CN102719526A - MicroRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize fluorescent nano silver cluster probe - Google Patents

Abstract

The invention provides an analysis method for quantitatively detecting microRNA by synthesizing fluorescent nano-silver cluster probes through constant temperature amplification reaction. The method is to design a DNA amplification template containing three functional sequences: binding sequence to target microRNA, nicking endonuclease cutting sequence and DNA complementary sequence for synthesizing fluorescent nanoclusters. When the target microRNA is combined with the DNA amplification template, the constant temperature amplification reaction and the specific enzyme cleavage reaction of the nicking endonuclease are induced to obtain a large number of single-stranded DNA products, in which the DNA sequence of the synthetic fluorescent nano-silver cluster is mixed with silver nitrate solution Fluorescent nano-silver cluster probes were prepared under the reduction of sodium borohydride, the fluorescence signal of the reaction system was measured and the fluorescence change value was calculated, compared with the standard working curve, the concentration of the target microRNA was calculated. The method has the characteristics of high sensitivity, strong specificity, wide linear detection range, low background signal, simple operation, etc., and can be widely used in the detection of microRNA in biological samples of tissue, blood or cells.

Description

An analytical method for the quantitative detection of microRNA by synthesizing fluorescent nanosilver cluster probes using isothermal amplification reaction

technical field

The invention relates to an analysis method for detecting microRNA with a fluorescent nano-silver cluster probe, belonging to the field of detection and analysis.

Background technique

MicroRNA is a kind of endogenous non-coding RNA with regulatory function found in eukaryotes, and its size is about 18-25 nucleotides in length. It is involved in a wide variety of regulatory pathways, including development, viral defense, hematopoietic processes, organ formation, cell proliferation and apoptosis, fat metabolism, etc. Recent studies have found that microRNA plays a vital role in the process of tumorigenesis and has become an ideal tumor marker. Therefore, quantitative detection of microRNA is of great significance for understanding its biological function, early diagnosis of cancer and development of new drugs. Compared with traditional nucleic acid detection, the unique characteristics of microRNA, such as short sequence, high family sequence homology and low abundance expression, increase the difficulty of its detection. At present, the methods for detecting microRNA mainly include Northern blot analysis, microarray chip technology, polymerase chain reaction (PCR) and so on. Although Northern blot is the current standard method for microRNA analysis, the operation is cumbersome, time-consuming, and low-sensitivity. The analysis and detection requires a large number of samples and separation and enrichment steps, and is very sensitive to contamination. Improper operation of each step in the experiment will affect the analysis results. . Although microarray chip technology can achieve high throughput and simultaneous detection of multiple components, its production and detection costs are high; the sample volume available on the chip is small, resulting in low sensitivity; in addition, the microRNA sequence is short and has high sequence similarity , cannot simultaneously optimize all microRNA hybridization environments to be tested, so the selectivity is not high. Polymerase chain reaction (PCR) is now the main method for quantitative detection of microRNA, including primer extension RT-PCR, stem-loop primer RT-PCR and microRNA tailing RT-PCR and other methods. Although the polymerase chain reaction-based microRNA detection method has the characteristics of rapidity, strong specificity, and high sensitivity, it requires reverse transcription, which increases the cost of the experiment and the complexity of the design. With the continuous discovery of new microRNA sequences and the gradual deepening of microRNA function research, new requirements have been put forward for the analysis and detection of microRNA. Therefore, it is still a challenge to develop simple and direct analytical techniques with high sensitivity and specificity for accurate detection of microRNAs.

Contents of the invention

The present invention relates to an analytical method for the quantitative detection of microRNA by using a constant temperature amplification reaction to synthesize a fluorescent nano-silver cluster probe, which has the advantages of simple operation, high sensitivity, strong specificity, low background signal, wide linear detection range, and wide application range. It is a simple and practical analytical technique.

The present invention utilizes target microRNA to induce isothermal amplification reaction to synthesize fluorescent nano-silver cluster probes for quantitative detection of microRNA. The analysis method is as follows. Design a DNA amplification template containing three functional sequences: the target microRNA binding sequence, the nicking endonuclease digestion sequence and the DNA complementary sequence for the synthesis of fluorescent nanoclusters. The sequence from 3' end to 5' end is target microRNA binding sequence, nicking endonuclease cleavage sequence, target microRNA binding sequence, nicking endonuclease cleavage sequence, and DNA complementary sequence for synthesizing fluorescent nanoclusters. When the target microRNA is combined with the DNA amplification template, a large number of single-stranded DNA products are obtained by inducing the constant temperature amplification reaction and the specific cleavage reaction of the nickelase, and one of the single-stranded DNA products can be combined with the DNA amplification template. Continue to act as a primer for the target microRNA, thereby greatly improving the efficiency of amplification. Another single-stranded DNA product is the synthetic DNA sequence of fluorescent nano-silver clusters, which can be combined with the added silver nitrate ion solution under sodium borohydride reduction to synthesize fluorescent nano-silver cluster probes. , emit the corresponding fluorescence, by measuring the fluorescence signal in the reaction system and calculating the fluorescence change value, compared with the standard working curve, the concentration of the target microRNA is calculated. In addition, the functional sequences in the DNA amplification template can also have different combinations and arrangements, such as the sequence from the 3' end to the 5' end is the target microRNA binding sequence, the nicking endonuclease digestion sequence, and the synthesis of fluorescent nanoclusters DNA complementary sequences, nicking endonuclease cleavage sequences, and DNA complementary sequences for the synthesis of fluorescent nanoclusters.

