Method for detecting miRNA by magnetic bead-assisted isothermal amplification

Abstract

The present invention discloses a method for detecting miRNA by isothermal amplification assisted by magnetic beads, comprising the following steps: coupling an HP probe to the surface of a streptavidin-coated magnetic bead; mixing the HP probe modified by the magnetic beads, a target Target, and a target miRNA sequence to form a reaction system, and performing an isothermal amplification reaction; and collecting the fluorescent signal of the modified magnetic beads. The present invention provides a method for detecting miRNA by isothermal amplification assisted by magnetic beads, which uses magnetic beads to separate the target to be detected from a complex environment, uses isothermal amplification to realize the circulation of miRNA, and realizes the amplified detection of miRNA.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a method for detecting miRNA by isothermal amplification assisted by magnetic beads. Background Art

[0002] A biomarker is a biochemical indicator, and its changes can indicate changes or possible changes in the structure or function of systems, organs, tissues, cells, and subcellular structures. The detection of biomarkers can reflect changes in human physiological processes, thereby making early diagnosis of diseases; it can also be used to determine the degree of intervention of the drugs used on the disease and to determine drug targets. There are many types of biomarkers, and common biomarkers include proteins and nucleic acids.

[0003] MicroRNA is a small RNA with a length of about 19 to 24 nucleotides. It can regulate the expression of specific genes by binding to messenger RNA, thereby acting on mRNA and inhibiting its translation. Therefore, it has an important impact on the physiological processes of organisms. Studies have shown that abnormal expression of miRNA is related to the occurrence and development of various cancers, and can be used as a disease marker for cancer, for the detection and diagnosis of cancer. However, they are usually present in complex samples such as cells, exosomes or body fluids at a low abundance, so their detection faces the problem of "whether it can be measured" and "whether it can be measured accurately".

[0004] Therefore, in order to ensure the smooth detection of complex samples, it is necessary to separate the target from other interfering components. Common separation methods for complex biological samples include magnetic separation, chromatographic separation, capillary electrophoresis separation, etc. Magnetic beads have a high magnet content and respond quickly to external magnetic fields. They can be bio-modified on the surface of magnetic beads to combine with a variety of analytes and are easy to operate. They are widely used in sample processing and biological analysis.

[0005] The extremely short length, extremely low content and high sequence homology of miRNA pose great challenges in detection. The traditional gold standard for nucleic acid detection is quantitative PCR (qPCR), but it requires complex reaction procedures and specific instruments. Isothermal amplification has become a powerful nucleic acid quantification method for miRNA detection. Compared with PCR, isothermal amplification does not require precise control of temperature cycles, and can quickly and effectively amplify target nucleic acids or recognition signals under simple conditions, thereby improving detection sensitivity. Summary of the invention

[0006] The technical problem to be solved by the present invention is to address the problem of low abundance of miRNA and interference from other substances, and to provide a method for detecting miRNA by isothermal amplification assisted by magnetic beads. The method uses magnetic beads to separate the target to be detected from a complex environment, and uses isothermal amplification to achieve the circulation of miRNA, thereby realizing the amplified detection of miRNA.

[0007] In order to achieve the above object, the present invention provides a method for detecting miRNA by isothermal amplification assisted by magnetic beads, comprising the following steps:

[0008] S1, coupling the HP probe to the surface of streptavidin-coated magnetic beads;

[0009] S2, mixing the HP probe modified with magnetic beads, the target Target, and the target miRNA sequence to form a reaction system, and performing an isothermal amplification reaction;

[0010] S3. Collect the fluorescence signal of the modified magnetic beads.

[0011] In the above method, further, the DNA sequence of the HP probe is shown as SEQ ID NO.1.

[0012] In the above method, further, the HP probe is modified with biotin that can be coupled to streptavidin-coated magnetic beads.

[0013] In the above method, further, the HP probe is modified with a FAM fluorescent group and a BHQ quenching group in the middle.

[0014] In the above method, further, the DNA sequence of the target Target sequence is shown as SEQ ID NO.2.

[0015] In the above method, further, the conditions of the isothermal amplification reaction in S2 are:

[0016] Incubate at 37°C for 150 to 240 min respectively.

[0017] In the above method, further, in S3, the fluorescence signal of the modified magnetic beads is collected, the excitation wavelength is set to 488 nM, the collection wavelength range is 505-525 nm, and the fluorescence signal of the magnetic beads is collected.

[0018] The above method further comprises adding 5-10 mM Mg to the reaction system of S2. 2+ Solution.

