A dual-target miRNA detection method based on crisper and rolling circle amplification

Abstract

The application discloses a double-target miRNA detection method based on CRISPR and rolling circle amplification, which comprises the following steps: (1) mixing a padlock probe, a connecting DNA and miRNA-21 and miRNA-155, carrying out annealing treatment, incubating the mixture in a DNA ligase reaction buffer system by using a DNA ligase, then terminating the reaction by heat treatment, and obtaining a circular DNA template; (2) incubating the circular DNA template with a DNA polymerase, dNTPs, recombinant albumin, a primer and a polymerase reaction buffer, then terminating the reaction by heat treatment, and obtaining an RCA product; (3) adding pre-assembled Cas12a-crRNA, the RCA product and FQ fluorescent probes into a buffer, and incubating; and (4) measuring a fluorescent signal. Through optimization of a detection system, the application successfully realizes detection of double-target miRNA, and has high detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and specifically relates to a dual-target miRNA detection method based on CRISPR and rolling circle amplification. Background Technology

[0002] In clinical practice, biomarkers are of great value for disease diagnosis, treatment, and prognosis. The discovery of circulating miRNAs in the blood has brought new hope to the research of tumor markers. Studies have found that miR-155 is higher in the serum of HCC patients than in normal controls, but its level decreases in the serum of postoperative patients; while miR-21 is highly expressed in the serum of HCC patients, but there is no significant difference between normal individuals and hepatitis patients. Therefore, miR-21 and miR-155 alone can be used to diagnose liver cancer, but simultaneous detection of both can improve the accuracy of detection and reduce the occurrence of false positives.

[0003] The CRISPR / Cas system is a highly efficient gene-editing tool, and detection methods based on the CRISPR / Cas system represent a promising new approach for nucleic acid detection. Combining the CRISPR / Cas system with nucleic acid amplification technologies (such as PCR, SDA, RCA, and loop-mediated isothermal amplification) allows for accurate and sensitive detection of low-concentration samples. Rolling circle amplification (RCA) is a simple and efficient isothermal enzymatic amplification technique with advantages such as high efficiency, high fidelity, and high sensitivity. Methods for detecting miRNA based on rolling circle amplification and CRISPR / Cas12a have been reported, for example, in patent document CN 117646069 A; however, currently, only single-target detection is possible, and multi-target detection has not been reported. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, this invention proposes a dual-target miRNA detection method based on CRISPR and rolling circle amplification, which achieves the goal of simultaneously detecting two miRNAs.

[0005] The technical solution of the present invention is as follows:

[0006] A dual-target miRNA detection method based on CRISPR and rolling circle amplification includes the following steps:

[0007] (1) The padlock probe, ligation DNA, miRNA-21, and miRNA-155 were mixed, annealed, and incubated with DNA ligase in a ligase reaction buffer system. The reaction was then terminated by heat treatment to obtain a circular DNA template. The sequence of the padlock probe is shown in SEQ ID NO.1. The sequence of the ligation DNA is shown in SEQ ID NO.2.

[0008] (2) The circular DNA template was incubated with DNA polymerase, dNTPs, recombinant albumin, primers and polymerase reaction buffer, and then the reaction was terminated by heat treatment to obtain the RCA product; the sequence of the primers is shown in SEQ ID NO.5;

[0009] (3) Add pre-assembled Cas12a-crRNA, RCA product and FQ fluorescent probe to buffer solution and incubate; the sequence of the crRNA is shown in SEQ ID NO.6;

[0010] (4) Measurement of fluorescence signal.

[0011] Preferably, in step (1), the annealing conditions are 95°C for 1 min; 80°C for 1 min; 70°C for 1 min; 60°C for 1 min; 50°C for 1 min; 40°C for 1 min; and 30°C for 10 min.

[0012] Preferably, in step (1), the incubation conditions are: incubation at 25°C for 10 to 50 minutes.

[0013] Preferably, in step (2), the incubation conditions are: incubation at 30°C for 1 to 3 hours.

[0014] Preferably, in step (3), the incubation conditions are: incubation at 37°C for 20 to 120 minutes.

