Dual-target mirna detection method based on crispr and rolling circle amplification

Abstract

A dual-target miRNA detection method based on CRISPR and rolling circle amplification, comprising the following steps: (1) mixing a padlock probe, a ligation DNA, miRNA-21, and miRNA-155, performing annealing, incubating with a DNA ligase in a ligase reaction buffer system, and then terminating the reaction by means of heat treatment to obtain a circular DNA template; (2) incubating the circular DNA template with a DNA polymerase, dNTPs, recombinant albumin, a primer, and a polymerase reaction buffer, and then terminating the reaction by means of heat treatment to obtain an RCA product; (3) adding pre-assembled Cas12a-crRNA, the RCA product, and an FQ fluorescent probe to a buffer, and incubating; and (4) measuring fluorescence signals. By means of optimizing the detection system, the detection of dual-target miRNAs is successfully achieved, and the detection sensitivity is high.

Description

A double-target miRNA detection method based on CRISPR and rolling circle amplification TECHNICAL FIELD

[0001] The present application belongs to the technical field of gene detection, and particularly relates to a double-target miRNA detection method based on CRISPR and rolling circle amplification. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, especially in Asia. The incidence and mortality rate in southern China is relatively high. In clinical practice, biomarkers play a very important role in the diagnosis, treatment and prognosis of diseases. The discovery of circulating miRNA in blood brings new hope for the research of tumor markers. Studies have found that miR-155 is higher in the serum of HCC patients than in normal controls, but it will decrease 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 people and hepatitis patients. Therefore, single miR-21 and miR-155 can be used to diagnose liver cancer, but the simultaneous detection of the two 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. The detection method based on the CRISPR / Cas system is a promising new nucleic acid detection method. Combining the CRISPR / Cas system with nucleic acid amplification technology (such as PCR and SDA, RCA, loop-mediated isothermal amplification) for detection can achieve accurate and sensitive detection of low-concentration samples. Rolling circle amplification (RCA) is a simple and efficient isothermal enzyme amplification technology with high efficiency, high fidelity and high sensitivity. Methods for detecting miRNA based on rolling circle amplification and CRISPR / Cas12a have been reported, such as patent document CN 117646069 A, but currently only single-target detection can be achieved, and no multi-target detection has been reported. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a double-target miRNA detection method based on CRISPR and rolling circle amplification, which realizes the simultaneous detection of two miRNAs.

[0005] The technical scheme of the present application is as follows:

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

[0007] (1) Mix the padlock probe, the connecting DNA and the miRNA-21 and the miRNA-155, anneal, incubate with the 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;

[0008] (2) Incubate the circular DNA template with the DNA polymerase, the dNTPs, the recombinant albumin, the primer and the 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;

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

[0010] (4) Measure the fluorescent signal.

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

[0012] Preferably, in step (1), the incubation conditions are 25℃ for 10-50 min.

[0013] Preferably, in step (2), the incubation conditions are 30℃ for 1-3 hours.

[0014] Preferably, in step (3), the incubation conditions are 37℃ for 20-120 min.

[0015] Preferably, step (1) is:

[0016] Mix 100nM padlock probe, 100nM connecting DNA and 10pM miRNA-21 and 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.

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

[0018] Preferably, step (2) is:

[0019] Incubate 5 μL of circular DNA template with 3-15 U of DNA polymerase, 3 μL of dNTPs, 1 μL of recombinant albumin, 3 μL of primers, and 2 μL of polymerase reaction buffer, and then terminate the reaction 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] Further, the present application also provides a kit for detecting miRNA-21 and miRNA-155, which comprises: a padlock probe, a connecting DNA, miRNA-21, miRNA-155, a Splint R ligase, dNTPs, recombinant albumin, primers, phi29 DNA polymerase, Cas12a-crRNA, and a FQ fluorescent probe.

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

[0024] The present application has the following beneficial effects:

[0025] Since CRISPR / Cas12a and rolling circle amplification can be used to detect miRNA155 and miRNA-21 respectively, in order to realize the simultaneous detection of both in the same system, it is necessary to ensure that no fluorescence appears when only one of the miRNAs exists, which is negative, and to avoid mutual interference between the two, so as to ensure the accuracy of the results when detecting miRNA155 and miRNA-21 at the same time. The present application successfully realizes the detection of double-target miRNA through the optimization of the detection system.

[0026] In order to test the detection ability of the method of the present application in complex biological systems, the inventors tested it in human serum. The results showed that the method of the present application can realize the detection of double-target miRNA at the level of fM in complex biological systems. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1: Fluorescence spectra obtained with 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 2: Optimization results of the reaction system. (A) Optimization results of the amount of Splint R ligase; (B) Optimization results of the amount of phi29 polymerase; (C) Results of Cas12a concentration; (D) Optimization results of cyclization time; (E) Optimization results of RCA time; (F) Optimization results of CRISPR / Cas12a cleavage time. Error bars represent the standard deviation of three measurements.

[0029] Figure 3: Sensitivity detection results. 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: Selectivity 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 the interfering agents (miRNA-122 and miRNA-10b) was almost identical to that of the individual target miRNA-21 and miRNA-155. 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] Note: In the sequence listing, the base T in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.6 represents U.

[0038] The steps for this method are as follows:

[0039] (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 at the same time, 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.

[0040] (2) Formation of the ring template

[0041] 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, 10 min) to obtain the circular DNA template (CDT). The CDT was stored at -20 °C for use in step (3).