[0019] The above method further comprises adding 0.1-0.2 U / μL polymerase solution to the reaction system of S2.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] The present invention provides a method for isothermal amplification and detection of miRNA assisted by magnetic beads, wherein magnetic beads are used to separate and enrich miRNA from a complex environment, and an isothermal amplification reaction is used to realize the circulation of miRNA to achieve signal amplification. Compared with the traditional method, the isothermal amplification reaction uses a hairpin probe that can form a primer by itself as a template, and isothermal amplification can be performed without adding an external primer, which can more simply and conveniently realize sensitive and specific detection of miRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0023] Figure 1 Schematic diagram of the process of detecting miRNA by isothermal amplification assisted by magnetic beads of the present invention.

[0024] Figure 2 (a) is a gel electrophoresis diagram of the isothermal amplification reaction in Experiment 1 of the present invention, and (b) is a fluorescence spectrum diagram of the isothermal amplification reaction in Experiment 2 of the present invention.

[0025] Figure 3 This is the UV spectrum of the modified magnetic beads in Experiment 3 of the present invention.

[0026] Figure 4 These are the flow cytometer characterization diagram and laser confocal microscopy characterization diagram of the magnetic bead-assisted isothermal amplification reaction in Experiment 4 of the present invention.

[0027] Figure 5 This is the reaction time optimization result of the magnetic bead-assisted isothermal amplification reaction in Experiment 5 of the present invention.

[0028] Figure 6 This is the optimization result of the magnesium ion concentration in the magnetic bead-assisted isothermal amplification reaction in Experiment 6 of the present invention.

[0029] Figure 7 This is the optimization result of the polymerase concentration in the magnetic bead-assisted isothermal amplification reaction in Experiment 7 of the present invention.

[0030] Figure 8 This is a flow cytometer characterization diagram of the sensitivity of detecting miRNA by the magnetic bead-assisted isothermal amplification reaction in Experiment 8 of the present invention.

[0031] Fig. 9 This is a flow cytometer characterization diagram of detecting miRNA specificity by magnetic bead-assisted isothermal amplification reaction in Experiment 9 of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0033] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0034] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0035] Example

[0036] The materials and instruments used in the following examples are all commercially available.

[0037] Embodiment 1:

[0038] A Hairpin probe (abbreviated as HP), the DNA sequence of HP is shown in SEQ ID NO.1; specifically: Biotin-TTTTT-BHQ-GCTGTAAACTGTTCCTGCTGAACTGAGCCAC AGTTTACAG-FAM-TAAACT .

[0039] The probe includes three modifications, among which Biotin is a biotin modification that can be coupled to streptavidin-coated magnetic beads, and the FAM fluorescent group and the BHQ quenching group are used for signal output; the probe sequence includes two parts, the bold font part is the target binding part that can hybridize with the target, and the single underlined part is the stem-loop structure of the self-primer, which can form a new hairpin structure after a configuration conversion and serve as a primer for an isothermal amplification reaction.

[0040] Example 2

[0041] A method for detecting miRNA by isothermal amplification assisted by magnetic beads, the detection principle diagram of which is shown in Figure 1 , the detection method comprises the following steps:

[0042] (1) Modification of labeled probe magnetic beads: The HP probe is coupled to the surface of streptavidin-coated magnetic beads; the 5' end of the HP probe is modified with biotin, and the middle of the oligonucleotide sequence is modified with a FAM fluorescent group and a BHQ quenching group. In the initial state, the probe is in a fluorescence quenching state.

[0043] (2) The magnetic bead-modified HP probe, target Target, target miRNA sequence, 5mM MgCl2 solution, and 0.1U / μL polymerase solution were mixed to form a reaction system, and incubated at 37°C for 150 minutes. Under the triggering of the target Target sequence, the target miRNA sequence hybridized with the probe on the surface of the magnetic bead, the probe underwent a configuration change, and an isothermal amplification reaction was performed on the surface of the magnetic bead; the amplified oligonucleotide sequence complementary to the probe underwent strand displacement to achieve target circulation. The target Target sequence is the miR-24 sequence; the Target is shown in the sequence SEQ ID NO.2, specifically: tggctcagttcagcaggaacag.

[0044] (3) Use flow cytometry to collect and analyze the fluorescent signals of the modified magnetic beads. After the target and the probe hybridize, the FAM fluorescent group and the BHQ quenching group move away, the fluorescent signal is restored, and after the target circulates, multiple probes are triggered to undergo isothermal amplification reactions, and the fluorescent signal is enhanced; miRNA detection is achieved by detecting the fluorescent signal of the modified magnetic beads.

