CN111763713A - Method and kit for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes - Google Patents

Abstract

The invention belongs to the field of miRNA detection, and particularly relates to a method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnosis purposes. Comprises the following steps of 1) preparing colloidal gold nanoparticles with uniform particle size by a hydrothermal method; 2) activating sulfhydryl group h-DNA by using TCEP, and reacting with colloidal gold to form a gold-sulfur bond for preparing a colloidal gold nucleic acid probe; 3) complementary pairing is carried out on the colloidal gold nucleic acid probe and the miRNA to be detected, and double-strand specific nuclease is added to realize target recycling and signal amplification; after the reaction is finished, adding a stop solution to inactivate the enzyme to obtain a reaction solution; 4) assembling and detecting the nucleic acid test strip: adding the reaction solution generated in the step 3) into the assembled nucleic acid test strip, and observing the detection result. The invention can obviously improve the detection sensitivity of miRNA.

Description

Non-diagnostic purpose based on target isothermal cyclic amplification and nucleic acid test strip technology detection Methods and kits for miRNA-21

technical field

The invention belongs to the field of miRNA detection, and in particular relates to a method for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes.

Background technique

MicroRNA-21 (miRNA-21) is a class of endogenous non-coding RNA small molecules, and the significant abnormal expression of miRNA-21 is closely related to the occurrence and development of various diseases, especially human tumors. There have been a lot of literature studies on the function of miRNA-21 in cancer. Many studies have shown that the miRNA expression profile in peripheral blood of cancer patients is specifically altered. More and more evidences suggest that miRNA-21 can be used as a novel molecular marker. It can be used in cancer diagnosis and prognosis evaluation. Traditional miRNA-21-based detections such as gene sequencing, fluorescence quantitative PCR, etc., but these detection methods all require large-scale instruments without exception, which are not only time-consuming and labor-intensive, but also only suitable for hospitals, etc. Some testing institutions cannot benefit every family to achieve portability and popularization. Therefore, the early and simple detection of miRNA-21 has become a research hotspot.

Target isothermal cyclic amplification technology usually refers to the in vitro isothermal amplification of nucleic acid based on enzymatic reaction, which mainly uses the ability of nucleases to cleave DNA recognition sites, and promotes the binding and release of targets under isothermal conditions, thereby producing corresponding The signal achieves signal accumulation and amplification. Among them, double-strand specific nuclease (DSN) is one of the most common nucleases suitable for miRNA detection. DSN can efficiently identify and digest DNA double-stranded DNA that is completely complementary paired or DNA/RNA hybrid double-stranded DNA strands. While nucleases have little effect on single-stranded DNA and single/double-stranded RNA, in addition, this degradation is only directed against at least 12 perfectly matched nucleotide sequences, so it is highly specific.

The colloidal gold nucleic acid test strip is a solid-phase immunoassay method for the detection of target substances in complex samples. Capillary action surges on the solid-phase chromatography membrane and is captured by the DNA sequence on the detection line, forming a T-line visible to the naked eye. Under certain conditions, the strength of the T-line signal is positively correlated with the concentration of the target nucleic acid. The method of combining the colloidal gold immunochromatographic detection test strip with the colloidal gold reader, according to the principle of reflection photometry, can quickly, accurately and simply measure the correlation. For quantitative or semi-quantitative detection of substances, colloidal gold nucleic acid test strips have become a point-of-care testing (POCT) method.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the defects in the prior art, and provide a non-diagnostic method for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology.

The present invention adopts the following technical solutions to achieve the above object:

A method for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes, comprising the following steps:

1) use the hydrothermal method to prepare the colloidal gold nanoparticles of uniform particle size;

2) after using TCEP to activate sulfhydryl h-DNA, react with colloidal gold to form a gold sulfide bond for the preparation of colloidal gold nucleic acid probe; Wherein, sulfhydryl h-DNA is 5'-SH-TTTTTAGCTTATCAACTACTGATCAACATCAGTCTGATAAGCTA-3';

3) complementary pairing of the colloidal gold nucleic acid probe and the miRNA to be detected, adding a double-stranded specific nuclease to achieve target recycling and signal amplification; after the reaction is completed, adding a stop solution to inactivate the enzyme to obtain a reaction solution;

4) Assembly and detection of nucleic acid test strips: add the reaction solution generated in the above step 3) to the assembled nucleic acid test strips, and observe the detection results; the nucleic acid test strips contain detection pads; the nucleic acid test strips contain detection pads; The detection pad contains a detection line, and the detection line is protected with sd-DNA at a concentration of 5-20 μM; the base sequence number of the sd-DNA is 5'-biotin-AAAACATGGTTACCGATCCAAGTTCAGTAGTTGATA-3'.

