阅读说明：本技术 非诊断目的基于靶标等温循环扩增及核酸试纸条技术检测miRNA-21的方法及试剂盒 (Method and kit for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnosis purpose ) 是由 常津 陈明慧 宫晓群 王汉杰 罗冉 于 2020-03-23 设计创作，主要内容包括：本发明属于miRNA检测领域,具体涉及一种非诊断目的基于靶标等温循环扩增及核酸试纸条技术检测miRNA-21的方法。包括下述步骤:1)使用水热法制备均一粒径的胶体金纳米粒子；2)使用TCEP活化巯基h-DNA后,与胶体金反应形成金硫键用于制备胶体金核酸探针；3)将胶体金核酸探针和待检miRNA互补配对,加入双链特异性核酸酶,实现靶标循环回收及信号放大；反应结束后,加入终止液,使酶失活,得到反应液；4)核酸试纸条的组装和检测：将上步骤3)产生的反应液加入到组装好的核酸试纸条上,观察检测结果。本发明能够显著提高miRNA的检测灵敏度。(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.)

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.

Technical Field

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.

Background

Micro RNA-21(miRNA-21) is a kind of endogenous non-coding RNA micromolecule, and the obvious abnormal expression of miRNA-21 is closely related to the occurrence and development of various diseases, particularly human tumors. The traditional detection based on miRNA-21, such as gene sequencing, fluorescence quantitative PCR and the like, requires large-scale instrument matching without exception, is time-consuming and labor-consuming, is only suitable for detection mechanisms such as hospitals and the like, and cannot benefit every family to realize portability and popularization. Therefore, early and convenient detection of miRNA-21 has become a research hotspot nowadays.

The target isothermal cycle amplification technology generally refers to an enzymatic reaction-based nucleic acid in vitro isothermal amplification technology, and mainly utilizes the capability of nuclease to cut DNA recognition sites to promote the combination and release of a target object under an isothermal condition, so as to generate a corresponding signal to realize signal accumulation and amplification. Among them, double-strand specific nuclease (DSN), which is one of the most common nucleases suitable for miRNA detection, efficiently recognizes and cleaves DNA strands in a complete complementary pair of DNA double strands or DNA/RNA hybrid double strands, and has little effect on single-stranded DNA and single/double-stranded RNA, and in addition, the degradation is only directed to at least 12 completely matched nucleotide sequences, and thus it has high specificity.

The colloidal gold nucleic acid test strip is a solid-phase immunoassay method for detecting target substances in complex samples, and the main principle is as follows: a short DNA sequence is marked by colloidal gold, and reacts with target substance nucleic acid to form a compound, and due to the fact that capillary action surges on a solid-phase chromatographic membrane, the DNA sequence on a detection line is captured to form a visible T line. Under a certain condition, the strength of a T-line signal is positively correlated with the concentration of target nucleic acid, and the method of combining a colloidal gold immunochromatography detection test strip with a colloidal gold reader can quickly, accurately and simply carry out quantitative or semi-quantitative detection on related substances according to the principle of a reflection photometry, and the colloidal gold nucleic acid test strip becomes a Point-of-care testing (POCT) method.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a method for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnosis purposes.

In order to achieve the purpose, the invention adopts the following technical scheme:

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:

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.

The specific steps of the step 2) are 10-50 mul of sulfhydryl stem-loop h-DNA probe with the concentration of 10-50 mul, 10-50 mul of tris (2-carboxyethyl) phosphine TCEP solution with the concentration of 10-50mM is added, 1-5mL of nano particles with uniform particle diameter are added after 1-3h of activation, and the rotary culture reaction is carried out for 16-24 h; then 4-10 μ L of 1mg/mL murine monoclonal primary antibody Ab1 (Beijing Boolson Biotechnology, bsm-2027M) 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-.

And 3-7 mu L of colloidal gold nucleic acid probe and miRNA to be detected are mixed in phosphate buffer according to the volume ratio of 1-3:1, 0.1-0.6U of double-strand specific nuclease and 10 multiplied by double-strand specific nuclease buffer are added, the mixture is uniformly mixed by rotating on a vortex instrument at low speed, and then the mixed solution is placed in a metal bath and reacts for 20-60 minutes at 35-60 ℃ to realize target circulation recovery and signal amplification. After the reaction, the enzyme was inactivated by adding a stop solution and reacting at 45 ℃ for 5 minutes.

The nucleic acid test strip in the step 4) comprises a test strip card shell, and a sample pad, a colloidal gold pad, a detection pad and an absorption pad which are arranged in the test strip card shell in sequence; 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.