[0045] Experiment 1: Gel electrophoresis characterization of isothermal amplification reaction:

[0046] Prepare a polyacrylamide gel with a mass concentration ratio of 12%. Take 5mL of acrylamide solution, 3.93mL of ultrapure water, 2mL of 5×TBE buffer, 70μL of ammonium persulfate and 7μL of TEMED solution in a small beaker, mix well and let stand until there are no bubbles. Draw the solution with a syringe and inject it into the installed electrophoresis device. Insert the comb vertically and let stand until the gel solidifies. Pull out the comb, put the device into the electrophoresis tank, and add an appropriate amount of 1×TBE solution. Take 10μL of sample and mix it with 2μL SYBR Gold dye and 2μL 6×loading buffer. Use an injector to take the mixed solution and add it to the gel sample well. Set the voltage to 90V and run for 2.5h. Take out the gel plate and analyze the bands using a gel imaging system.

[0047] The results are as follows Figure 2 As shown in (a), lanes 1, 2, 3, and 4 are HP, Target, HP+Target, and HP+Polymerase, respectively, and lane 5 is the isothermal amplification reaction HP+Target+Polymerase, wherein lane 5 produces a new bright band, indicating the successful implementation of the isothermal amplification reaction.

[0048] Experiment 2: Fluorescence spectroscopy characterization of isothermal amplification reaction

[0049] Four groups of samples, HP, HP+Target, HP+Polymerase, and HP+Target+Polymerase, were prepared, and the latter three groups were reacted overnight at 37° C. The excitation wavelength was set to 488 nm, and the collection range was 510 nm-650 nm to detect the fluorescence intensity.

[0050] The results are as follows Figure 2 As shown in (b), the fluorescence intensity of the fourth group of samples HP+Target+Polymerase is significantly stronger than that of the first three groups, indicating the successful implementation of the isothermal amplification reaction.

[0051] Experiment 3: Modification of magnetic beads

[0052] The magnetic beads were shaken and mixed on a vortex oscillator, and the magnetic beads were placed in an ep tube, and an appropriate amount of Buffer I was added, and the mixture was shaken and mixed. The mixture was placed on a magnetic stand, and the supernatant was removed, and the washing was repeated three times. HP probe was added to the magnetic beads, and the mixture was shaken and mixed, and then placed in a constant temperature metal shaking bath, and the parameters were set to 37°C for incubation. After magnetic separation, the supernatant was retained, and its ultraviolet absorption spectrum was measured with an ultraviolet-visible spectrophotometer; the magnetic beads were washed three times with Buffer I buffer, and then dispersed in PBS buffer to obtain MB-HP.

[0053] The results are as follows Figure 3 As shown: the ultraviolet absorption intensity of the HP probe decreased before and after the magnetic beads were modified, indicating that magnetic beads modified with the HP probe were obtained.

[0054] Experiment 4: Flow cytometric characterization of magnetic bead-assisted isothermal amplification reaction and confocal laser scanning

[0055] Four groups of samples were prepared: MB-HP, MB-HP+Target, MB-HP+Polymerase, and MB-HP+Target+Polymerase. The last three groups of samples were reacted at 37°C. The samples were collected and analyzed using a flow cytometer. The excitation wavelength was set to 488 nM, the collection wavelength range was 505-525 nm, and 1×10 4 Fluorescence signal of the magnetic beads.

[0056] After diluting the above four groups of samples with PBS buffer, appropriate amounts of samples were taken and transferred to confocal dishes. The samples were placed under a laser confocal microscope for imaging, with the excitation wavelength set to 488 nm, and the fluorescence signals of 490-530 nm were collected. The FAM fluorescence signals on the magnetic beads were observed using a 100x oil objective.

[0057] The results are as follows Figure 4As shown, flow cytometry and laser confocal microscopy results showed that the fluorescence intensity of MB-HP+Target+Polymerase was higher than that of the other three groups, proving that the reaction of the entire system occurred successfully.

[0058] Experiment 5: Investigation of reaction time of magnetic bead-assisted isothermal amplification reaction

[0059] MB-HP, Target, Polymerase, and dNTP were mixed and incubated at 37°C for 0, 30, 60, 90, 120, 150, 180, 210, and 240 min, respectively. The excitation wavelength was set to 488 nM, the collection wavelength range was 505-525 nm, and 1×10 4 Fluorescence signal of the magnetic beads.

[0060] The results are as follows Figure 5 As shown, the flow cytometer results showed that after the reaction time reached 150 minutes, the fluorescence intensity of the system basically reached a platform, so 150 minutes was selected as the optimal time for the system reaction.