2. the non-diagnostic purpose according to claim 1 detects the method for miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology, it is characterized in that, the concrete steps of step 1) are:

The sodium citrate solution was added to the reactor, heated to boiling under magnetic stirring, and the chloroauric acid solution was added for the first time; the molar ratio of chloroauric acid and sodium citrate was 1:10-20; the solution turned light pink Then stop heating, the solution is cooled to 90-95 ℃, add the same amount of chloroauric acid solution added for the first time to react for 20-30min, and then add the same amount of chloroauric acid solution added for the first time to react to obtain gold seeds solution;

The gold seed solution and the separately prepared sodium citrate solution are mixed as the growth solution for the next step of the reaction, the growth solution is heated to 90-95 ° C, and then the chloroauric acid solution is added to react for 20-30min, and the same amount of chloroauric acid solution is added again to obtain the following: A large-diameter colloidal gold nanoparticle solution for one-step reaction; wherein, the molar ratio of gold seed solution: chloroauric acid: sodium citrate is 1:1:13.75; the diameter of the prepared colloidal gold nanoparticle solution is 13-40 nm.

The specific steps of step 2) are as follows: 10-50 μL of a thiol stem-loop h-DNA probe with a concentration of 10-50 μM, adding 10-50 μL of a tris(2-carboxyethyl) phosphine TCEP solution with a concentration of 10-50 mM, and activated After 1-3h, add 1-5mL of uniform particle size nanoparticles, and rotate for 16-24 hours; then add 4-10μL of 1mg/mL mouse monoclonal primary antibody Ab1 (Beijing Boaosen Biotechnology, bsm -2027M), react for 1-3h; then add 0.1-0.5mL of sodium chloride solution with a concentration of 3M, put it at 4°C to react overnight, centrifuge at 8000-15000 revolutions for 15-30min, remove the supernatant, and use Resuspend the pellet in 0.1-0.5mL of 0.01M phosphate buffered solution PBS to obtain a colloidal gold nucleic acid probe.

The specific steps of step 3) are to mix 3-7 μL of colloidal gold nucleic acid probe and miRNA to be detected in phosphate buffer solution according to the volume ratio of 1-3:1, add 0.1-0.6U of double-stranded specific nuclease and 10 ×Double-strand specific nuclease buffer, rotate at low speed on a vortexer to mix evenly, then place the above mixed solution in a metal bath and react at 35-60°C for 20-60 minutes to achieve target recycling and signal amplification . After the reaction was completed, a stop solution was added and the reaction was carried out at 45°C for 5 minutes to inactivate the enzyme.

In step 4), the nucleic acid test strip includes a test strip jam and sequentially arranged sample pads, glue gold pads, detection pads, and absorption pads arranged in the test strip jam; wherein the length of the sample pad is 1.1- Between 2cm; the length of the glue gold pad is 0.3-0.8cm; the length of the detection pad is 2.5-4.5cm; the length of the absorption pad is 1.1-2cm.

The detection pad comprises a detection line and a quality control line, wherein the quality control line contains the secondary antibody Ab2 (Thermo Fisher Scientific, A32723) of anti-Ab1 ((Beijing Boaosen Biotechnology, bsm-2027M)), The concentration is 0.8-2mg.

The specific step of step 4) is to add the reaction solution obtained in step 3) to the test strip, and the signal collection time of the test strip is 5-15min. relationship to establish the standard curve of the detection technology of this invention.

The present invention also includes a kit for detecting miRNA-21, comprising a colloidal gold nucleic acid probe, a nucleic acid test strip and a double-strand specific nuclease.

Compared with the prior art, the beneficial effects of the present invention are:

1. Significantly improve the detection sensitivity of miRNA. Mainly through the organic combination of amplifying the detection signal and reducing the background noise, that is, based on the DSN-assisted target recycling and signal enrichment amplification strategy, to provide more d-DNA to be detected for the detection of nucleic acid test strips ; Through the aggregation of colloidal gold nucleic acid probes as the detection signal, the colloidal gold signal does not need additional light excitation and overcomes the interference of background noise; thereby significantly improving the detection sensitivity.