[0015] Preferably, step (1) is as follows:

[0016] 100 nM padlock probe, 100 nM ligation DNA, 10 pM miRNA-21, and 10 pM miRNA-155 were mixed, annealed, and incubated with 3–15 U DNA ligase in a 20 μL ligase reaction buffer system. The reaction was then terminated by heat treatment to obtain a circular DNA template. The sequence of the padlock probe is shown in SEQ ID NO.1, and the sequence of the ligation DNA is shown in SEQ ID NO.2.

[0017] Preferably, in the annealing system, 2 μL each of the padlock probe and L-DNA (100 nM), 2 μL each of the target miRNA-21 and miRNA-155, and then 8 μL of water treated with DEPC (diethyl pyrocarbonate) are added.

[0018] Preferably, step (2) is as follows:

[0019] 5 μL of circular DNA template was incubated with 3–15 U DNA polymerase, 3 μL of dNTPs, 1 μL of recombinant albumin, 3 μL of primers, and 2 μL of polymerase reaction buffer. The reaction was then terminated by heat treatment to obtain the RCA product. The sequence of the primers is shown in SEQ ID NO.5.

[0020] Preferably, in step (3), the concentration ratio of Cas12a to crRNA is 20-100 nM:100 nM.

[0021] Preferably, the sequence of the FQ fluorescent probe is 6-FAM-TTTTTT-BHQ1.

[0022] Furthermore, the present invention also provides a kit for detecting miRNA-21 and miRNA-155, the kit comprising: a padlock probe, ligated DNA, miRNA-21, miRNA-155, Splint R ligase, dNTPs, recombinant albumin, primers, phi29 DNA polymerase, Cas12a-crRNA, and FQ fluorescent probe.

[0023] The sequence of the padlock probe is shown in SEQ ID NO.1, the sequence of the DNA linker is shown in SEQ ID NO.2, the sequence of miRNA-155 is shown in SEQ ID NO.3, the sequence of miRNA-21 is shown in SEQ ID NO.4, the sequence of the primer is shown in SEQ ID NO.5, and the sequence of the crRNA is shown in SEQ ID NO.6.

[0024] The beneficial effects of this invention are:

[0025] Since CRISPR / Cas12a and rolling circle amplification can be used to detect miRNA155 and miRNA-21 separately, to achieve simultaneous detection of both in the same system, it is essential to ensure that no fluorescence appears when only one miRNA is present, indicating a negative result. Simultaneously, interference between the two must be avoided to ensure the accuracy of results when simultaneously detecting miRNA155 and miRNA-21. This invention successfully achieves the detection of dual-target miRNAs through optimization of the detection system.

[0026] To verify the detection capability of the method of this invention in complex biological systems, the inventors conducted tests in human serum. The results showed that the method of this invention can achieve the determination of dual-target miRNAs at the femtosecond (fM) level in complex biological systems. Attached Figure Description

[0027] Figure 1 Fluorescence spectra obtained using different reaction components for the analysis of miRNA-21 and miRNA-155. The concentrations of miRNA-21 and miRNA-155 were both 10 pM.

[0028] Figure 2Figure 1 shows the optimized results of the reaction system. (A) Optimized results of the amount of Splint R ligase; (B) Optimized results of the amount of phi29 polymerase; (C) Results of Cas12a concentration; (D) Optimized results of cyclization time; (E) Optimized results of RCA time; (F) Optimized results of CRISPR / Cas12a cleavage time. Error bars represent the standard deviation of three measurements.

[0029] Figure 3 : Sensitivity detection results graph. Error bars represent the standard deviation of three measurements. The results show a linear relationship between the fluorescence intensity of the solution and the concentrations of miRNA-21 (A) and miRNA-155 (B), with both miRNA-21 and miRNA-155 concentrations ranging from 1 fM to 10 pM.