The detection pad comprises a detection line and a quality control line, wherein the quality control line comprises a second antibody Ab2 (Saimer fly science, A32723) of an anti-Ab 1 ((Beijing Boaosen Biotechnology, bsm-2027M)), and the concentration of the second antibody Ab2 is 0.8-2 mg.

The concrete step of the step 4) is that the reaction solution obtained in the step 3) is added into the test strip, the signal collection time of the test strip is 5-15min, and a standard curve of the detection technology is established according to the relation between the accumulation of the colloidal gold signal on the biological detection line in the corresponding time and the concentration of the corresponding miRNA.

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

Compared with the prior art, the invention has the beneficial effects that:

1. the detection sensitivity of miRNA is obviously improved. The detection signal is amplified, so that the organic combination of the two aspects of background noise is reduced, namely more d-DNA to be detected is provided by a strategy based on DSN auxiliary target circulation recovery and signal enrichment amplification, and the method is used for detecting the nucleic acid test strip; the colloidal gold nucleic acid probe is gathered to be used as a detection signal, and the interference of background noise is overcome due to the characteristic that the colloidal gold signal does not need extra light excitation; thereby significantly improving the sensitivity of detection.

2. The rapid and sensitive direct detection of the miRNA in the sample is realized, and the detection time of the miRNA in the sample is remarkably prolonged to about 1 hour due to the high specificity of the DSN enzyme and the rapidity of the nucleic acid test strip aiming at the sample.

3. The high-efficiency specificity detection of miRNA in the sample is realized, and the target miRNA can be detected from the sample to be detected in a high-specificity manner through the synergistic effect of the molecular stem-loop probe and the DSN enzyme, so that the high-specificity detection is realized.

Drawings

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

FIG. 2 is a diagram of the result of agarose gel electrophoresis characterization based on DSN assisted target isothermal cycle amplification;

FIG. 3 is a selection of h-DNA for the present method using different h-DNA color development under negative conditions;

FIG. 4 is a sensitivity experimental diagram for detecting miRNA based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes.

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings and preferred embodiments.

The principle of the kit for detecting miRNA-21 is shown in figure 1, and the kit is characterized in that firstly, colloidal gold nanoparticles with uniform particle size are prepared by a hydrothermal method, and then the colloidal gold nanoparticles are reacted with a sulfhydrylation h-DNA molecular stem-loop probe to prepare a high-specificity colloidal gold nucleic acid probe. Under the condition that a target object exists, firstly, h-DNA on the colloidal gold nucleic acid probe can be combined with target miRNA-21 in a clinical sample in a highly efficient and specific manner; then, through the capability of the DSN to efficiently recognize and enzyme-cut the DNA double strand in the completely complementary paired DNA double strand or DNA/RNA hybrid double strand, releasing the target miRNA-21, and cutting the h-DNA to generate colloidal gold d-DNA; the detection line on the nucleic acid test strip fixes sd-DNA through biotin streptavidin, and the quality control line fixes goat anti-mouse antibody; d-DNA generated in the presence of miRNA-21 is finally complementarily paired with sd-DNA on a detection line on a nucleic acid test strip to generate a detection signal, and meanwhile, a nucleic acid probe generates a corresponding quality control signal when flowing through a quality control line. The feasibility of the method is verified as shown in FIG. 2, when the target miRNA-21 and the DSN exist at the same time, the h-DNA is cut, and the band is weakened. When the target substance does not exist, the colloidal gold nucleic acid probe cannot be combined with the target substance from the sample, so that the subsequent reaction cannot be carried out, and a colloidal gold signal of the nucleic acid test strip is not generated. The detection sensitivity curve result of the method is shown in FIG. 4, and the sensitivity can reach nM level. The primers, DNA and RNA sequences used in the kit are synthesized by Shanghai.

Design of specific h-DNA: through sequence analysis of target miRNA, a specific h-DNA stem-loop probe is creatively designed according to the difference between a paired Tm temperature value and a delta G energy value. The temperature value of the miRNA-21 pairing Tm is 57 ℃, the energy value of delta G is-12.3, therefore, the Tm value of the designed hDNA probe is higher than 57 ℃, the energy value of delta G is higher than-12.3, and the h-DNA probe can efficiently and specifically combine with the target miRNA. Due to the selective cutting property of DSN, as shown in FIG. 3, three stem-loop probes, h-DNA1, h-DNA2 and h-DNA3, were designed; the nucleic acid sequences used therein are shown in table 1: TABLE 1