The method of the present invention can be further described as follows:

After the target microRNA is combined with the DNA amplification template, the constant temperature amplification reaction is induced and the specific enzyme cleavage reaction of the nicking endonuclease is used to obtain a single-stranded DNA product, and the single-stranded DNA product is reduced with the added silver nitrate solution in sodium borohydride Fluorescent nano-silver cluster probes are prepared under the action, the fluorescent signal in the reaction system is measured and the change value of the fluorescent signal is calculated, compared with the standard working curve, the concentration of the target microRNA is calculated.

The particle diameter of the fluorescent nano-silver cluster probe is 1-10 nm, the excitation wavelength is 500-680 nm, and the emission wavelength is 510-900 nm.

Target microRNA detection method of the present invention comprises the following steps:

Step 1: adding the target microRNA to the DNA amplification template solution to incubate the reaction;

Step 2: Add a constant temperature amplification mixture to the reaction solution obtained in step 1, and after incubating the reaction, heat-inactivate the enzyme to terminate the reaction;

Step 3 Add silver nitrate solution and citric acid buffer solution to the reaction solution obtained in step 2, mix well, centrifuge to take the supernatant, then add silver nitrate solution and freshly prepared sodium borohydride solution, react in the dark to synthesize fluorescence nanosilver clusters;

Step 4 measures the fluorescence signal of the fluorescent nano-silver cluster solution obtained in step 3.

The target microRNA in the step 1 comes from biological samples of tissue, blood or cells.

The incubation reaction in step 1 is carried out at 37° C. to 55° C. in a reaction buffer solution, the volume ratio of the DNA amplification template reaction solution in step 1 to the target microRNA is 0.1 to 10, and the reaction time is 5 to 50 minutes. The concentration of the DNA amplification template is 10 to 800nM; the concentration of the target microRNA is 1aM to 100nM; the DNA amplification template contains three functional sequences: a binding sequence to the target microRNA, and a cleavage sequence by the nicking endonuclease and the DNA complementary sequence of the synthesized fluorescent nano-cluster, the reaction buffer solution is 25mM Tris-HNO ₃ , 50mM sodium nitrate, 5mM magnesium nitrate, 0.5mM dithiothreitol and the pH is 6.0-8.9.

The incubation reaction in step 2 is at 35°C to 55°C and reaction buffer, the volume ratio of the reaction solution obtained in step 1 to the constant temperature amplification mixture is 0.1 to 10, and the reaction is 20 to 200 minutes, and the constant temperature amplification mixture is Contains constant temperature polymerase, nicking endonuclease, RNase inhibitor and dNTP; the concentration of the constant temperature polymerase is 0.01UμL ^-1 ～1.0U μL ^-1 , the concentration of nicking endonuclease is 0.1U μL ^-1 ～ 1.0UμL ^-1 , the concentration of RNase inhibitor is 0.1U μL ^-1 ~1.0UμL ^-1 , the concentration of dNTP solution is 80 ~ 500μM; the reaction buffer is 10mM sodium nitrate, 20mM ammonium nitrate, 20mM Tris-HNO ₃ , 2mM magnesium nitrate and 0.1% Triton X-100 and pH 6.0-8.9.

(exo-)DNA聚合酶、phi29DNA聚合酶或Klenow Fragment聚合酶。The thermostatic polymerase is commercially available

(exo-)DNA polymerase, phi29 DNA polymerase or Klenow Fragment polymerase.

The nicking endonuclease is commercially available Nt.CviPII, Nb.BbvCI, Nb.BtsI, Nt.BsmAI, Nt.BbvCI, Nt.BspQI, Nt.AlwI or Nt.BstNBI.

The standard working curve is a standard solution of target microRNA with a known concentration. After reacting according to the operation steps, measure the fluorescence signal in the reaction system and calculate the fluorescence change value, and then according to the concentration of microRNA in the standard solution and the concentration in the system, Fluorescence change value to make a standard working curve.

In the step 3, the silver nitrate concentration is 10-800 μM, the sodium borohydride solution is freshly prepared, and its concentration is 10-10 mM, the reaction temperature is 25°C-55°C, and the reaction time is 20-120 minutes.

In the method of the present invention, the design of double-nicking endonuclease cutting sequence is adopted in the DNA amplification template, and the single-stranded DNA sequence obtained by the amplification is combined with the DNA amplification template to act as a primer, which greatly improves the amplification efficiency. The design of DNA complementary sequences for the synthesis of fluorescent nanoclusters in DNA amplification templates enables the application of a novel fluorescent nanoprobe. Compared with traditional fluorescent probes, fluorescent nanoprobes have excellent optical properties, such as high fluorescence intensity, resistance to photobleaching, tunable emission color, and tunable excitation and emission wavelengths.

The advantages of the present invention are:

(1) High sensitivity: In the DNA amplification template, the double microRNA binding sequence (or the DNA complementary sequence of the double synthetic fluorescent nanocluster) and the design of the nicking endonuclease cutting sequence make the amplification product the same as the target microRNA, and can Combined with the DNA amplification template, the constant temperature amplification reaction is started to achieve the effect of exponential signal amplification, thereby greatly improving the detection sensitivity.

(2) Good selectivity: Based on the principle of specific complementary base pairing between the target miRNA and the DNA amplification template, the interference of other molecules is very small.

