阅读说明：本技术 基于恒温无酶多级扩增的miRNA化学发光检测试剂盒 (miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification ) 是由 王慧敏 杨慧然 于 2021-06-25 设计创作，主要内容包括：本发明公开了一种基于恒温无酶多级扩增的miRNA化学发光检测试剂盒,通过核酸互补杂交,目标miRNA特异性地打开发卡探针,发生催化发卡自组装反应,生成大量双链DNA产物,双链产物继续引发杂交链式反应,形成含有多个G-四链体的DNA纳米线,G-四链体与氯化血红素结合形成具有类辣根过氧化物酶催化活性的G-四链体/氯化血红素DNAzyme。在过氧化氢的存在下,DNAzyme催化鲁米诺的氧化反应,产生多级扩增的化学发光信号。本发明试剂盒可以检测低至16 pM的miRNA；该检测方法无需荧光基团修饰、外部光源和蛋白酶介入,通过多级扩增反应即可有效提高miRNA检测的灵敏度,从而解决了现有检测方法由于自发荧光、荧光漂白、串光以及稳定性低导致的灵敏度有限的难题。(The invention discloses a miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification, wherein a hairpin probe is specifically opened by a target miRNA through nucleic acid complementary hybridization to generate a catalytic hairpin self-assembly reaction to generate a large amount of double-strand DNA products, the double-strand products continuously initiate a hybridization chain reaction to form a DNA nanowire containing a plurality of G-quadruplexes, and the G-quadruplexes are combined with hemin to form a G-quadruplex/hemin DNAzyme with horseradish peroxidase-like catalytic activity. In the presence of hydrogen peroxide, DNAzyme catalyzes the oxidation reaction of luminol, producing a multi-stage amplified chemiluminescent signal. The kit can detect miRNA with the size of 16 pM; the detection method does not need fluorescent group modification, external light source and protease intervention, and can effectively improve the sensitivity of miRNA detection through multistage amplification reaction, thereby solving the problem of limited sensitivity caused by autofluorescence, fluorescence bleaching, cross-luminescence and low stability of the existing detection method.)

1. The miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification is characterized by consisting of hairpin nucleic acid and a reaction substrate, wherein the hairpin nucleic acid comprises hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5, hairpin H6 and hydroxyethyl piperazine ethanethionate buffer solution;

h1 nucleic acid molecule sequence is shown in SEQ ID NO: 1,

h2 nucleic acid molecule sequence is shown in SEQ ID NO: 2,

h3 nucleic acid molecule sequence is shown in SEQ ID NO: 3,

h4 nucleic acid molecule sequence is shown in SEQ ID NO: 4,

h5 nucleic acid molecule sequence is shown in SEQ ID NO: 5,

h6 nucleic acid molecule sequence is shown in SEQ ID NO: 6;

the reaction substrate comprises hemin powder, luminol powder and 30% hydrogen peroxide solution.

2. The miRNA chemiluminescence detection kit based on isothermal enzyme-free multistage amplification of claim 1, wherein the hairpin further comprises H7, H7 nucleic acid molecule sequence is shown in SEQ ID NO: 7.

3. the miRNA chemiluminescence detection kit based on isothermal enzyme-free multistage amplification of claim 2, wherein the molar concentration ratio of hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5 and hairpin H6 is 1-2: 1-2: 6-7: 8-9: 7-8: 8-9; or the molar concentration ratio of the hairpin H1, the hairpin H2, the hairpin H3, the hairpin H4, the hairpin H5, the hairpin H6 and the hairpin H7 is 1-2: 1-2: 6-7: 8-9: 7-8: 8-9: 0.3-0.8.

4. The miRNA chemiluminescence detection kit based on isothermal enzyme-free multistage amplification of claim 3, wherein the molar concentration ratio of hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5, hairpin H6 is 2: 2: 6: 8: 7: 8; or the molar concentration ratio of the hairpin H1, the hairpin H2, the hairpin H3, the hairpin H4, the hairpin H5, the hairpin H6 and the hairpin H7 is 2: 2: 6: 8: 7: 8: 0.5.

