example 1.

[0027] Figure 3 This is the amplification curve after ligation using T4 DNA ligase in Example 2.

[0028] Figure 4 As used in Example 2 E. coli Amplification curve after DNA ligase ligation.

[0029] Figure 5 This is the amplification curve from Example 3. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0031] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0032] This invention develops a reassembly connection detection scheme, designing specific hairpin primers, which include a first hairpin primer and a second hairpin primer. A schematic diagram of the structure and working principle is shown below. Figure 1 As shown.

[0033] The first hairpin primer includes, from 5' to 3', a universal primer sequence, a first stem sequence, a target recognition region, a dangling base, and a second stem sequence, wherein: a) the first and second stem sequences are reverse complementary sequences to form a hairpin structure, and the hairpin length should be at least 6 bp. When the hairpin opens, the second stem sequence serves as the connecting end, complementing the second stem sequence recognition region in the second hairpin primer to form a T-shaped double strand; b) the target recognition region is used to recognize and bind the 12-18 nt sequence at the 3' end of the target miRNA; c) the dangling base should be mismatched with the next base of the target miRNA bound by the target recognition region, so that the second stem sequence has free activity after the first hairpin primer binds to the target miRNA, and the number of dangling bases can be 1-3 nt; the universal primer sequence is used for one-end primer binding in subsequent qPCR.

[0034] The second hairpin primer, from its 5' to 3' end, includes a third stem sequence, a universal primer loop sequence, a fourth stem sequence, a second stem sequence recognition region, a dangling base, and a target recognition region, wherein: a) the target recognition region is used to recognize and bind the 3-7 nt sequence at the 5' end of the target miRNA; b) the dangling base should be mismatched with the next base of the target miRNA bound to the target recognition region, so that the stem-loop structure of the second hairpin primer has free mobility after binding to the target miRNA, and the number of dangling bases can be 1-3 nt; c) the second stem sequence recognition region should have at least a 5 bp complementary region to the second stem sequence in the first hairpin primer; d) the third and fourth stem sequences form a stem structure, and the fourth stem sequence and the second stem sequence recognition region together serve as the DNA template for the ligation reaction, with the third stem sequence serving as the ligation end for ligation with the first hairpin primer; e) the universal primer loop sequence is used for one-end primer binding in subsequent qPCR; f) the ends of the second hairpin primer can be modified with phosphate groups to ensure subsequent ligation reactions while avoiding self-ligation.

[0035] qPCR primers and probes: qPCR primers are designed in the universal primer sequence of the first hairpin primer and the universal primer loop sequence of the second hairpin primer. The fluorescent probe sequence design should include the target recognition region sequence in the first hairpin primer. When used for multiplex fluorescence detection, different fluorescent groups and corresponding quenching groups can be used.

[0036] During the detection process, the first hairpin primer recognizes and binds to the target miRNA, causing its loop structure to be specifically bound by the target miRNA and opening the stem structure. After opening, the stem structure and the remaining miRNA sequence jointly and specifically attract the second hairpin primer for reassembly, forming a T-shaped double-stranded structure. The two primers, using the DNA strand as a clamp, are then ligated using T4 DNA ligase, forming long fragments containing both the miRNA's reverse-specific sequence and a universal sequence for subsequent qPCR detection. This reassembly ligation method improves ligation specificity through stem-loop design, eliminates Splint ligase dependence, reduces reaction costs and improves ligation efficiency by replacing T4 DNA ligase, and, moreover, introduces both specific and longer universal sequences after ligation, facilitating multiplex qPCR detection.

[0037] Example 1 This embodiment performs a structural feasibility test on the first hairpin primer and the second hairpin primer.

[0038] 1. Experimental setup and primer design: Taking miR-21 as the target miRNA as an example, the first and second hairpin primer sequences were designed. By comparing the stem lengths of different first hairpin primers and the recognition region length of the second stem sequence of the second hairpin primer, the appropriate structure required for ligation was initially determined. The specific sequences are shown in Table 1.

[0039] Table 1 Note: In the first hairpin primer, the bolded part is the first / second stem sequence, the underlined part is the target recognition region, and the italicized part is the dangling base; in the second hairpin primer, the bolded part is the second stem sequence recognition region, the underlined part is the target recognition region, and the italicized part is the dangling base; the first and second hairpin primers in the table are divided into three primer combinations: a, b, and c. The first hairpin primer-b and the first hairpin primer-c have the same sequence. The stem lengths of the three first hairpin primers (4, 5, and 6) and the second stem sequence recognition region lengths of the two second hairpin primers (4 and 5) were tested.