(3) Low background signal: the synthesis of fluorescent nano-silver clusters is directly related to the base composition of DNA sequences, and only DNA sequences with specific base composition can synthesize fluorescent nano-silver clusters with stable fluorescent signals.

(4) Multiplex detection: By using the differences in the optical properties of silver nanocluster probes synthesized by DNA sequences with different base compositions, and designing different DNA amplification templates for different target microRNAs, multiple microRNA detections can be realized.

(5) High-throughput detection: 96 or 384 microplates are used, which can realize the simultaneous production of standard working curves and detection of samples to be tested, reducing detection errors.

(6) The operation process is simple and fast: the whole detection process is simple.

(7) No pollution: The whole detection process does not need to use organic solvents or toxic reagents.

Description of drawings

Figure 1 is a schematic diagram of the principle of the analytical method for the quantitative detection of miR-141 by using the isothermal amplification reaction to synthesize fluorescent nano-silver cluster probes.

Figure 2 Working curve preparation. (A) Fluorescence spectra of reaction solutions with different concentrations of miR-141; (B) Working curve of fluorescence change (F/F ₀ -1) and logarithm value of miR-141 concentration in reaction solutions with different concentrations of miR-141.

Figure 3 Specificity experiments. (A) Fluorescence spectra of different target microRNA reaction solutions; (B) Histogram of experimental data comparison (F/F ₀ -1).

Figure 4 is a schematic diagram of the principle of the analytical method for the quantitative detection of miR-21 by using the isothermal amplification reaction to synthesize fluorescent nano-silver cluster probes.

Fig. 5 is a schematic diagram of the principle of an analysis method for the quantitative detection of let-7a synthesized by a fluorescent nano-silver cluster probe using an isothermal amplification reaction.

Fig. 6 is a schematic diagram of the principle of an analysis method for the quantitative detection of miR-646 by using a constant temperature amplification reaction to synthesize a fluorescent nano-silver cluster probe.

Fig. 7 is a schematic diagram of the principle of an analysis method for multiplex detection of miR-141 and miR-21 by synthesizing fluorescent nano-silver cluster probes by constant temperature amplification reaction.

Symbol Description

(exo-)DNA聚合酶。In Figure 1, miR-141 is 5'-UAACACUGUCUGGUAAAGAUGG-3', A is the target miR-141 binding sequence (5'-CCATCTTTACCAGACAGTGTTA-3'), and X is the Nt.B stNBI restriction sequence (5'-TCTTGACTC- 3'), B is the DNA complementary sequence (5'-TTGGGCGGGTGGGTGGG-3') of the synthetic fluorescent nano-silver cluster, T is the DNA sequence consistent with miR-14 generated by amplification (5'-TAACACTGTCTGGTAAAGATGG-3'), R is The DNA sequence of the synthetic fluorescent nano silver cluster (5'-CCCACCCACCCGCCCAA-3'), C is Nt.BstNBI endonuclease, D is

(exo-)DNA polymerase.

In accompanying drawing 2, F is the fluorescence value of miR-141 standard solution at 644nm, F ₀ is the fluorescence value of blank sample (not containing miR-141) at 644nm, (F/F ₀ -1) represents miR-141 The fluorescence change value of the standard solution.

In accompanying drawing 3, F is the fluorescence value of the target microRNA solution at 644nm, F ₀ is the fluorescence value of the blank sample (without microRNA) at 644nm, (F/F ₀ -1) represents the fluorescence change value of the microRNA solution.

In Figure 4, miR-21 is 5'-UAGCUUAUCAGACUGAUGUUGA-3', A is the target miR-21 binding sequence (5'-TCAACATCAGTCTGATAAGCTA-3'), X is the Nt.BstNBI restriction sequence (5'-ACGTGACTC-3 '), B is the DNA complementary sequence of the synthetic fluorescent nanosilver cluster (5'-TTGGGCGGGTGGGTGGG-3'), T is the DNA sequence consistent with miR-21 (5'-TAGCTTATCAGACTGATGTTGA-3'), R is the synthetic DNA sequence of fluorescent nanosilver cluster (5'-CCCACCCACCCGCCCAA-3'), C is Nt.BstNBI endonuclease, D is (exo-)DNA polymerase.

(exo-)DNA聚合酶。In Figure 5, let-7a is 5'-UGAGGUAGUAGGUUGUAUAGUU-3', A is the target let-7a binding sequence (5'-AACTATACAACCTACTACCTCA-3'), and X is the Nb.BbvCI restriction sequence (5'-GCTGAGG-3 '), B is the DNA complementary sequence of the synthetic fluorescent nanosilver cluster (5'-TTGGGCGGGTGGGTGGG-3'), T is the DNA sequence consistent with let-7a produced by amplification (5'-TGAGGTAGTAGGTTGTATAGTT-3'), R is the synthetic The DNA sequence of the fluorescent nanosilver cluster (5'-CCCACCCACCCGCCCAA-3'), C is Nb.BbvCI endonuclease, D is

(exo-)DNA polymerase.