[0030] Figure 4 Selective results. Using miRNA-122 and miRNA-10b as controls, when the concentration of miRNA-21(A) or miRNA-155(B) was fixed at 10 pM, a significant increase in fluorescence signal intensity was only observed when the other target was miRNA-155 or miRNA-21. Furthermore, the signal intensity of the mixture of target miRNA-21 and miRNA-155 with interfering agents (miRNA-122 and miRNA-10b) was almost the same as that of target miRNA-21 and miRNA-155 alone. Detailed Implementation

[0031] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0032] 1. Dual-target miRNA detection method

[0033] The oligonucleotide sequences required for this method are listed in Table 1-1 and were synthesized by Sangon Biotech Co., Ltd., as detailed in the table below:

[0034] The sequences used in this invention:

[0035] Table 1-1

[0036]

[0037]

[0038] Note: In the sequence listing, the base T in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.6 represents U.

[0039] The steps for this method are as follows:

[0040] (1) Rolling circle amplification (RCA) is an efficient enzymatic isothermal reaction that uses a circular probe as a template to generate long tandem single-stranded DNA or RNA products under the initiation of short primers. When the target miRNA-21 and miRNA-155 are both absent, or only one of them is present, Padlock and L-DNA are two independent single-stranded structures, which cannot initiate the subsequent rolling circle amplification reaction, and the reaction terminates. When the target miRNA-21 and miRNA-155 are present simultaneously, Padlock and L-DNA can be ligated into a circular template by annealing under the action of Splint R ligase. With the help of primer DNA and phi29 polymerase, a single-stranded DNA product containing thousands of molecules that can excite the trans-cleavage activity of the CRISPR / Cas12a system can be amplified along the circular template. Cas12a is used to cleave the FQ fluorescent probe to produce detectable fluorescence.

[0041] (2) Formation of the ring template

[0042] To synthesize a circular DNA template in a 20 μL system, prior to the template circularization process, 2 μL of a 100 nM padlock probe, 2 μL of a 100 nM ligation DNA (L-DNA), and 2 μL each of target miRNA-21 and miRNA-155 were mixed with 8 μL of DEPC-treated water and annealed under the following conditions: 95 °C for 1 min; 80 °C for 1 min; 70 °C for 1 min; 60 °C for 1 min; 50 °C for 1 min; 40 °C for 1 min; and 30 °C for 10 min. The template was then ligated into a circular form by incubation with 9 U of Splint R ligase at 25 °C for 40 min in 1×Splint R Buffer. The process was terminated by heat treatment (65 °C for 10 min) to obtain the circular DNA template (CDT). The CDT was stored at -20 °C for use in step (3).

[0043] (3) Generation of RCA amplification products

[0044] For the RCA reaction, 5 μL of the CDT prepared in step (2) above was incubated with phi29 DNA polymerase (6 μL, 1.5 U / μL), dNTPs (3 μL, 5 mM), recombinant albumin (1 μL, 2 mg / ml), primer DNA (3 μL, 100 nM), and polymerase reaction buffer (2 μL, 10×). The reaction mixture was incubated at 30°C for 2.5 h, and then terminated by heat treatment (65°C, 10 min). Store at -20°C for use in step (3).

[0045] (4) Activation of CRISPR / Cas12a reverse enzyme digestion function

[0046] Cas12a proteins (LbCpf1, FnCpf1, AsCpf1) possess both cis and trans cleavage activities on single-stranded DNA. When Cas12a forms a ternary complex with a specific crRNA and its target DNA, this complex acquires strong trans cleavage activity, breaking the single-stranded DNA into 2-4 nucleotide fragments. Utilizing this property of Cas12a, we first converted the signal detection of dual-target miRNAs into DNA signal detection via rolling circle amplification (rolling circle amplification). Then, the single-stranded repeat unit of the rolling circle amplification reaction is complementary to the designed crRNA and excites the trans cleavage activity of Cas12a, which is used to cleave the FQ signal probe, generating detectable fluorescence.

[0047] CRISPR / Cas12a reverse digestion activation reaction conditions: CRISPR / Cas12a digestion assay was performed in 1×NEBuffer 2.1. Pre-assembled Cas12a-crRNA (80 / 100 nM) was added along with 10 μL of activator (RCA product) and 0.5 μM FQ fluorescent probe to a total volume of 20 μL, and incubated at 37°C for 60 min. This was reserved for use in step (5).