2. Realize the rapid, sensitive and direct detection of miRNA in the sample. For samples, due to the university specificity of DSN enzyme and the rapidity of nucleic acid test strips, the detection time of miRNA in the sample is significantly improved, and the miRNA detection time is shortened to 1 hour about.

3. Realize the efficient and specific detection of miRNA in the sample. Through the synergistic effect of the molecular stem-loop probe and DSN enzyme, the target miRNA can be detected with high specificity from the sample to be tested, and high-specificity detection can be achieved.

Description of drawings

Figure 1 is a schematic diagram of the detection principle of a method for detecting miRNA-21 based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes;

Figure 2 is a graph showing the results of agarose gel electrophoresis characterization results based on isothermal cyclic amplification of DSN-assisted targets;

Figure 3 shows the color development of different h-DNA under negative conditions, which is used for the selection of h-DNA in this method;

Figure 4 is a graph showing the sensitivity of miRNA detection based on target isothermal cyclic amplification and nucleic acid test strip technology for non-diagnostic purposes.

Detailed ways

In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention will be described in further detail below in conjunction with the accompanying drawings and the best embodiments.

The principle of the kit of the present invention for detecting miRNA-21 is shown in Figure 1. First, colloidal gold nanoparticles with uniform particle size are prepared by hydrothermal method, and then the colloidal gold nanoparticles are combined with thiolated h-DNA molecular stem-loop probes. reaction to prepare a highly specific colloidal gold nucleic acid probe. In the presence of the target, first, h-DNA on the colloidal gold nucleic acid probe can efficiently and specifically bind to the target miRNA-21 in clinical samples; then, it can efficiently recognize and digest complete complementation through DSN The ability of paired DNA double strands or DNA/RNA hybridization double strands to release the target miRNA-21, h-DNA is cleaved to generate colloidal gold d-DNA; the detection line on the nucleic acid test strip passes through biotin Streptavidin was used to immobilize sd-DNA, and goat anti-mouse antibody was immobilized on the quality control line; the d-DNA generated in the presence of miRNA-21 was finally complementary to the sd-DNA on the detection line on the nucleic acid test strip to generate a detection signal , and at the same time, when the nucleic acid probe flows through the quality control line, a corresponding quality control signal will be generated. The feasibility verification of this method is shown in Figure 2. When the target miRNA-21 and DSN are present at the same time, it can be seen that h-DNA is cleaved and the band becomes weaker. In the absence of the target substance, the colloidal gold nucleic acid probe cannot bind to the target substance from the sample, so the subsequent reaction cannot proceed, and the colloidal gold signal of the nucleic acid test strip will not be generated. The detection sensitivity curve results of this method are shown in Figure 4, and its sensitivity can reach the nM level. The primers, DNA and RNA sequences used in this kit were synthesized by Shanghai Shenggong.

Design of specific h-DNA: creatively design specific h-DNA stem-loop probes according to the difference of the paired Tm temperature value and ΔG energy value through the sequence analysis of the target miRNA. The Tm temperature value of miRNA-21 pairing is 57°C, and the ΔG energy value is -12.3. Therefore, the designed hDNA probe has a Tm value higher than 57°C and a ΔG energy value higher than -12.3. The h-DNA probe can be highly specific and specific. binding target miRNA. Due to the selective cleavage of DSN, as shown in Figure 3, three stem-loop probes were designed, namely h-DNA1, h-DNA2 and h-DNA3; the nucleic acid sequences used are shown in Table 1: Table 1

Embodiment 1: a kind of non-diagnostic purpose is based on the method of target isothermal cyclic amplification and nucleic acid test strip technology detection miRNA-21, comprises the following steps:

1) Use the hydrothermal method to prepare colloidal gold nanoparticles of uniform particle size; add sodium citrate solution to the reactor, heat to boiling under magnetic stirring conditions, and add chloroauric acid solution for the first time; The molar ratio of sodium citrate is 1:15; after the solution becomes light pink, the heating is stopped, the solution is cooled to 90-95°C, and the same amount of chloroauric acid solution as the first added amount is added to react for 20-30min, and then added with The first time adding the same amount of chloroauric acid solution reaction to obtain gold seed solution;

The gold seed solution and the separately prepared sodium citrate solution are mixed as the growth solution for the next step of the reaction, the growth solution is heated to 90-95 ° C, and then the chloroauric acid solution is added to react for 20-30min, and the same amount of chloroauric acid solution is added again to obtain the following: A large-diameter colloidal gold nanoparticle solution for one-step reaction; wherein, the molar ratio of gold seed solution: chloroauric acid: sodium citrate is 1:1:13.75; the diameter of the prepared colloidal gold nanoparticle solution is 13-40 nm.