In Figure 6, miR-646 is 5'-AAGCAGCUGCCUCUGAGGC-3', A is the target miR-646 binding sequence (5'-GCCTCAGAGGCAGCTGCTT-3'), and X is the Nb.BbvCI restriction sequence (5'-GCTGAGG-3 '), B is the DNA complementary sequence of the synthetic fluorescent nanosilver cluster (5'-TTGGGCGGGTGGGTGGG-3'), T is the DNA sequence consistent with miR-646 generated by amplification (5'-AAGCAGCTGCCTCTGAGGC-3'), R/R ': DNA sequence (5'-CCCACCCACCCGCCCAA-3') of the synthesized fluorescent nanosilver cluster, C is Nb.BbvCI endonuclease, D is phi29 DNA polymerase.

(exo-)DNA聚合酶。In accompanying drawing 7, miR-141 is 5'-UAACACUGUCUGGUAAAGAUGG-3', miR-21 sequence is 5'-UAGCUUAUCAGACUGAUGUUGA-3', A is target miR-21 binding sequence (5'-UAGCUUAUCAGACUGAUGUUGA-3'), X is Nt.B stNBI restriction sequence (5'-TCTTGACTC-3'), E is the target miR-141 binding sequence (5'-UAACACUGUCUGGUAAAGAUGG-3'), B is the DNA complementary sequence for the synthesis of fluorescent nanosilver clusters (5'- TTGGGCGGGTGGGTGGG-3'), F is the DNA complementary sequence (5'-ATCGGGGGCGA-3') of the synthetic fluorescent nano-silver cluster, T is the DNA sequence consistent with miR-21 produced by amplification (5'-TAGCTTATCAGACTGATGTTGA-3'), U is the amplified DNA sequence consistent with miR-141 (5'-TAACACTGTCTGGTAAAGATGG-3'), R is the DNA sequence of the synthetic fluorescent nano-silver cluster (5'-ATCGCCCCCGAT-3'), S is the synthetic fluorescent nano-silver cluster The DNA sequence (5'-CCCACCACCCGCCCAA-3'), C is Nt.BstNBI endonuclease, D is

(exo-)DNA polymerase.

Specific implementation examples:

Will help to understand the present invention by following embodiment, but content of the present invention can not be limited

Implementation example 1:

(exo-)DNA聚合酶和Nt.BstNBI核酸内切酶。DNA扩增模板上的序列排布为靶标miR-141结合序列(5’-CCATCTTTACCAGACAGTGTTA-3’)，Nt.BstNBI酶切序列(5’-TCTTGACTC-3’)，miR-141结合序列(5’-CCATCTTTACCAGACAGTGTTA-3’)，Nt.BstNBI酶切序列(5’-TCTTGACTC-3’)，以及合成荧光纳米银簇的DNA互补序列(5’-TTGGGCGGGTGGGTGGG-3’)，3’端磷酸化。The analysis method for the quantitative detection of miR-141 (5'-UAACACUGUCUGGUAAAGAUGG-3') synthesized by a constant temperature amplification reaction with a fluorescent nano-silver cluster probe, the detection principle is shown in Figure 1. use

(exo-)DNA polymerase and Nt.BstNBI endonuclease. The sequence arrangement on the DNA amplification template is the target miR-141 binding sequence (5'-CCATCTTTACCAGACAGTGTTA-3'), the Nt.BstNBI restriction sequence (5'-TCTTGACTC-3'), the miR-141 binding sequence (5'-CCATCTTTACCAGACAGTGTTA-3'), Nt.BstNBI restriction sequence (5'-TCTTGACTC-3'), and the DNA complementary sequence for the synthesis of fluorescent nanosilver clusters (5'-TTGGGCGGGTGGGTGGG-3'), phosphorylated at the 3' end.

(exo-)DNA聚合酶(0.05U μL^-1)、Nt.BstNBI核酸内切酶(0.4U μL^-1)、RNase抑制剂(0.8U μL^-1)和dNTP(250μM)，反应缓冲液为10mM硝酸钠、20mM硝酸铵、20mM Tris-HNO₃、2mM硝酸镁和0.1％Triton X-100，pH为8.8。于53℃孵育反应50分钟，然后于90℃孵育5分钟终止反应。向上述反应液中加入8μL硝酸银溶液(497.5μM)和100μL柠檬酸钠缓冲溶液(20mM，pH 7.0)，震荡混匀后，12,000转离心10分钟后取上清溶液，于黑暗中放置15分钟后，加入新鲜配制的1μL硼氢化钠(3.6mM)溶液，混匀，于黑暗中放置1小时，测定溶液的荧光信号。The following specific implementation of miR-141 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The room temperature in the present invention refers to the laboratory temperature of 23-28° C. for carrying out the operation. The specific operation steps are as follows: add 1 μL target miR-141 solution to 4 μL DNA amplification template solution (200 nM), the reaction buffer solution is 25 mM Tris-HNO ₃ , 50 mM sodium nitrate, 5 mM magnesium nitrate, 0.5 mM dithiothreitol, pH is 7.9. After incubating at 88°C for 10 minutes, add 5 μL of constant temperature amplification mixture, including

(exo-)DNA polymerase (0.05U μL ^-1 ), Nt.BstNBI endonuclease (0.4U μL ^-1 ), RNase inhibitor (0.8U μL ^-1 ) and dNTP (250μM), the reaction buffer is 10 mM sodium nitrate, 20 mM ammonium nitrate, 20 mM Tris- _HNO3 , 2 mM magnesium nitrate and 0.1% Triton X-100, pH 8.8. Reactions were incubated at 53°C for 50 minutes and then terminated by incubation at 90°C for 5 minutes. Add 8 μL silver nitrate solution (497.5 μM) and 100 μL sodium citrate buffer solution (20 mM, pH 7.0) to the above reaction solution, shake and mix well, then centrifuge at 12,000 rpm for 10 minutes, take the supernatant solution, and place it in the dark for 15 minutes Afterwards, 1 μL of a freshly prepared sodium borohydride (3.6 mM) solution was added, mixed evenly, and left in the dark for 1 hour to measure the fluorescence signal of the solution.