5. a miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification is characterized by comprising the following steps:

(1) respectively annealing hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5, hairpin H6 and hairpin H7 dissolved in HEPES, mixing in molar ratio, adding miRNA and HEPES, incubating for 12 hours, adding a hemin solution, incubating for 1 hour, and diluting the reaction solution by 20 times;

(2) adding a luminol solution into the reaction solution diluted by 20 times, placing the reaction solution in a light-emitting cup, quickly injecting a hydrogen peroxide solution, and detecting a chemiluminescence signal by using a weak chemiluminescence measuring instrument to realize the detection of miRNA;

the sequence of the H1 nucleic acid molecule is shown as SEQ ID NO: 1,

h2 nucleic acid molecule sequence is shown in SEQ ID NO: 2,

h3 nucleic acid molecule sequence is shown in SEQ ID NO: 3,

h4 nucleic acid molecule sequence is shown in SEQ ID NO: 4,

h5 nucleic acid molecule sequence is shown in SEQ ID NO: 5,

h6 nucleic acid molecule sequence is shown in SEQ ID NO: 6.

6. the miRNA chemiluminescence detection kit based on isothermal enzyme-free multistage amplification of claim 5, wherein the hairpin further comprises H7, H7 nucleic acid molecule sequence is shown in SEQ ID NO: 7.

7. the miRNA chemiluminescence detection kit based on isothermal enzyme-free multistage amplification of claim 6, wherein the molar concentration ratio of hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5, hairpin H6 is 1-2: 1-2: 6-7: 8-9: 7-8: 8-9;

or the molar concentration ratio of the hairpin H1, the hairpin H2, the hairpin H3, the hairpin H4, the hairpin H5, the hairpin H6 and the hairpin H7 is 1-2: 1-2: 6-7: 8-9: 7-8: 8-9: 0.3-0.8.

8. The miRNA chemiluminescence detection kit based on isothermal enzyme-free multistage amplification of claim 7, wherein the molar concentration ratio of hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5 and hairpin H6 is 2: 2: 6: 8: 7: 8; or the molar concentration ratio of the hairpin H1, the hairpin H2, the hairpin H3, the hairpin H4, the hairpin H5, the hairpin H6 and the hairpin H7 is 2: 2: 6: 8: 7: 8: 0.5.

9. use of the constant temperature enzyme-free multistage amplification-based miRNA chemiluminescence detection kit of any one of claims 1-4 in the preparation of a medicament for detecting the content of miRNA-21 in a cell lysate.

10. The use of claim 9, wherein the miRNA chemiluminescence assay kit based on isothermal enzyme-free multistage amplification is used for preparing a medicament for differentiating tumor cells from normal cells by detecting the miRNA-21 content in a cell lysate, wherein the tumor cells comprise HepG2 or HL-7702 cells.

Technical Field

The invention belongs to the field of molecular biology and medical detection, and particularly relates to a miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification.

Background

MicroRNA (miRNA) is small RNA which is endogenous and has the length of about 20-24 nucleotides, has a plurality of important regulation effects in cells, has the abnormal expression closely related to the progress of cancer, can be used as a tumor marker, and provides important information for early diagnosis, prognosis and related biological function research of cancer.

However, due to the characteristics of short miRNA sequence, low content, high homology, etc., the detection application of miRNA is limited. Therefore, the development of a miRNA detection method with high sensitivity and high specificity has important significance for realizing early diagnosis of cancer. The nucleic acid amplification technology is an important technology in the fields of biology, medicine and the like, is widely applied to target object detection, and can identify specific pathogens or disease markers. The kit is characterized in that the requirements of a constant-temperature enzyme-free nucleic acid amplification technology on an instrument are greatly simplified, the reaction time is greatly shortened, the reaction conditions are not limited by an enzyme reaction environment, the kit mainly comprises a catalytic hairpin self-assembly reaction (CHA), a Hybrid Chain Reaction (HCR) and a nuclease (DNAzyme) mediated reaction, the applicability is stronger, and the kit is simpler and more sensitive compared with the traditional detection means. However, at present, a miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification does not exist.

Disclosure of Invention

The invention aims to provide a miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification.

The purpose of the invention is realized by the following technical scheme: a miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification is disclosed, wherein the sequence of miRNA-21 is shown in Table 1, and specifically is UAGCUUAUCAGACUGAUUGAUUGA.

The miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification consists of DNA powder, reaction substrates and buffer solution, wherein the DNA powder comprises hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5, hairpin H6 and hairpin H7, and the specific sequences are as follows:

h1 nucleic acid molecule sequence is shown in SEQ ID NO: 1,

h2 nucleic acid molecule sequence is shown in SEQ ID NO: 2,

h3 nucleic acid molecule sequence is shown in SEQ ID NO: 3,

h4 nucleic acid molecule sequence is shown in SEQ ID NO: 4,

h5 nucleic acid molecule sequence is shown in SEQ ID NO: 5,

h6 nucleic acid molecule sequence is shown in SEQ ID NO: 6,

and/or H7 nucleic acid molecule sequence as set forth in SEQ ID NO: 7.

the reaction substrate comprises hemin, luminol and hydrogen peroxide solution;

the buffer solution was HEPES buffer (10mM HEPES buffer solution, pH 7.5, containing 600mM sodium chloride and 50mM potassium chloride).

The molar concentration ratio of hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5 and hairpin H6 is 1-2: 1-2: 6-7: 8-9: 7-8: 8-9; or the molar concentration ratio of the hairpin H1, the hairpin H2, the hairpin H3, the hairpin H4, the hairpin H5, the hairpin H6 and the hairpin H7 is 1-2: 1-2: 6-7: 8-9: 7-8: 8-9: 0.3-0.8.

Preferably, the molar concentration ratio of the hairpin H1 to the hairpin H2 to the hairpin H3 to the hairpin H4 to the hairpin H5 to the hairpin H6 is 2: 2: 6: 8: 7: 8; or the molar concentration ratio of the hairpin H1, the hairpin H2, the hairpin H3, the hairpin H4, the hairpin H5, the hairpin H6 and the hairpin H7 is 2: 2: 6: 8: 7: 8: 0.5.

a method for detecting miRNA-21 by using a miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification comprises the following steps:

(1) respectively preparing 100 mu M of hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5, hairpin H6 and hairpin H7 powder by using HEPES buffer solution, oscillating and dispersing uniformly, then annealing respectively, mixing according to a molar concentration ratio, adding a target miRNA-21, incubating for 12 hours together, adding a hemin solution, incubating for 1 hour to obtain a reaction solution, and diluting the reaction solution by 20 times;

(2) adding a luminol solution into the reaction solution diluted by 20 times, placing the reaction solution into a light-emitting cup, quickly injecting a hydrogen peroxide solution, and detecting a chemiluminescence signal by using a weak chemiluminescence measuring instrument to realize the detection of miRNA-21;

the sequence of the H1 nucleic acid molecule is shown as SEQ ID NO: 1,

h2 nucleic acid molecule sequence is shown in SEQ ID NO: 2,

h3 nucleic acid molecule sequence is shown in SEQ ID NO: 3,

h4 nucleic acid molecule sequence is shown in SEQ ID NO: 4,

h5 nucleic acid molecule sequence is shown in SEQ ID NO: 5,

h6 nucleic acid molecule sequence is shown in SEQ ID NO: 6,

and/or H7 nucleic acid molecule sequence as set forth in SEQ ID NO: 7.

in the step (1), the molar concentration ratio of hairpin H1, hairpin H2, hairpin H3, hairpin H4, hairpin H5 and hairpin H6 is 1-2: 1-2: 6-7: 8-9: 7-8: 8-9; or the molar concentration ratio of the hairpin H1, the hairpin H2, the hairpin H3, the hairpin H4, the hairpin H5, the hairpin H6 and the hairpin H7 is 1-2: 1-2: 6-7: 8-9: 7-8: 8-9: 0.3-0.8.

Preferably, the molar concentration ratio of the hairpin H1 to the hairpin H2 to the hairpin H3 to the hairpin H4 to the hairpin H5 to the hairpin H6 is 2: 2: 6: 8: 7: 8; or the molar concentration ratio of the hairpin H1, the hairpin H2, the hairpin H3, the hairpin H4, the hairpin H5, the hairpin H6 and the hairpin H7 is 2: 2: 6: 8: 7: 8: 0.5.

the invention also provides the application of the miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification in the preparation of medicines for detecting the miRNA-21 content in cell lysate.

The miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification is applied to the preparation of a medicine for distinguishing tumor cells and normal cells by detecting the content of miRNA-21 in cell lysate, wherein the tumor cells comprise HepG2 or HL-7702 cells.

The invention has the following advantages: in the kit, miRNA-21 can initiate an upstream CHA reaction, the generated product can continuously initiate a downstream HCR, and DNAzyme is activated at the same time to generate a multiple cascade amplified chemiluminescent signal, so that the kit has the advantages of good stability, high sensitivity and strong specificity. When miRNA-21 exists, a DNA nanowire containing a plurality of G-quadruplex structures can be formed, and the catalytic activity is greatly improved after the DNA nanowire is compounded with hemin; in addition, the utilization rate of reactants can be greatly increased based on the domain limiting effect of the nucleic acid nanostructure. The kit does not need an external excitation light source, can avoid the problems of autofluorescence, fluorescence bleaching, crosstalk and the like, and has lower background signals; the hairpin probe used by the kit does not need to be modified by a fluorescent group, so that the detection cost is greatly reduced; the kit disclosed by the invention is based on a constant-temperature enzyme-free multistage tandem CHA-HCR-DNAzyme nucleic acid amplification reaction, can effectively improve the specificity and sensitivity of miRNA detection, and provides an efficient new method for early diagnosis, treatment and prognosis judgment of cancers. By changing the sequence of the functional hairpin H7, other biomolecules such as nucleic acids and proteins can be detected.

Table 1: sequences of nucleic acid molecules referred to herein

Drawings

FIG. 1 is a schematic diagram of the detection of a target object according to the present invention.

FIG. 2 is an optimization plot of the CHA to HCR ratio.

FIG. 3 is a diagram showing the mechanism verification.

FIG. 4 is a graph of the concentration of the optimized hairpin H3.

FIG. 5 is a graph of the concentration of the optimized hairpin H5.

FIG. 6 is a graph of the concentration of the optimized hairpin H1.

FIG. 7 is a graph showing the effect of signal amplification by the CHA-HCR-DNAzyme, CHA-DNAzyme and HCR-DNAzyme.

FIG. 8 is a diagram of miRNA chemiluminescence detection kit based on isothermal enzyme-free multistage amplification for detecting miRNA-21 at different concentrations.

FIG. 9 is a diagram of miRNA-21 detection in different cell lysates by miRNA chemiluminescence detection kit based on isothermal enzyme-free multistage amplification.

Detailed Description

The invention is further described below with reference to the drawings and examples, without limiting the scope of the invention to the following.

The reagents used in the examples of the invention were as follows:

HEPES (high efficiency particulate air): purchased from Sigma, 99.5%;

HEPES sodium salt: purchased from Sigma, 96%;

sodium chloride: purchased from Sigma, 99%;

potassium chloride: purchased from chemical reagents of national medicine group, and the content is more than or equal to 99.5 percent;

luminol: purchased from Sigma, ≧ 97% (HPLC);

hemin: purchased from Sigma, ≧ 97% (HPLC);

30% hydrogen peroxide solution: purchased from kyoto congo chemicals, inc;

dimethyl sulfoxide: purchased from national drug group chemical agents, ltd;

DEPC water: water treated with paraformaldehyde and diethylpyrocarbonate was purchased from bio-engineering (shanghai) inc.

HEPES buffer (10mM HEPES buffer solution, pH 7.5, containing 600mM sodium chloride and 50mM potassium chloride) preparation method: weighing 0.834g of HEPES, 0.2603g of HEPES sodium salt, 17.49g of sodium chloride and 1.8638g of potassium chloride in a beaker, adding DEPC water to dissolve, then fixing the volume to 500mL, and then adjusting the pH to 7.5 by using 1M of sodium hydroxide solution at the temperature of 25 ℃;

preparation method of DNA mother liquor (100. mu.M): the DNA powder was dissolved by TE buffer (pH 8.0) in the amount indicated by the label on the DNA tube and stored at-20 ℃.

