CN102719526A - MicroRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize fluorescent nano silver cluster probe - Google Patents
MicroRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize fluorescent nano silver cluster probe Download PDFInfo
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Abstract
The invention provides a microRNA quantitative detection analytic method by utilizing isothermal amplification to synthesize a fluorescent nano silver cluster probe. The method is as below: a DNA amplification template containing three kinds of functional sequences is designed: a sequence binding with a target microRNA, a nicking endonuclease enzyme sequence and a DNA complementary sequence for synthesis of fluorescent nano cluster; when the target microRNA and the DNA amplification template are combined, isothermal amplification reaction and specific enzyme reaction of nicking endonuclease are induced to obtain a large number of single-stranded DNA products; the DNA sequence for synthesis of fluorescent nano silver cluster and a silver nitrate solution are employed to prepare the fluorescent nano silver cluster probe under the reduction of sodium borohydride; fluorescence signal of the reaction system are determined, and a fluorescence change value is calculated; the value is compared with a standard working curve to calculate the concentration of the target microRNA. The method has the advantages of high sensitivity, strong specificity, wide linear detection range, low background signal and simple operation, and can be widely applied to microRNA detection of biological samples such as tissue, blood or cells.
Description
Technical field
The present invention relates to the analytical procedure that a kind of fluorescence nano silver aggregate probe detects microRNA, belonged to the check and analysis field.
Background technology
MicroRNA is one type of endogenic non-coding RNA with adjusting function in eukaryote, finding, and its size is about 18～25 Nucleotide.It participates in various adjusting approach, comprises growth, virus defense, hematopoiesis, organ formation, cell proliferation and apoptosis, metabolism of fat etc.Recent research finds that microRNA plays crucial effects in the tumour generating process, become one type of ideal tumor marker.So detection by quantitative microRNA is to understanding its biological function, the early diagnosis of cancer and the exploitation of new drug all have crucial meaning.Compare with traditional detection of nucleic acids, its exclusive characteristics of microRNA, short like sequence, the high and low abundance of family's sequence homology is expressed, and has increased the difficulty of its detection.The method that detects microRNA at present mainly contains the Northern engram analysis, micro-array chip technology, polymerase chain reaction (PCR) etc.Though the Northern trace is the standard method that current microRNA analyzes, complex operation, length consuming time; Sensitivity is low; Need a large amount of sample and separation and concentration step during analyzing and testing, and very responsive to polluting, and each step misoperation all can the impact analysis result in the experiment.Though the micro-array chip technology can realize high-throughput, polycomponent detects simultaneously, and it is made and the testing cost height; Available sample volume is very little on the chip, causes sensitivity not high; In addition, the microRNA sequence is short, and sequence similarity is high, can not optimize all microRNA to be measured hybridization environment simultaneously, so selectivity is not high.Polymerase chain reaction (PCR) is the main method of present detection by quantitative microRNA, comprises primer extension RT-PCR, methods such as stem ring primer RT-PCR and microRNA tailing RT-PCR.Though characteristics such as the microRNA detection method based on the polymerase chain reaction has fast, high specificity, sensitivity height need the rt operation, have increased the cost of experiment and the complicacy of design.Along with new microRNA sequence constantly find and to the microRNA functional study progressively deeply, the analyzing and testing of microRNA has been proposed new demand.Therefore, exploitation is directly simple, and is highly sensitive, high specificity, and the analytical technology that accurately detects microRNA remains a challenge.
Summary of the invention
The present invention relates to a kind of analytical procedure of utilizing the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative microRNA; Have simple to operate, highly sensitive, high specificity; Advantages such as background signal is low, linear detection range is wide, applied widely are a kind of simple and practical analytical technologies.