Preparation of the working curve: prepare miR-141 standard solutions of known concentrations, respectively, with concentrations of 1aM, 10aM, 100aM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, and 1nM, and blank samples (not containing miR-141 , that is, the concentration of miR-141 is 0), and the above-mentioned steps are followed to measure the fluorescence spectrum of each reaction solution, as shown in FIG. 2(A). Calculate its fluorescence change value (F/F ₀ -1), that is, the fluorescence value (F) of the miR-141 standard solution at 644nm and the fluorescence value (F ₀ ) of the blank sample (without miR-141) at 644nm Ratio minus 1, then according to the concentration of miR-141 standard solution and its fluorescence change value (F/F ₀ -1) make a standard working curve, as shown in Figure 2 (B), the concentration logarithmic value of miR-141 standard solution and its The fluorescence change value (F/F ₀ -1) has a linear relationship in the range of 1aM-1nM, and the linear equation is Y=10.57+1.25lgX.

Figure 3 shows the specificity experiment of the method of the present invention, select miR-429, miR-200b, let-7d, miR-21 as miR-141 control sample (detection concentration is 1pM), and blank sample (do not contain miR -141, that is, the concentration of miR-141 is 0), the sequence of miR-429 is 5'-UAAUACUGUCUGGUAAAACCGU-3', the sequence of miR-200b is 5'-UAAUACUGCCUGGUAAUGAUGA-3', and the sequence of let-7d is 5'-AGAGGUAGUAGGUUGCAUAGUU-3' , the miR-21 sequence is 5'-UAGCUUAUCAGACUGAUGUUGA-3', as shown in Figure 3 (A) is the fluorescence spectrum of each target reaction solution, and Figure 3 (B) is the fluorescence change value (F/F ₀ - 1), the experimental results show that the method has strong specificity.

Implementation example 2:

The analytical method for the quantitative detection of miR-21 (5'-UAGCUUAUCAGACUGAUGUUGA-3') synthesized by a fluorescent nano-silver cluster probe using a constant temperature amplification reaction, the detection principle is shown in Figure 4. use (exo-)DNA polymerase and Nt.BstNBI endonuclease. The sequence arrangement on the DNA amplification template is the target miR-21 binding sequence (5'-TCAACATCAGTCTGATAAGCTA-3'), the Nt.BstNBI restriction sequence (5'-ACGTGACTC-3'), the miR-21 binding sequence (5'-TCAACATCAGTCTGATAAGCTA-3'), Nt.BstNBI restriction sequence (5'-ACGTGACTC-3'), and the DNA complementary sequence for the synthesis of fluorescent nanosilver clusters (5'-TTGGGCGGGTGGGTGGG-3'), phosphorylated at the 3' end.

(exo-)DNA聚合酶(0.05U μL^-1)、Nt.BstNBI核酸内切酶(0.4U μL^-1)、RNase抑制剂(0.8U μL^-1)和dNTP(250μM)，反应缓冲液为10mM硝酸钠、20mM硝酸铵、20mMTris-HNO₃、2mM硝酸镁和0.1％Triton X-100，pH为8.8。于53℃孵育反应50分钟，然后于90℃孵育5分钟终止反应。向上述反应液中加入8μL硝酸银溶液(497.5μM)和100μL柠檬酸钠缓冲溶液(20mM，pH 7.0)，震荡混匀后，12,000转离心10分钟后取上清溶液，于黑暗中放置15分钟后，加入新鲜配制的1μL硼氢化钠(3.6mM)溶液，混匀，于黑暗中放置1小时，测定溶液的荧光信号。The following specific implementation of miR-21 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The room temperature in the present invention refers to the laboratory temperature of 23-28° C. for carrying out the operation. The specific operation steps are as follows: add 1 μL target miR-21 solution to 4 μL DNA amplification template solution (200 nM), the reaction buffer solution is 25 mM Tris-HNO ₃ , 50 mM sodium nitrate, 5 mM magnesium nitrate, 0.5 mM dithiothreitol, and the pH is 7.9. After incubating at 88°C for 10 minutes, add 5 μL of constant temperature amplification mixture, including

(exo-)DNA polymerase (0.05U μL ^-1 ), Nt.BstNBI endonuclease (0.4U μL ^-1 ), RNase inhibitor (0.8U μL ^-1 ) and dNTP (250μM), the reaction buffer is 10 mM sodium nitrate, 20 mM ammonium nitrate, 20 mM Tris-HNO ₃ , 2 mM magnesium nitrate and 0.1% Triton X-100, pH 8.8. Reactions were incubated at 53°C for 50 minutes and then terminated by incubation at 90°C for 5 minutes. Add 8 μL silver nitrate solution (497.5 μM) and 100 μL sodium citrate buffer solution (20 mM, pH 7.0) to the above reaction solution, shake and mix well, then centrifuge at 12,000 rpm for 10 minutes, take the supernatant solution, and place it in the dark for 15 minutes Afterwards, 1 μL of a freshly prepared sodium borohydride (3.6 mM) solution was added, mixed evenly, and left in the dark for 1 hour to measure the fluorescence signal of the solution.