[0048] (5) Measurement of fluorescence signal

[0049] Because one end of the FQ probe is modified with the fluorescent group FAM and the other end with the quenching group BHQ, the quenching group can quench the fluorescence of the fluorescent group when the probe is intact. However, activated Cas12a can cleave the FQ probe into 2-4 nt fragments, separating the fluorescent group and the quenching group, thus restoring the fluorescence.

[0050] Measurement conditions for fluorescence signal: Add 80 μL of enzyme-free water to the reaction solution in step (4), and then use a fluorescence meter to excite the fluorescence intensity with light at a wavelength of 475 nm.

[0051] The feasibility of this method was investigated by measuring the fluorescence intensity detected using a fluorescence meter. Figure 1 As shown, RCA amplification can only be completed when the target miRNA-21 and miRNA-155 are present simultaneously, thereby activating the trans-cleavage activity of Cas12a and cleaving the FQ probe to restore the fluorescence intensity.

[0052] 2. Optimization of the reaction system

[0053] like Figure 2 As shown, the concentrations of ligase Splint R, polymerase phi29, and Cas12a, as well as the time required for circular template formation, RCA amplification, and incubation for Cas12a cleavage, were optimized to obtain the optimal reaction conditions.

[0054] Example 1: Sensitivity Detection

[0055] The linear response to the analyte is crucial for the analysis; therefore, we measured the changes in fluorescence intensity in the presence of different concentrations of miRNA-21 and miRNA-155, and used the fluorescence spectrum value at 528 nm as the basis for judgment. Figure 3 The results showed that the fluorescence intensity at 528 nm gradually increased with increasing concentrations of miRNA-21 and miRNA-155, remaining within the range of 1 fM to 10 pM. The fluorescence intensity gradually decreased with decreasing concentrations of miRNA-21 and miRNA-155. The linear regression equations for miRNA-21 and miRNA-155 were Y = 89.62X - 92.06 and Y = 84.50X - 74.20, respectively. These results indicate that this biosensor can achieve highly sensitive detection of miRNA-21 and miRNA-155.

[0056] Example 2: Accuracy Detection

[0057] The detection capability of this method in complex biological samples was studied by spiked recovery rate analysis. Specifically, a quantitative amount of standard substance was added to a sample matrix without the analyte, and the sample was analyzed according to the sample processing steps. The ratio of the obtained result to the theoretical value was then compared. The specific process is as follows: First, 3% human serum, 1 fM standard miRNA-21 sample, and 1 fM standard miRNA-155 sample were added to the buffer for rolling circle amplification reaction. After rolling circle amplification reaction, the dual-target miRNA detection method of this invention described above was used for detection. The results showed that the detected miRNA-21 concentration was 1.002 fM, the recovery rate was 100.2%, and the relative standard deviation (RSD) was 0.4%. The detected miRNA-155 concentration was 1.070 fM, the recovery rate was 107.0%, and the relative standard deviation (RSD) was 3.7%.

[0058] Example 3: Specificity Detection

[0059] Using miRNA-122 and miRNA-10b as controls, the concentration of miRNA-21 or miRNA-155 was fixed at 10 pM, and then different concentrations of another target were added. The dual-target miRNA detection method of this invention described above was used for detection. The results showed ( Figure 4 There was a significant increase in fluorescence signal intensity only when the other target was miRNA-155 or miRNA-21, and the signal intensity of the mixture of target miRNA-21 and miRNA-155 with interfering agents (miRNA-122 and miRNA-10b) was almost the same as that of target miRNA-21 and miRNA-155 alone.

[0060] Example 4: A kit for detecting miRNA-21 and miRNA-155

[0061] The kit includes: padlock probe, ligation DNA, miRNA-21, miRNA-155, Splint R ligase, dNTPs, recombinant albumin, primers, phi29 DNA polymerase, Cas12a-crRNA, and FQ fluorescent probe.

[0062] The sequence of the padlock probe is shown in SEQ ID NO.1, the sequence of the DNA linker is shown in SEQ ID NO.2, the sequence of miRNA-155 is shown in SEQ ID NO.3, the sequence of miRNA-21 is shown in SEQ ID NO.4, the sequence of the primer is shown in SEQ ID NO.5, and the sequence of the crRNA is shown in SEQ ID NO.6.

[0063] In practice, the specific dosage of each component in the kit should refer to the content described in point 1 above.