Preparation method of hemin solution (4. mu.M): weighing 0.0326g of hemin powder, dissolving in 5mL of dimethyl sulfoxide to obtain 10mM hemin solution, and gradually diluting to 4 μ M with dimethyl sulfoxide;

preparation method of luminol solution (250. mu.M): weighing 0.025g of luminol powder, dissolving in 2.5ml of concentrated ammonia water, adding 25ml of DEPC water for dilution to obtain 5mM luminol solution, and adding 1 mu L of 5mM luminol solution into 19 mu L of LHEPES solution to obtain 250 mu M luminol solution;

H₂O₂preparation of the solution (1.5 mM): 2 μ L of 30% H₂O₂The solution was dissolved in 17.58. mu.L HEPES solution to give 1M H₂O₂Solution, 1. mu.L of 1M H₂O₂The solution was dissolved in 9. mu.L of HEPES solution to give 100mM H₂O₂The solution was finally diluted with 1.8. mu.L of 100mM H₂O₂The solution was dissolved in 118.2. mu.L of HEPES solution to give 1.5mM H₂O₂And (3) solution.

The design concept of the hairpin probe is as follows: design related hairpin probes by using NUPACK software and synthesis of related nucleic acid sequences by committing the institute of Biotechnology (Shanghai) corporationAnd (4) columns. FIG. 1 is a schematic diagram of a multi-stage amplification reaction for target detection, in which a target I firstly opens a hairpin H1 through a strand displacement reaction to obtain an I-H1 intermediate, then the hairpin H2 replaces I in I-H1 to generate a product H1-H2, and the replaced I continuously participates in reactions of H1 and H2 to generate a plurality of double-stranded products H1-H2. The products H1-H2 bring the two-part sequences of T into close proximity, thereby priming the downstream HCR. T opens the hairpin H3, H4, H5 and H6 in turn through strand displacement reaction to generate an intermediate product T-H3-H4-H5-H6, exposes the same sequence as T, continues to trigger the sequential opening of the hairpin, and finally generates a long double-stranded DNA nanowire, namely T- (H3-H4-H5-H6)_N. The DNA nanowire can be drawn to the distance of two parts of G4 sequences modified on hairpin H3 and H5 to form complete G4, hemin is added to form G4/hemin DNAzyme with horseradish peroxidase-like catalytic activity, the DNAzyme catalyzes luminol to emit light in the presence of hydrogen peroxide, and the light emitting intensity is in positive correlation with the concentration of a target object, so that the aim of detecting the target object is fulfilled by the method.

Example 1

To increase the signal amplification effect, the ratio of the concentrations of the CHA (including hairpin H1, H2) and HCR (including hairpin H3, H4, H5, H6) reactants in the DNA incubation solution was optimized. Specifically, the concentration ratio of the CHA to HCR hairpin reactants is 4: 1. 2: 1. 1: 1. 1: 2. 1: 4.

firstly, uniformly mixing 1 mu L of single hairpin mother solution and 15.67 mu L of HEPES buffer solution to obtain a single hairpin solution with the concentration of 6 mu M, and then respectively annealing the single hairpin solution by a PCR instrument under the conditions of keeping the temperature at 95 ℃ for 5min and keeping the temperature at 25 ℃ for 2 h.

The reactant hairpins are then mixed at different concentration ratios, for example, a ratio of CHA to HCR concentration of 4: the specific procedure of 1 was to add 25. mu.L of HEPES buffer to 5.83. mu.L of hairpin H1 and H2 and 1.46. mu.L of hairpin H3, H4, H5 and H6 to obtain a nucleic acid incubation solution with a CHA/HCR concentration ratio of 4: 1. Other proportion relations are converted according to the above, the nucleic acid incubation solutions with different CHA and HCR hairpin concentration ratios and 1.25 mu L I (target substances capable of initiating the CHA reaction and the CHA-HCR-DNAzyme reaction) are incubated for 12 hours, 9.85 mu L of chlorhematin solution with the concentration of 4 mu M is added, reaction solution containing G-quadruplex/chlorhematin DNAzyme is obtained after 1 hour, the reaction solution is diluted by 20 times, 13.13 mu L of luminol solution with the concentration of 250 mu M is added into the reaction solution diluted by 20 times, the reaction solution is mixed evenly and put into a luminescent cup, 108.27 mu L of hydrogen peroxide solution with the concentration of 1.5mM is injected, and a chemiluminescent signal is detected immediately by a weak chemiluminescence measuring instrument. As shown in FIG. 2, by comparing the signal-to-back ratios at different CHA and HCR hairpin concentration ratios, it was found that the signal-to-back ratio was the greatest when the ratio was 1:4, and thus it was finally determined that 1:4 was the optimal CHA and HCR hairpin concentration ratio.