Preparation of the working curve: Prepare miR-21 standard solution and blank sample (without miR-21, i.e. the concentration of miR-21 is 0) of known concentration respectively, operate according to the above steps, measure the fluorescence spectrum of each reaction solution, and calculate Its fluorescence change value (F/F ₀ -1), that is, the ratio of the fluorescence value of the miR-21 standard solution at 644nm to the fluorescence value of the blank sample at 644nm minus 1, and then according to the concentration of the miR-21 standard solution and its Fluorescence change value (F/F ₀ -1) to make a standard working curve.

Implementation example 3:

The analytical method for the quantitative detection of let-7a (5'-UGAGGUAGUAGGUUGUAUAGUU-3') synthesized by a constant temperature amplification reaction with a fluorescent nano-silver cluster probe, the detection principle is shown in Figure 5. Using phi29 DNA polymerase and Nb.BbvCI endonuclease. The sequence arrangement on the DNA amplification template is the target let-7a binding sequence (5'-AACTATACAACCTACTACCTCA-3'), the Nb.BbvCI restriction sequence (5'-GCTGAGG-3'), the let-7a binding sequence (5' -AACTATACAACCTACTACCTCA-3'), Nb.BbvCI cleavage sequence (5'-GCTGAGG-3'), and the DNA complementary sequence of synthetic fluorescent nanosilver clusters (5'-TTGGGCGGGTGGGTGGG-3'), phosphorylated at the 3' end.

The following specific implementation of let-7a detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The room temperature in the present invention refers to the laboratory temperature of 23-28° C. for carrying out the operation. The specific operation steps are as follows: add 1 μL target let-7a solution to 4 μL DNA amplification template solution (200 nM), the reaction buffer solution is 25 mM Tris-HNO ₃ , 50 mM sodium nitrate, 5 mM magnesium nitrate, 0.5 mM dithiothreitol, The pH is 7.9. After incubating at 88°C for 10 minutes, add 5 μL of constant temperature amplification mixture, including phi29 DNA polymerase (0.05U μL ^-1 ), Nb.BbvCI endonuclease (0.4U μL ^-1 ), RNase inhibitor (0.8U μL ⁻¹ ) and dNTP (250 μM), the reaction buffer was 10 mM sodium nitrate, 20 mM ammonium nitrate, 20 mM Tris-HNO ₃ , 2 mM magnesium nitrate and 0.1% Triton X-100, pH 8.8. Reactions were incubated at 37°C for 50 minutes and then terminated by incubation at 90°C for 5 minutes. Add 8 μL silver nitrate solution (497.5 μM) and 100 μL sodium citrate buffer solution (20 mM, pH 7.0) to the above reaction solution, shake and mix well, then centrifuge at 12,000 rpm for 10 minutes, take the supernatant solution, and place it in the dark for 15 minutes Afterwards, 1 μL of a freshly prepared sodium borohydride (3.6 mM) solution was added, mixed evenly, and left in the dark for 1 hour to measure the fluorescence signal of the solution.

Preparation of working curve: Prepare let-7a standard solution and blank sample with known concentration respectively (do not contain let-7a, that is, let-7a concentration is 0), operate according to the above steps, measure the fluorescence spectrum of each reaction solution, and calculate Its fluorescence change value (F/F ₀ -1), i.e. the ratio of the fluorescence value of the let-7a standard solution at 644nm to the fluorescence value of the blank sample at 644nm minus 1, then according to the concentration of the let-7a standard solution and its Fluorescence change value (F/F ₀ -1) to make a standard working curve.

Implementation example 4:

The analytical method for the quantitative detection of miR-646 (5'-AAGCAGCUGCCUCUGAGGC-3') synthesized by a constant temperature amplification reaction with a fluorescent nano-silver cluster probe, the detection principle is shown in Figure 6. Using phi29 DNA polymerase and Nb.BbvCI endonuclease. The sequence arrangement on the DNA amplification template is the target miR-646 binding sequence (5'-GCCTCAGAGGCAGCTGCTT-3'), the Nb.BbvCI restriction sequence (5'-GCTGAGG-3'), and the DNA complementation of the synthetic fluorescent nanosilver cluster Sequence (5'-GGGTGGGTGGGCGGGTT-3'), Nb.BbvCI restriction sequence (5'-GCTGAGG-3'), and DNA complementary sequence (5'-GGGTGGGTGGGCGGGTT-3') for the synthesis of fluorescent nanosilver clusters, 3' end Phosphorylation.

The following specific implementation of miR-646 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The room temperature in the present invention refers to the laboratory temperature of 23-28° C. for carrying out the operation. The specific operation steps are as follows: 1 μL target miR-646 solution was added to 4 μL DNA amplification template solution (200 nM), the reaction buffer solution was 25 mM Tris-HNO ₃ , 50 mM sodium nitrate, 5 mM magnesium nitrate, 0.5 mM dithiothreitol, and the pH was 7.9. After incubating at 88°C for 10 minutes, add 5 μL of constant temperature amplification mixture, including phi29 DNA polymerase (0.05U μL ^-1 ), Nb.BbvCI endonuclease (0.4U μL ^-1 ), RNase inhibitor (0.8U μL -1 ^-1 ) and dNTP (250 μM), the reaction buffer was 10 mM sodium nitrate, 20 mM ammonium nitrate, 20 mM Tris-HNO ₃ , 2 mM magnesium nitrate and 0.1% Triton X-100, pH 8.8. Reactions were incubated at 37°C for 50 minutes and then terminated by incubation at 90°C for 5 minutes. Add 8 μL silver nitrate solution (497.5 μM) and 100 μL sodium citrate buffer solution (20 mM, pH 7.0) to the above reaction solution, shake and mix well, then centrifuge at 12,000 rpm for 10 minutes, take the supernatant solution, and place it in the dark for 15 minutes Afterwards, 1 μL of a freshly prepared sodium borohydride (3.6 mM) solution was added, mixed evenly, and left in the dark for 1 hour to measure the fluorescence signal of the solution.