2) After using TCEP to activate sulfhydryl h-DNA1, it reacts with colloidal gold to form gold-sulfur bonds for the preparation of colloidal gold nucleic acid probes; 20 μL of 10-50 μM sulfhydryl stem-loop h-DNA probe is added, and 20 μL of the concentration is 20mM tris(2-carboxyethyl)phosphine TCEP solution, after activation for 1-3h, add 2mL of uniform particle size nanoparticles, and rotate for 16-24 hours; then add 6μL of 1mg/mL mouse monoclonal first Antibody Ab1, react for 1-3h; then add 0.2mL of 3M sodium chloride solution, put it at 4°C for overnight reaction, centrifuge at 8000-15000 revolutions for 15-30min, remove the supernatant, and use 0.3mL of The precipitate was resuspended in 0.01M phosphate buffered solution PBS to obtain colloidal gold nucleic acid probes.

3) The colloidal gold nucleic acid probe and the miRNA to be detected are complementary paired, and double-stranded specific nuclease is added to realize target recycling and signal amplification; after the reaction is completed, a stop solution is added to inactivate the enzyme to obtain a reaction solution; Take 5μL of colloidal gold nucleic acid probe and the miRNA to be tested and mix them in phosphate buffer in proportion, add 0.3U double-strand specific nuclease and 10× double-strand specific nuclease buffer, colloidal gold nucleic acid probe: miRNA to be tested : The volume ratio of double-strand specific nuclease is 1:1:1; rotate at low speed on a vortexer to make it evenly mixed, then place the above mixed solution in a metal bath and react at 50°C for 50 minutes to achieve target recycling and recovery. Signal amplification. After the reaction was completed, a stop solution was added, and the reaction was carried out at 45°C for 5 minutes to inactivate the enzyme. The concentrations of the miRNAs to be tested are respectively prepared at 0M, ^0.5x10-9M , ^1x10-9M , ^2x10-9M , ^4x10-9M , and ^10x10-9M , respectively.

4) Assembly and detection of nucleic acid test strips: add the reaction solution generated in the above step 3) to the assembled nucleic acid test strips, and observe the detection results; the nucleic acid test strips contain detection pads; the nucleic acid test strips contain detection pads; The paper strip includes a test strip jam shell and a sample pad, a glue gold pad, a detection pad, and an absorption pad arranged in sequence in the test strip jam shell; wherein the length of the sample pad is between 1.1-2 cm; the glue gold The length of the pad is 0.3-0.8 cm; the length of the detection pad is 2.5-4.5 cm; the length of the absorbent pad is 1.1-2 cm. The detection pad comprises a detection line and a quality control line, wherein the detection line contains deoxyribonucleotide (sd-DNA) for detection at a concentration of 5-20 μM; the quality control line contains a secondary antibody against Ab1 Ab2 ((Thermo Scientific, A32723), the concentration is 0.15mg-1.0mg/mL.

The specific step of step 4) is to add the reaction solution obtained in step 3) to the assembled nucleic acid test strip, and the collection time of the test strip signal is 10 min. The relationship between the corresponding miRNA concentrations was established to establish a standard curve for the detection technology of the invention. The results are shown in Figure 4, the color development of nucleic acid test strip colloidal gold is proportional to the concentration of miRNA-21, the greater the concentration, the greater the color intensity, so the present invention can be applied to the detection of miRNA-21 in samples

In step 2) of comparative example 1 and comparative example 2, h-DNA2 and h-DNA3 colloidal gold were respectively used to form gold-sulfur bonds for the preparation of colloidal gold nucleic acid probes; the results showed that they could not specifically bind to the target miRNA.

The above contents are only preferred embodiments of the present invention. For those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific embodiments and application scope. limits.