Example 2

In this example, control experiments in which a single hairpin is absent in the reaction system in sequence are designed, and chemiluminescent signals generated by different experimental groups are detected, and the method and the amount of each raw material are the same as those in example 1.

The results of the experiment are shown in FIG. 3. When hairpin H is removed from upstream CHA₁Or H₂Or by eliminating hairpins H3, H from the downstream HCR₄And H5, the fluorescence of the target-initiated reaction was low, demonstrating that when either the upstream CHA or the downstream HCR is blocked, the CHA-HCR-DNAzyme is unable to amplify the signal. When hairpin H is removed from downstream HCR₆The target-initiated reaction will then produce less fluorescence, since the reaction is now equivalent to the CHA reaction, lacking hairpin H₆The HCR of (a) can only be used as a signal output tool, and does not amplify the signal any more. Similarly, fluorescence is lower in the hairpin deficient system without target. Thus, it was demonstrated that the CHA-HCR-DNAzyme reaction produces a multi-stage amplified chemiluminescent signal only after the addition of the target in the presence of both the upstream CHA and downstream HCR reactants.

Example 3

This example optimizes the hairpin concentration. Through the hairpin-lacking test, the background signals generated by the hairpin H1, H3 and H5 are larger, the concentration of a single hairpin of H1, H3 or H5 is further optimized to obtain the optimal reaction concentration, and the specific operation steps and the amount of other reagents are the same as those in example 1. The specific adjustment contents are as follows:

h3: 500nM, 600nM, 700nM, 800nM, results are shown in FIG. 4.

H5: 500nM, 600nM, 700nM, 800nM, results are shown in FIG. 5.

H1: 50nM, 100nM, 150nM, 200nM, results are shown in FIG. 6.

By changing the hairpin concentrations of H1, H3 and H5, the signal-to-back ratios were found to be highest when the hairpin H1 was 200nM, H3 was 600nM and H5 was 700nM, so that the concentrations of the hairpins H1, H2, H3, H4, H5 and H6 in the nucleic acid incubation solution were finally determined to be 200nM, 600nM, 800nM, 700nM and 800nM in this order.

Example 4

In order to verify the amplification effect and detection capability of the multi-stage signal, the secondary CHA-DNAzyme and HCR-DNAzyme reactions are designed and compared with the detection effect of the multi-stage CHA-HCR-DNAzyme reaction.

The multi-stage CHA-HCR-DNAzyme reaction consisted of 42.5. mu.L of DNA incubation solution and 131.25. mu.L of reaction substrate solution. The DNA incubation solution comprises 1.46 μ L hairpin H1, 1.46 μ L hairpin H2, 4.38 μ L hairpin H3, 5.83 μ L hairpin H4, 5.11 μ L hairpin H5, 5.83 μ L hairpin H6 and 18.43 μ L HEPES buffer; the 131.25. mu.L of reaction substrate solution included 9.85. mu.L of hemin solution, 13.13. mu.L of luminol solution, and 108.27. mu.L of hydrogen peroxide solution. mu.L of 100. mu.M single hairpin stock solution was taken and mixed well with 15.67. mu.L of HEPES buffer to give a 6. mu.M single hairpin solution. mu.M of the single hairpin solution was annealed by a PCR instrument at a temperature of 95 ℃ for 5min and 25 ℃ for 2 h. After annealing, mixing was performed according to the above hairpin volumes, and after mixing, 1.25. mu.LI (target that can initiate both the CHA-DNAzyme reaction and the CHA-HCR-DNAzyme reaction) was added, finally ensuring that the concentrations of hairpin H1 and H2 were 200nM, hairpin H3 was 600nM, H4 was 800nM, H5 was 700nM and H6 was 800nM throughout the nucleic acid incubation. After 6 hairpins and I are incubated for 12h, 4 mu M of hemin solution is added, reaction is carried out for 1h to obtain reaction liquid, the reaction liquid is diluted by 20 times, 13.13 mu L of 250 mu M luminol solution is added, the mixture is uniformly mixed and then put into a luminescent cup, 108.27 mu L of 1.5mM hydrogen peroxide solution is injected, and a chemiluminescence signal is immediately detected by a weak chemiluminescence measuring instrument.