Preparation of the working curve: prepare miR-646 standard solution and blank sample with known concentration respectively (does not contain miR-646, i.e. miR-646 concentration is 0), operate according to the above steps, measure the fluorescence spectrum of each reaction solution, and calculate Its fluorescence change value (F/F ₀ -1), that is, the ratio of the fluorescence value of the miR-646 standard solution at 644nm to the fluorescence value of the blank sample at 644nm minus 1, and then according to the concentration of the miR-646 standard solution and its Fluorescence change value (F/F ₀ -1) to make a standard working curve.

Implementation example 5:

(exo-)DNA聚合酶和Nt.BstNBI核酸内切酶。DNA扩增模板1上的序列排布为靶标miR-141结合序列(5’-CCATCTTTACCAGACAGTGTTA-3’)，Nt.BstNBI  酶切序列(5’-TCTTGACTC-3’)，miR-141结合序列(5’-CCATCTTTACCAGACAGTGTTA-3’)，Nt.BstNBI酶切序列(5’-TCTTGACTC-3’)，以及合成荧光纳米银簇的DNA互补序列(5’-TTGGGCGGGTGGGTGGG-3’)，3’端磷酸化。DNA扩增模板2上的序列排布为靶标miR-21结合序列(5’-TCAACATCAGTCTGATAAGCTA-3’)，Nt.BstNBI酶切序列(5’-ACGTGACTC-3’)，miR-21结合序列(5’-TCAACATCAGTCTGATAAGCTA-3’)，Nt.B stNBI酶切序列(5’-ACGTGACTC-3’)，以及合成荧光纳米银簇的DNA互补序列(5’-TTGGGCGGGTGGGTGGG-3’)，3’端磷酸化。Synthesis of fluorescent nano-silver cluster probes for multiple detection of miR-141 (5'-UAACACUGUCUGGUAAAGAUGG-3') and miR-21 (5'-UAGCUUAUCAGACUGAUGUUGA-3') analysis method using constant temperature amplification reaction, the detection principle is shown in Figure 7 Show. use

(exo-)DNA polymerase and Nt.BstNBI endonuclease. The sequence arrangement on the DNA amplification template 1 is the target miR-141 binding sequence (5'-CCATCTTTACCAGACAGTGTTA-3'), the Nt.BstNBI restriction sequence (5'-TCTTGACTC-3'), the miR-141 binding sequence (5 '-CCATCTTTACCAGACAGTGTTA-3'), Nt.BstNBI restriction sequence (5'-TCTTGACTC-3'), and the DNA complementary sequence of synthetic fluorescent nanosilver clusters (5'-TTGGGCGGGTGGGTGGG-3'), phosphorylated at the 3' end. The sequence arrangement on the DNA amplification template 2 is the target miR-21 binding sequence (5'-TCAACATCAGTCTGATAAGCTA-3'), the Nt.BstNBI restriction sequence (5'-ACGTGACTC-3'), the miR-21 binding sequence (5'- '-TCAACATCAGTCTGATAAGCTA-3'), Nt.B stNBI restriction sequence (5'-ACGTGACTC-3'), and DNA complementary sequence for synthesizing fluorescent nanosilver clusters (5'-TTGGGCGGGTGGGTGGG-3'), phosphorylated at the 3' end .

The following specific implementation of miR-141 and miR-21 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The room temperature in the present invention refers to the laboratory temperature of 23-28° C. for carrying out the operation. The specific operation steps are as follows: add 1 μL of target miR-141 or miR-21 or a mixture of the two into 4 μL of the mixed solution of DNA amplification template 1 and DNA amplification template 2 (each 200 nM), and the reaction buffer solution is 25 mM Tris-HNO ₃ , 50mM sodium nitrate, 5mM magnesium nitrate, 0.5mM dithiothreitol, pH 7.9. After incubating at 88°C for 10 minutes, add 5 μL of constant temperature amplification mixture, including (exo-)DNA polymerase (0.05U μL ^-1 ), Nt.BstNBI endonuclease (0.4U μL ^-1 ), RNase inhibitor (0.8U μL ^-1 ) and dNTP (250μM), the reaction buffer is 10 mM sodium nitrate, 20 mM ammonium nitrate, 20 mM Tris-HNO ₃ , 2 mM magnesium nitrate and 0.1% Triton X-100, pH 8.8. Reactions were incubated at 53°C for 50 minutes and then terminated by incubation at 90°C for 5 minutes. Add 8 μL of silver nitrate solution (497.5 μM) and 100 μL of sodium citrate buffer solution (20 mM, pH 7.0) to the above reaction solution, shake and mix well, centrifuge at 12,000 rpm for 10 minutes, take the supernatant solution, and place it in the dark for 15 minutes , add freshly prepared 1 μL of sodium borohydride (3.6 mM) solution, mix well, place in the dark for 1 hour, and measure the fluorescence signal of the solution.