[0040] 2. Hairpin Structure Preparation and Reassembly Ligation Test: Three sets of first hairpin primers were placed in 50 mM Tris-HCl (pH 7.5) buffer and subjected to a denaturation-annealing program (e.g., 95℃ for 5 min; 1 h followed by slow temperature reduction to 23℃) to form hairpin structures. The three hairpin primer combinations (a, b, and c) were tested with 0 and 0.5 μM miR-21 samples, respectively. The concentrations of the first and second hairpin primers in the reaction solution were each 200 nM. Ligation was performed using 0.5 μL T4 DNA ligase (NEB, MO202L) at 25℃ for 30 min, followed by denaturation at 95℃ for 5 min.

[0041] Electrophoretic evaluation of ligation products: The denatured products after ligation in the three groups of experiments were analyzed by electrophoresis using an Agilent 4150 instrument and the D1000 ScreenTape electrophoresis reagent. The results are as follows: Figure 2 As shown in the figure, in group a, no ligation bands were observed in either the 0 or 0.5 μM detection products; in group b, no ligation band was observed in the 0 μM detection product, while the 0.5 μM detection product showed a clear ligation sequence fragment; and in group c, a small number of ligation sequence fragments were present in the 0 μM detection product. The comparison results between group a and either group b or c reflect that, to achieve ligation, the second stem sequence recognition region in the second hairpin primer should have at least a 5 bp complementary region to the second stem sequence in the first hairpin primer to ensure that the T-type double-stranded structure has the ability to stably complete ligation; at the same time, the stem length in the first hairpin primer should not be less than 6 bp. If the stem length is insufficient, the hairpin structure is not stable enough, resulting in non-specific hairpin opening and ligation, which leads to the false positive detection phenomenon in group c.

[0042] Example 2 This embodiment conducts a comparative test on the connection system and connection temperature.

[0043] 1. Experimental setup: Test comparison E. coliUnder two conditions—ligation with DNA ligase (NEB, M0205L) at 16℃ and ligation with T4 DNA ligase at 25℃—the hairpin primers in group b were used to detect miR-21 at concentrations of 0 and 100 nM. The ligation efficiency and the difference between positive and negative fluorescence signals were determined by fluorescent qPCR.

[0044] 2. Ligation and qPCR experiments: Using the first / second hairpin primers of group b prepared in Example 1, E. coli DNA ligase and T4 DNA ligase were used to ligate miR-21 at concentrations of 0 and 100 nM. The reaction system and reaction procedure are shown in Table 2.

[0045] Table 2 After ligation, the ligation products were detected by fluorescent qPCR. The enzyme reaction solution used was Pro Taq HS premixed probe qPCR reagent (Aikerui, AG11704). The qPCR primer and probe sequences used are shown in Table 3.

[0046] Table 3 The preparation of the qPCR reaction system and the reaction procedure are shown in Table 4.

[0047] Table 4 3. Analysis of qPCR detection results The results of qPCR detection of fluorescence amplification curves are as follows: Figure 3 and Figure 4 As shown.

[0048] The amplification curves in qPCR detection effectively reflected the amount of miR-21 used and the reassembly ligation effect. Both T4 and E. coli ligases could achieve the reassembly ligation of 100 nM miR-21 at their respective reaction systems and temperatures, and the detection results showed good specificity. However, after using T4 ligase... Figure 3 The negative dosage and the 100 nM dosage of miR-21 showed a higher final amplification inflection point (ΔCt) compared to the final ΔCt value of E. coli ligase. Figure 4 The value is close to 20, thus having the advantage of a greater difference between positive and negative signals, indicating that the T4 ligase system has stronger detection performance for low-concentration samples.

[0049] Example 3 This embodiment evaluates the detection performance of miR-21.

[0050] 1. Experimental setup: A miR-21 dosage gradient was set up in every 10-fold increments from 0.1 fM to 10 nM. The method of this invention was used to detect the dosage of miR-21 at each gradient and the negative dosage. The fluorescence signal of each reaction in the fluorescent qPCR was used to evaluate the ligation status of each gradient and the ability of this invention to detect low concentrations of miRNA.

[0051] 2. Ligation and qPCR experiments: Using the first / second hairpin primer combination, T4 ligase ligation reaction system and program, qPCR reaction system and program, and primers and probes, miR-21 at concentration gradients of 0, 0.1 fM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, and 1 μM were detected.

[0052] 3. Analysis of qPCR test results: The results of qPCR detection of fluorescence amplification curves are as follows: Figure 5 As shown in the results, the inflection points of the fluorescence amplification curves at different miR-21 detection levels exhibit a clear gradient. The inflection point of the amplification curve at a dosage of 0.1 fM is approximately 35, which is clearly distinguishable from the negative control. Therefore, this invention can effectively detect miRNA at a minimum concentration of 0.1 fM. Based on the signal difference between this and the negative sample, we believe that there is room for further reduction in the minimum detection concentration.