Example 4-1

To prove that the detection kit has higher sensitivity and amplification effect, the detection kit is compared with other two kits: namely a chemiluminescence detection kit based on a secondary CHA-DNAzyme nucleic acid amplification reaction: the target was I (target capable of initiating the reaction of CHA-DNAzyme and CHA-HCR-DNAzyme), consisting of 42.5. mu.L of DNA incubation solution and 131.25. mu.L of reaction substrate solution; the DNA incubation solution comprises 1.46 mu L of hairpin H1, 1.46 mu L of hairpin H2, 4.38 mu L of hairpin H3, 5.83 mu L of hairpin H4, 5.11 mu L of hairpin H5 and 24.26 mu L of HEPES buffer solution; the hairpin annealing concentration and the reaction substrate solution are the same as the miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage nucleic acid amplification reaction.

Example 4 to 2

A chemiluminescence detection kit based on a two-stage HCR-DNAzyme nucleic acid amplification reaction comprises: the target was T (target capable of initiating HCR-DNAzyme reaction) and consisted of 42.5. mu.L of DNA incubation solution and 131.25. mu.L of reaction substrate solution; the DNA incubation solution comprises 4.38 mu L of hairpin H3, 5.83 mu L of hairpin H4, 5.11 mu L of hairpin H5, 5.83 mu L of hairpin H6 and 21.35 mu L of HEPES buffer solution, and the hairpin annealing concentration and the reaction substrate solution are the same as the miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage nucleic acid amplification reaction.

The results of detection of different concentrations of target by the three kits of example 4, example 4-1, and example 4-2 are shown in FIG. 7, and the test kit of example 4 can detect a lower concentration of target than the other two kits, and the signal amplification effect is more significant when detecting the same concentration of target, as shown in FIG. 5nM, the chemiluminescence detection kit based on the two-stage CHA-DNAzyme nucleic acid amplification reaction has a chemiluminescence intensity of only about 2300, the chemiluminescence detection kit based on the two-stage HCR-DNAzyme nucleic acid amplification reaction has a chemiluminescence intensity of only about 1600, and the chemiluminescence detection kit based on the multi-stage CHA-HCR-DNAzyme nucleic acid amplification reaction has a chemiluminescence intensity of about 6800, which proves that the multi-stage amplification kit of the present invention has a better signal amplification effect, moreover, the detection limits of the CHA-DNAzyme, the HCR-DNAzyme and the CHA-HCR-DNAzyme are 0.41nM, 2.67nM and 0.021nM respectively as can be obtained by linear curve calculation, and it is obvious that the detection sensitivity of the multistage amplification kit designed by the invention is higher.

Example 5

A miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification is used for detecting miRNA-21 in a solution.

The kit consists of 43.75 mu L of nucleic acid incubation solution and 131.25 mu L of reaction substrate solution; the nucleic acid incubation solution comprises 1.46 mu L of hairpin H1, 1.46 mu L of hairpin H2, 4.38 mu L of hairpin H3, 5.83 mu L of hairpin H4, 5.11 mu L of hairpin H5, 5.83 mu L of hairpin H6, 2.19 mu L of hairpin H7, 1.25 mu L of miRNA-21 and 16.24 mu L of HEPES buffer solution; the 131.25. mu.L of reaction substrate solution included 9.85. mu.L of hemin solution, 13.13. mu.L of luminol solution, and 108.27. mu.L of hydrogen peroxide solution. Hairpin H1-H6 at a concentration of 6 μ M and hairpin H7 at a concentration of 1 μ M were annealed at 95 ℃ for 5min and 25 ℃ for 2H, mixed according to the above hairpin volumes after annealing, and 1.25 μ L of miRNA-21 at different concentrations were added to ensure that the concentrations of hairpin H1 and H2 in the nucleic acid incubation liquid were 200nM, hairpin H3 was 600nM, H4 was 800nM, H5 was 700nM, H6 was 800nM, and H7 was 50 nM. After the hairpin and miRNA-21 are incubated for 12h, 4 mu M of hemin solution is added, the reaction is carried out for 1h at room temperature, the reaction solution is diluted by 20 times, 13.13 mu L of 250 mu M luminol solution is added, the mixture is uniformly mixed and then put into a luminescent cup, 108.27 mu L of 1.5mM hydrogen peroxide solution is injected, and a chemiluminescent signal is immediately detected by a weak chemiluminescent measuring instrument. The detection effect of the detection kit is shown in figure 8, the chemiluminescence intensity and the miRNA-21 concentration present a good linear relationship, and a linear curve of y ═ 11738.11795+29.7558 × (R) is obtained within the miRNA-21 concentration range of 0-100pM²0.9939), the detection limit is calculated to be 16pM, and the concentration of miRNA-21 in the sample can be calculated by detecting the chemiluminescence intensity, thereby realizing the high-sensitivity detection of miRNA-21.