[0064] The above description is only a part of the embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for detecting double-target miRNA based on CRISPR and rolling circle amplification for non-diagnostic purposes, characterized by, Comprising the following steps: (1) Mix the padlock probe, the connecting DNA and miRNA-21, miRNA-155, anneal, incubate with DNA ligase in the ligase reaction buffer system, then terminate the reaction by heat treatment, and obtain the circular DNA template; the sequence of the padlock probe is shown as SEQ ID NO. 1; the sequence of the connecting DNA is shown as SEQ ID NO. 2; the DNA ligase is Splint R ligase; (2) Incubate the circular DNA template with DNA polymerase, dNTPs, recombinant albumin, primer and polymerase reaction buffer, then terminate the reaction by heat treatment, and obtain the RCA product; the sequence of the primer is shown as SEQ ID NO. 5; the DNA polymerase is phi29 DNA polymerase; (3) Add pre-assembled Cas12a-crRNA, RCA product and FQ fluorescent probe in the buffer, and incubate; the sequence of the crRNA is shown as SEQ ID NO. 6; the sequence of the FQ fluorescent probe is 6-FAM-TTTTTT-BHQ1; (4) Measure the fluorescence signal. 2.The dual-target miRNA detection method based on CRISPR and rolling circle amplification for non-diagnostic purposes according to claim 1, wherein, In step (1), the annealing condition is 95℃, 1min; 80℃, 1min; 70℃, 1min; 60℃, 1min; 50℃, 1min; 40℃, 1min; 30℃, 10min. 3.The dual-target miRNA detection method based on CRISPR and rolling circle amplification according to claim 1, characterized in that, In step (1), the incubation condition is 25℃ for 10-50min. 4.The method of claim 1, wherein the method is a method for detecting two miRNAs based on CRISPR and rolling circle amplification for non-diagnostic purposes, characterized by, In step (2), the incubation condition is 30℃ for 1-3h. 5.The dual-target miRNA detection method based on CRISPR and rolling circle amplification according to claim 1, characterized in that, In step (3), the incubation condition is 37℃ for 20-120min. 6.The dual-target miRNA detection method based on CRISPR and rolling circle amplification for non-diagnostic purposes according to claim 1, wherein, Step (1) is: Mix 100nM padlock probe, 100nM connecting DNA and 10pM miRNA-21, 10pM miRNA-155, anneal, incubate with 3-15U DNA ligase in 20μL ligase reaction buffer system, then terminate the reaction by heat treatment, and obtain the circular DNA template; the sequence of the padlock probe is shown as SEQ ID NO. 1; the sequence of the connecting DNA is shown as SEQ ID NO.

2. 7.The dual-target miRNA detection method based on CRISPR and rolling circle amplification for non-diagnostic purposes according to claim 1, wherein, Step (2) is: Incubate 5μL circular DNA template with 3-15U DNA polymerase, 3μL dNTPs, 1μL recombinant albumin, 3μL primer and 2μL polymerase reaction buffer, then terminate the reaction by heat treatment, and obtain the RCA product; the sequence of the primer is shown as SEQ ID NO.

5.

8. The non-diagnostic dual-target miRNA detection method based on CRISPR and rolling circle amplification according to claim 1, characterized in that, In step (3), the concentration ratio of Cas12a and crRNA is 20-100nM:100nM.

9. A kit for detecting miRNA-21 and miRNA-155, characterized in that, The kit comprises: padlock probe, connecting DNA, miRNA-21, miRNA-155, Splint R ligase, dNTPs, recombinant albumin, primer, phi29 DNA polymerase, Cas12a-crRNA and FQ fluorescent probe; The sequence of the padlock probe is shown as SEQ ID NO. 1, the sequence of the connecting DNA is shown as SEQ ID NO. 2, the sequence of the miRNA-155 is shown as SEQ ID NO. 3, the sequence of the miRNA-21 is shown as SEQ ID NO. 4, the sequence of the primer is shown as SEQ ID NO. 5, the sequence of the crRNA is shown as SEQ ID NO. 6, and the sequence of the FQ fluorescent probe is 6-FAM-TTTTTT-BHQ1.

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