Claims (8)

1. A method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnosis purposes is characterized by comprising the following steps:

1) preparing colloidal gold nanoparticles with uniform particle size by using a hydrothermal method;

2) activating sulfhydryl group h-DNA by using TCEP, and reacting with colloidal gold to form a gold-sulfur bond for preparing a colloidal gold nucleic acid probe; wherein, the sulfhydryl-H-DNA is 5 '-SH-TTTTTAGCTTATCAACTACTGATCAACATCAGTCTGATAAGCTA-3';

3) complementary pairing is carried out on the colloidal gold nucleic acid probe and the miRNA to be detected, and double-strand specific nuclease is added to realize target recycling and signal amplification; after the reaction is finished, adding a stop solution to inactivate the enzyme to obtain a reaction solution;

4) assembling and detecting the nucleic acid test strip: adding the reaction solution generated in the step 3) to the assembled nucleic acid test strip, and observing the detection result; the nucleic acid test strip comprises a detection pad; the detection pad comprises a detection line, and sd-DNA is protected on the detection line, and the concentration is 5-20 mu M; the base sequence of sd-DNA is 5 '-biotin-AAAACATGGTTACCGATCCAAGTTCAGTAGTTGATA-3'.

2. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes according to claim 1, wherein the specific steps of step 1) are as follows:

adding a sodium citrate solution into a reactor, heating to boil under the condition of magnetic stirring, and adding a chloroauric acid solution for the first time; the molar ratio of the chloroauric acid to the sodium citrate is 1: 10-20 parts of; stopping heating after the solution turns into light pink, cooling the solution to 90-95 ℃, adding chloroauric acid solution with the same amount as the first addition amount for reaction for 20-30min, and adding chloroauric acid solution with the same amount as the first addition amount again for reaction to obtain gold seed solution;

mixing the gold seed solution and an additionally prepared sodium citrate solution to serve as a growth solution for the next reaction, heating the growth solution to 90-95 ℃, adding a chloroauric acid solution to react for 20-30min, and adding an equivalent chloroauric acid solution again to obtain a large-particle-size colloidal gold nanoparticle solution for the next reaction; wherein, the gold seed solution: gold chloride acid: the molar ratio of the sodium citrate is 1:1: 13.75; the grain diameter of the prepared colloidal gold nano particle solution is 13-40 nm.

3. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes according to claim 1, wherein the specific steps of step 2) are 10-50 μ L of a sulfhydryl stem-loop h-DNA probe with a concentration of 10-50 μ M, 10-50 μ L of a tris (2-carboxyethyl) phosphine TCEP solution with a concentration of 10-50mM is added, 1-5mL of nanoparticles with uniform particle size are added after activation for 1-3h, and the rotation culture reaction is carried out for 16-24 h; then 4-10 μ L of 1mg/mL murine monoclonal primary antibody Ab1 is added for reaction for 1-3 h; then 0.1-0.5mL of 3M sodium chloride solution is added, after reaction at 4 ℃ overnight, the mixture is centrifuged for 15-30min at 8000-.

4. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes as claimed in claim 1, wherein the specific steps of step 3) are to take 3-7 μ L of the colloidal gold nucleic acid probe and miRNA to be detected to mix in phosphate buffer according to the volume ratio of 1-3:1, add 0.1-0.6U of double-stranded specific nuclease and 10 Xdouble-stranded specific nuclease buffer, rotate on a vortex apparatus at low speed to mix them uniformly, then place the mixed solution in a metal bath, react for 20-60 minutes at 35-60 ℃, and realize target recycling and signal amplification. After the reaction, the enzyme was inactivated by adding a stop solution and reacting at 45 ℃ for 5 minutes.

5. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid strip technology for non-diagnostic purposes as claimed in claim 1, wherein the nucleic acid strip in step 4) comprises a strip shell and a sample pad, a gold pad, a detection pad and an absorption pad arranged in sequence in the strip shell; wherein the length of the sample pad is between 1.1-2 cm; the length of the rubber gold pad is 0.3-0.8 cm; the length of the detection pad is 2.5-4.5 cm; the length of the absorption pad is 1.1-2 cm.

6. The method of claim 5 for detecting miRNA-21 for non-diagnostic purposes based on target isothermal amplification and nucleic acid dipstick technology, wherein the detection pad comprises a detection line and a quality control line, wherein the quality control line comprises the second antibody Ab2 of anti Ab1 at a concentration of 0.8-2 mg.

7. The method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes according to claim 6, wherein the specific step of step 4) is to add the reaction solution obtained in step 3) to a test strip, the test strip signal collection time is 5-15min, and a standard curve of the detection technology is established according to the relation between the accumulation of colloidal gold signals on a raw detection line and the concentration of the corresponding miRNA in the corresponding time.

8. A kit for detecting miRNA-21 according to any one of claims 1-7, comprising a colloidal gold nucleic acid probe, a nucleic acid strip, and a double-strand specific nuclease.

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