[0061] Experiment 6: Effect of magnesium ion concentration on magnetic bead-assisted isothermal amplification reaction

[0062] In order to improve the efficiency of isothermal amplification reaction, MB-HP, Target, Polymerase, and dNTP were mixed, and 0, 0.5, 1, 2, 5, and 10 mM MgCl2 solutions were added respectively, and incubated at 37°C for 150 min. The excitation wavelength was set to 488 nM, the collection wavelength range was 505-525 nm, and 1×10 4 Fluorescence signal of the magnetic beads.

[0063] The results are as follows Figure 6 As shown, the flow cytometry results showed that different concentrations of magnesium ions had a certain effect on the signal-to-background ratio of the isothermal amplification reaction, and a platform was reached when the magnesium ion concentration was 5 mM, which was selected as the optimal magnesium ion concentration for the system reaction.

[0064] Experiment 7: Effect of polymerase concentration on magnetic bead-assisted isothermal amplification reaction

[0065] In order to improve the efficiency of isothermal amplification reaction, MB-HP, Target, dNTP, 5mM MgCl2 solution were mixed, and 0.05, 0.075, 0.1, and 0.2U / μL polymerase solution were added respectively, and incubated at 37°C for 150min. The excitation wavelength was set to 488nM, the collection wavelength range was 505-525nm, and 1×10 4 Fluorescence signal of the magnetic beads.

[0066] The results are as follows Figure 7 As shown, the flow cytometry results showed that different concentrations of polymerase had a certain effect on the signal-to-background ratio of the isothermal amplification reaction, among which 0.1U / μL polymerase concentration was the optimal polymerase concentration for the system reaction.

[0067] Experiment 8: Detection of miRNA sensitivity by magnetic bead-assisted isothermal amplification reaction

[0068] MB-HP, dNTP, MgCl2, and Polymerase were mixed, and different concentrations of Target (0, 0.05, 0.1, 0.15, 0.25, and 0.375 nM) were added, and incubated at 37°C for 150 min. The excitation wavelength was set to 488 nM, the collection wavelength range was 505-525 nm, and 1×10 4 The fluorescence signal of each magnetic bead was used to draw a standard curve.

[0069] The results are as follows Figure 8 As shown, in the range of 0-0.375nM, the fluorescence intensity of the magnetic beads is linearly related to the Target concentration, the detection limit is 0.95pM, and the correlation coefficient is 0.9954.

[0070] Experiment 9: Detection of miRNA specificity by magnetic bead-assisted isothermal amplification reaction

[0071] MB-HP, dNTP, MgCl2, and Polymerase were mixed, and different miRNAs (miR-24, miR-21, miR-31, miR-17-5p, and miR-155) of the same concentration were added, and incubated at 37°C for 150 min. The excitation wavelength was set to 488 nM, the collection wavelength range was 505-525 nm, and 1×10 4 Fluorescence signal of the magnetic beads.

[0072] The results are as follows Fig. 9 As shown, a strong fluorescence signal is generated only when the target miR-24 is present, indicating that this method has strong specificity for the detection of miRNA.

[0073] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention. Sequence Listing <110> Hunan University <120> Method for detecting miRNA by magnetic bead-assisted isothermal amplification <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 51 <212> DNA <213> Artificial Sequence <400> 1 tttttgctgt aaactgttcc tgctgaactg agccacagtt tacagtaaac t 51 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <400> 2 tggctcagtt cagcaggaac ag 22

Claims

1. Use of a magnetic bead-modified HP probe in preparing an isothermal amplification kit for detecting miRNA, characterized in that: The following steps are involved: S1, coupling the HP probe to the surface of streptavidin-coated magnetic beads; S2, mixing the HP probe modified with magnetic beads, the target Target, and the target miRNA sequence to form a reaction system, and performing an isothermal amplification reaction; S3, collecting the fluorescence signal of the modified magnetic beads; The DNA sequence of the HP probe is shown in SEQ ID NO.1; specifically: Biotin- TTTTT-BHQ-GCTGTAAACTGTTCCTGCTGAACTGAGCCACAGTTTACAG-FAM-TAAACT.

2. The use according to claim 1, characterized in that The DNA sequence of the target Target sequence is shown as SEQ ID NO.

2.

3. The use according to claim 1 or 2, characterized in that The conditions of the isothermal amplification reaction in S2 are: incubation at 37° C. for 150 to 240 min.

4. The use according to claim 1 or 2, characterized in that Add 5-10 mM mg into the reaction system of S2. 2+ Solution.

5. The use according to claim 1 or 2, characterized in that Add 0.1-0.2 U / μL of polymerase solution to the reaction system of S2.

6. The use according to claim 1 or 2, characterized in that: In the S3, the fluorescence signal of the modified magnetic beads is collected, the excitation wavelength is set to 488 nm, the collection wavelength range is 505-525 nm, and the fluorescence signal of the magnetic beads is collected.

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