[0053] In summary, this invention develops a reassembly ligation detection scheme for miRNA detection. It designs hairpin primers with specific structures and develops a two-step detection process. The loop structure of the first hairpin primer specifically binds to the target miRNA and opens the stem structure. After opening, the stem structure and the remaining miRNA sequence jointly and specifically attract the second hairpin primer for reassembly, forming a T-shaped double-stranded structure. The two hairpin primers are then ligated using a DNA strand as a clamp, forming long fragments containing both the miRNA's reverse-specific sequence and a universal sequence for subsequent qPCR detection. The reassembly ligation method improves ligation specificity through stem-loop design, eliminates Splint ligase dependence, reduces reaction costs and improves ligation efficiency by replacing DNA ligase. Furthermore, the long fragments after ligation contain both specific and longer universal sequences, facilitating multiplex qPCR detection. It can effectively detect miRNA at a minimum concentration of 0.1 fM, and based on the signal difference with negative samples, there is room for further reduction in the minimum detection concentration.

[0054] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A hairpin primer for detecting miRNA, characterized in that, The hairpin primers include a first hairpin primer and a second hairpin primer; The first hairpin primer includes, from 5' to 3' end, a universal primer sequence, a first stem sequence, a first target recognition region, a first dangling base sequence, and a second stem sequence, wherein the universal primer sequence is the sequence that binds to one end of the qPCR primer, the first stem sequence and the second stem sequence are reverse complementary sequences, the first target recognition region is the sequence that binds to the 3' end of the target miRNA, and the first dangling base sequence is the sequence of the next base mismatch of the target miRNA that binds to the first target recognition region; The second hairpin primer includes, from 5' to 3', a third stem sequence, a universal primer loop sequence, a fourth stem sequence, a second stem sequence recognition region, a second dangling base sequence, and a second target recognition region, wherein the third stem sequence and the fourth stem sequence are reverse complementary sequences, the universal primer loop sequence is a sequence that binds to the other end of the qPCR primer, the second stem sequence recognition region is a sequence that binds to the second stem sequence in the hairpin primer 1, the second target recognition region is a sequence that binds to the 5' end of the target miRNA, and the second dangling base sequence is a sequence with a mismatched next base of the target miRNA that binds to the second target recognition region.

2. The hairpin primer for detecting miRNA according to claim 1, characterized in that, In the first hairpin primer, the first stem sequence and the second stem sequence are reverse complementary to form a hairpin structure, and the length of the hairpin structure is at least 6 bp; Optionally, the length of the second stem sequence recognition region in the second hairpin primer that is complementary to the second stem sequence in the first hairpin primer is at least 5 bp.

3. The hairpin primer for detecting miRNA according to claim 1 or 2, characterized in that, The second hairpin primer has phosphate groups modified at its 5' and 3' ends; Optionally, the primer at one end of the qPCR and the primer at the other end of the qPCR are primer pairs used for performing qPCR.

4. The hairpin primer for detecting miRNA according to any one of claims 1-3, characterized in that, The length of the first target recognition region complementary to the 3' end of the target miRNA is at least 12-18 bp; Optionally, the length of the first suspended base sequence is 1-3 nt.

5. The hairpin primer for detecting miRNA according to any one of claims 1-4, characterized in that, The second target recognition region is complementary to the 5' end of the target miRNA and has a length of at least 3-7 bp; Optionally, the length of the second dangling base sequence is 1-3 nt.

6. The use of the hairpin primer for detecting miRNA as described in any one of claims 1-5 in the detection of miRNA.

7. A kit for detecting miRNA, characterized in that, The kit includes the hairpin primers and qPCR primers and probes for detecting miRNA as described in any one of claims 1-5; The qPCR primer at one end is a sequence that binds to the universal primer sequence in the first hairpin primer, and the primer at the other end is a sequence that binds to the universal primer loop sequence in the second hairpin primer; The probe sequence includes the sequence of the first target recognition region in the first hairpin primer, and one end of the probe is modified with a fluorescent group and the other end is modified with a quenching group.

8. A method for detecting miRNA, characterized in that, The method includes: mixing the hairpin primer for detecting miRNA as described in any one of claims 1-5 with the sample to be tested and DNA ligase to perform a ligation reaction; taking the ligation reaction product and performing qPCR detection using qPCR primers and probes; and determining the miRNA content in the sample to be tested by the fluorescence amplification curve.

9. The method for detecting miRNA according to claim 8, characterized in that, The DNA ligase includes T4 DNA ligase or E. coli DNA ligase.

10. The method for detecting miRNA according to claim 8, characterized in that, The conditions for the connection reaction are: 15~30℃, 25~65 min; 93~98℃, 3~10 min.

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