Example 6

A miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification is used for detecting miRNA-21 in cells.

Respectively adding 2X 10 of pancreatin⁶Personal liver cancer cell(HepG2) and human normal hepatocytes (HL-7702) were digested, dispersed in 1mL of PBS solution, centrifuged at 1000rpm for 5min, the PBS solution was removed, the cells were redispersed in 250. mu.L of Tris-HCl solution, crushed at 0 ℃ for 20min by an ultrasonic cell crusher, and finally centrifuged at 12000rpm for 20min at 4 ℃ to give a supernatant as a cell lysate.

The kit consists of 43.75 mu L of nucleic acid incubation solution and 131.25 mu L of reaction substrate solution; the nucleic acid incubation solution comprises 1.46 mu L of hairpin H1, 1.46 mu L of hairpin H2, 4.38 mu L of hairpin H3, 5.83 mu L of hairpin H4, 5.11 mu L of hairpin H5, 5.83 mu L of hairpin H6, 2.19 mu L of hairpin H7, 1.25 mu L of miRNA-21 and 16.24 mu L of HEPES buffer solution; the 131.25. mu.L of reaction substrate solution included 9.85. mu.L of hemin solution, 13.13. mu.L of luminol solution, and 108.27. mu.L of hydrogen peroxide solution. Hairpin H1-H6 at a concentration of 6 μ M and hairpin H7 at a concentration of 1 μ M were annealed at 95 ℃ for 5min and 25 ℃ for 2H, mixed according to the above hairpin volumes after annealing, and then 1.25 μ L of different cell lysates were added, ensuring concentrations of hairpin H1 and H2 of 200nM, hairpin H3 of 600nM, H4 of 800nM, H5 of 700nM, H6 of 800nM and H7 of 50nM in the nucleic acid incubation liquid. After incubating the hairpin and the cell lysate for 12h, adding 4 mu M hemin solution, reacting for 1h at room temperature, diluting the reaction solution by 20 times, adding 13.13 mu L of 250 mu M luminol solution, mixing uniformly, putting into a luminescent cup, injecting 108.27 mu L of 1.5mM hydrogen peroxide solution, and immediately detecting a chemiluminescence signal by a weak chemiluminescence measuring instrument, wherein the chemiluminescence intensity in different cells is shown in figure 9. The concentrations of miRNA-21 in HepG2 and HL-7702 cells obtained by conversion of the linear curves (figure 8) in example 5 are respectively 92.3pM and 19.8pM, and the concentration of miRNA-21 in HepG2 cells is higher than that in HL-7702 cells, so that the kit can calculate the concentration of miRNA-21 in the cells by detecting chemiluminescence intensity, and provides a certain data support for distinguishing cancer cells from normal cells according to the difference of the content of miRNA-21 in the cells.

SEQUENCE LISTING

SEQUENCE LISTING

<110> university of three gorges

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CTCTATCATTATCTTGCTTCATCTTCATCAAGATAATGATAGAGACCGACACTC

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GCTTCATCTTCATCTCCGGTTTTGCGGAGATGAAGATGAAGCACGATG

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GTGGGTCAAAACCGGAGATGAAGATGAAGCTTGCCTGCTTCATCTTCATCTCCG

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GCTTCATCTTCATCTCCGACACTCCGGAGATGAAGATGAAGCAGGCAA

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TCAACATCAGTCTGATAAGCTAAGAGTGTCGGTCTCTATCATTATCTTAGCTTATCAGACTG

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