Preparation of the working curve: prepare standard solutions of miR-141 and miR-21 of known concentrations and blank samples (not containing miR-141 and miR-21), operate according to the above steps, measure the fluorescence spectrum of each reaction solution, and calculate Its fluorescence change value (F/F ₀ -1), that is, the ratio of the fluorescence value of the miR-141 standard solution at its characteristic emission peak at 644nm to the fluorescence value of the blank sample at 644nm minus 1, the miR-21 standard solution at The ratio of the fluorescence value at 625nm of its characteristic emission peak to the fluorescence value of the blank sample at 625nm minus 1, and then according to the concentration of miR-141 and miR-21 standard solution and its fluorescence change value (F/F ₀ -1) Make standard working curves respectively.

Claims (10)

1. An analytical method utilizing a constant temperature amplification reaction to synthesize a fluorescent nano-silver cluster probe to detect microRNA, characterized in that, after the target microRNA is combined with a DNA amplification template, the constant temperature amplification reaction and the specificity of the nickel endonuclease are induced A large number of single-stranded DNA products were obtained by the digestion reaction of the enzyme, and the single-stranded DNA products and the added silver nitrate solution were reduced by sodium borohydride to prepare fluorescent nano-silver cluster probes, and the fluorescence signals in the reaction system were measured and the fluorescence signal changes were calculated. , compared with the standard working curve to calculate the concentration of the target microRNA. 2 . The method according to claim 1 , wherein the particle diameter of the fluorescent nano-silver cluster probe is 1-10 nm, the excitation wavelength is 500-680 nm, and the emission wavelength is 510-900 nm. 3. method as claimed in claim 1, is characterized in that, described target microRNA detection comprises the following operation steps: Step 1: adding the target microRNA to the DNA amplification template solution to incubate the reaction; Step 2: Add a constant temperature amplification mixture to the reaction solution obtained in step 1, and after incubating the reaction, heat-inactivate the enzyme to terminate the reaction; Step 3: adding silver nitrate solution to the reaction solution obtained in step 2, after mixing, centrifuging to get the supernatant, then adding silver nitrate solution and freshly prepared sodium borohydride solution, and reacting in the dark to synthesize fluorescent nano-silver clusters; Step 4 measures the fluorescence signal of the fluorescent nano-silver cluster solution obtained in step 3. 4. The method according to claim 3, wherein the target microRNA in the step 1 is from a biological sample of tissue, blood or cells. 5. The method according to claim 3, wherein the incubation reaction in the step 1 is at 37° C. to 55° C. and a reaction buffer solution, the volume of the DNA amplification template reaction solution and the target microRNA in the step 1 The ratio is 0.1 to 10, the reaction time is 5 to 50 minutes, the concentration of the DNA amplification template is 10 to 800nM; the concentration of the target microRNA is 1aM to 100nM; the DNA amplification template and the target microRNA are described The DNA amplification template contains three functional sequences: the target microRNA binding sequence, the nicking endonuclease digestion sequence and the DNA complementary sequence for the synthesis of fluorescent nanoclusters. The reaction buffer solution is 25mM Tris-HNO ₃ , 50mM nitric acid Sodium, 5mM magnesium nitrate, 0.5mM dithiothreitol and pH 6.0-8.9. 6. The method according to claim 3, wherein the incubation reaction in step 2 is at 35° C. to 55° C. and a reaction buffer, and the volume ratio of the reaction solution obtained in step 1 to the constant temperature amplification mixture is 0.1 to 10, react for 20 to 200 minutes, the constant temperature amplification mixture contains constant temperature polymerase, nicking endonuclease, RNase inhibitor and dNTP; the concentration of the constant temperature polymerase is 0.01UμL ^-1 ~1.0U μL ^{- 1.} The concentration of nicking endonuclease is 0.1U μL ^-1 ~1.0U μL ^-1 , the concentration of RNase inhibitor is 0.1U μL ^-1 ~1.0U μL ^-1 , the concentration of dNTP solution is 80 ~ 500 μM; the reaction buffer It is 10mM sodium nitrate, 20mM ammonium nitrate, 20mM Tris-HN0 ₃ , 2mM magnesium nitrate and 0.1% TritonX-100 and the pH is 6.0-8.9. 7. The method according to claim 6, characterized in that, the thermostatic polymerase is a commercially available

(exo-)DNA polymerase, phi29 DNA polymerase or Klenow Fragment polymerase. 8. The method according to claim 6, wherein the nicking endonuclease is commercialized Nt.CviPII, Nb.BbvCI, Nb.BtsI, Nt.BsmAI, Nt.BbvCI, Nt.BspQI, Nt.AlwI or Nt.BstNBI. 9. The method according to claim 3, characterized in that, the concentration of silver nitrate in the step 3 is 10-800 μM, the sodium borohydride solution is freshly prepared, its concentration is 10 μM-10 mM, and the reaction temperature is 25°C-55°C. °C, the reaction time is 20 to 120 minutes. 10. The method according to claim 1, wherein the standard working curve is a standard solution of the target microRNA of known concentration, after reacting according to the operation steps, measure the fluorescent signal in the reaction system and Calculate the fluorescence change value, and then make a standard working curve according to the concentration of microRNA in the standard solution and the fluorescence change value in the system.

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