[0042] (3) Generation of RCA amplification products

[0043] 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).

[0044] (4) Activation of CRISPR / Cas12a reverse digestion function

[0045] 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.

[0046] 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).

[0047] (5) Measurement of fluorescence signal

[0048] Because one end of the FQ probe is modified with the fluorophore FAM and the other end with the quencher BHQ, the quencher can quench the fluorescence of the fluorophore when the probe is intact. However, activated Cas12a can cleave the FQ probe into 2-4 nt fragments, separating the fluorophore and the quencher, thus restoring the fluorescence.

[0049] 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.

[0050] The feasibility of this method was investigated by measuring the fluorescence intensity detected by a fluorescence meter. As shown in Figure 1, RCA amplification could only be completed when both target miRNA-21 and miRNA-155 were present, activating the trans-cleavage activity of Cas12a and cleaving the FQ probe to restore the fluorescence intensity.

[0051] 2. Optimization of the reaction system

[0052] As shown in Figure 2, the concentrations of ligase Splint R, polymerase phi29, and Cas12a, as well as the time required for forming a circular template, RCA amplification, and incubation for Cas12a cleavage, were optimized to obtain the optimal reaction conditions.

[0053] Example 1: Sensitivity Detection

[0054] Linear response to the analytes is crucial for the analysis; therefore, we measured the changes in fluorescence intensity at different concentrations of miRNA-21 and miRNA-155, using the fluorescence spectrum at 528 nm as the basis for judgment. As shown in Figure 3, the fluorescence intensity at 528 nm gradually increased with increasing miRNA-21 and miRNA-155 concentrations, remaining within the range of 1 fM to 10 pM. The fluorescence intensity gradually decreased with decreasing miRNA-21 and miRNA-155 concentrations. 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.

[0055] Example 2: Accuracy Detection

[0056] The detection capability of this method in complex biological samples was studied by spiked recovery rate analysis. Specifically, a quantitative 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%.

[0057] Example 3: Specificity Detection

[0058] 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 the present invention described above was used for detection. The results (Figure 4) show that a significant increase in fluorescence signal intensity only occurred 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 the interfering agents (miRNA-122 and miRNA-10b) was almost the same as that of the individual target miRNA-21 and miRNA-155.

[0059] Example 4: A kit for detecting miRNA-21 and miRNA-155

[0060] 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.

[0061] 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.

[0062] In practice, the specific dosage of each component in the kit should refer to the content described in point 1 above.

[0063] 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, characterized in that, The method comprises the following steps: (1) mixing the padlock probe, the connecting DNA and the miRNA-21 and the miRNA-155, annealing, incubating with the DNA ligase in the DNA ligase reaction buffer system, then terminating the reaction by heat treatment, and obtaining 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; (2) incubating the circular DNA template with the DNA polymerase, the dNTPs, the recombinant albumin, the primer and the polymerase reaction buffer, then terminating the reaction by heat treatment, and obtaining the RCA product; the sequence of the primer is shown as SEQ ID NO. 5; (3) adding the pre-assembled Cas12a-crRNA, the RCA product and the FQ fluorescent probe in the buffer, and incubating; the sequence of the crRNA is shown as SEQ ID NO. 6; (4) measuring the fluorescence signal. 2.The dual-target miRNA detection method based on CRISPR and rolling circle amplification according to claim 1, wherein, In step (1), the annealing condition is 95℃ for 1 minute, 80℃ for 1 minute, 70℃ for 1 minute, 60℃ for 1 minute, 50℃ for 1 minute, 40℃ for 1 minute and 30℃ for 10 minutes. 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 10-50 minutes. 4.The dual-target miRNA detection method based on CRISPR and rolling circle amplification according to claim 1, wherein, In step (2), the incubation condition is 30℃ for 1-3 hours. 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 20-120 minutes. 6.The dual-target miRNA detection method based on CRISPR and rolling circle amplification according to claim 1, wherein, Step (1) is: Mixing 100nM padlock probe, 100nM connecting DNA, 10pM miRNA-21 and 10pM miRNA-155, annealing, incubating with 3-15U DNA ligase in 20μL DNA ligase reaction buffer system, then terminating the reaction by heat treatment, and obtaining 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 according to claim 1, characterized in that, Step (2) is: Incubating 5μL circular DNA template with 3-15U DNA polymerase, 3μL dNTPs, 1μL recombinant albumin, 3μL primer and 2μL polymerase reaction buffer, then terminating the reaction by heat treatment, and obtaining the RCA product; the sequence of the primer is shown as SEQ ID NO.

5. 8.The dual-target miRNA detection method based on CRISPR and rolling circle amplification according to claim 1, wherein, In step (3), the concentration ratio of Cas12a and crRNA is 20-100nM:100nM. 9.The dual-target miRNA detection method based on CRISPR and rolling circle amplification according to claim 1, wherein, The sequence of the FQ fluorescent probe is 6-FAM-TTTTTT-BHQ1.

10. A kit for detecting miRNA-21 and miRNA-155, characterized in that, The kit comprises the padlock probe, the connecting DNA, the miRNA-21, the miRNA-155, the Splint R ligase, the dNTPs, the recombinant albumin, the primer, the phi29 DNA polymerase, the Cas12a-crRNA and the 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, and the sequence of the crRNA is shown as SEQ ID NO.

6. 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, and the sequence of the crRNA is shown as SEQ ID NO

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