The present invention utilizes target microRNA to induce the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative microRNA following.Design a DNA cloning template that contains three kinds of function sequences: with target microRNA binding sequence, nicking endonuclease enzyme is cut the DNA complementary sequence of sequence and synthetic fluorescence nano bunch.Hold 5 ' end to put in order and be target microRNA binding sequence from 3 ', nicking endonuclease enzyme is cut sequence, and target microRNA binding sequence, nicking endonuclease enzyme are cut the DNA complementary sequence of sequence and synthetic fluorescence nano bunch.When target microRNA with after the DNA cloning template combines; Induce isothermal amplification reactions and the specific endonuclease reaction of nicking endonuclease to obtain a large amount of single stranded DNA products; Wherein a kind of single stranded DNA product can combine with the DNA cloning template; Continue to serve as the primer effect of target microRNA, thereby greatly improve the efficient of amplification.Another kind of single stranded DNA product is the synthetic DNA sequence of silver-colored bunch of fluorescence nano; Can with the Silver Nitrate solion that adds under the sodium borohydride reduction effect, synthetic fluorescence nano silver aggregate probe is under the incident light irradiation of certain wavelength; Launch corresponding fluorescence; Through the fluorescent signal in the assaying reaction system and calculate fluorescence change value,, extrapolate the concentration of target microRNA with the standard working curve comparison.In addition; The function sequence also can have different combinations and pattern of rows and columns in the DNA cloning template; As holding 5 ' end to put in order and be target microRNA binding sequence from 3 ', nicking endonuclease enzyme is cut sequence, the DNA complementary sequence of synthetic fluorescence nano bunch; Nicking endonuclease enzyme is cut the DNA complementary sequence of sequence and synthetic fluorescence nano bunch.
Method of the present invention can further describe as follows:
After utilizing target microRNA and the DNA cloning template combining; Induce isothermal amplification reactions and the specific endonuclease reaction of nicking endonuclease to obtain the single stranded DNA product; The silver nitrate solution of this single stranded DNA product and adding prepares fluorescence nano silver aggregate probe under the sodium borohydride reduction effect; Fluorescent signal in the assaying reaction system also calculates fluorescent signal change value, with the standard working curve comparison, extrapolates the concentration of target microRNA.
Said fluorescence nano silver aggregate probe particle diameter is 1～10nm, and excitation wavelength is 500～680nm, and emission wavelength is 510～900nm.
Target microRNA detection method of the present invention comprises following operation steps:
Step 1 joins target microRNA in the DNA cloning template solution, carries out incubation reaction;
Add the constant-temperature amplification mixed solution in the reaction solution of step 2 gained in step 1, after the incubation reaction, heat inactivation enzyme, termination reaction;
Add silver nitrate solution and citric acid solution in the reaction solution of step 3 gained in step 2, behind the mixing, the centrifuging and taking supernatant adds silver nitrate solution and freshly prepared sodium borohydride solution then, and the synthetic fluorescence nano in reaction back is silver-colored bunch in the dark;
The fluorescent signal of the fluorescence nano of gained silver bunch solution in step 4 determination step 3.
Target microRNA comes from the biological specimen of tissue, blood or cell in the said step 1.
Incubation reaction in the said step 1 37 ℃～55 ℃ with the reaction buffered soln in; DNA cloning template reaction liquid in the step 1 and the volume ratio of target microRNA are 0.1～10; Reaction times is 5～50 minutes, and described DNA cloning template concentrations is 10～800nM; Described target microRNA concentration is 1aM～100nM; Described DNA cloning template contains three kinds of function sequences: with target microRNA binding sequence, nicking endonuclease enzyme is cut the DNA complementary sequence of sequence and synthetic fluorescence nano bunch, and described reaction buffered soln is 25mM Tris-HNO
3, 50mM SODIUMNITRATE, 5mM magnesium nitrate, 0.5mM WR 34678 and pH are 6.0～8.9.
The incubation reaction of said step 2 be 35 ℃～55 ℃ with reaction buffer in; The reaction solution of step 1 gained and constant-temperature amplification mixeding liquid volume ratio are 0.1～10; Reacted 20～200 minutes, and contained constant temperature polysaccharase, nicking endonuclease, RNase suppressor factor and dNTP in the constant-temperature amplification mixed solution; Described constant temperature polymerase concentration is 0.01U μ L
-1～1.0U μ L
-1, nicking endonuclease enzyme concn is 0.1U μ L
-1～1.0U μ L
-1, the RNase inhibitor concentration is 0.1U μ L
-1～1.0U μ L
-1, the dNTP strength of solution is 80～500 μ M; Described reaction buffer is 10mM SODIUMNITRATE, 20mM an ammonium nitrate, 20mM Tris-HNO
3, 2mM magnesium nitrate and 0.1%Triton X-100 and pH be 6.0～8.9.
Said constant temperature polysaccharase is commercial
(exo-) archaeal dna polymerase, phi29DNA polysaccharase or Klenow Fragment polysaccharase.
Said nicking endonuclease is commercial Nt.CviPII, Nb.BbvCI, Nb.BtsI, Nt.BsmAI, Nt.BbvCI, Nt.BspQI, Nt.AlwI or Nt.BstNBI.
Described standard working curve is the standardized solution by the target microRNA of concentration known; After said operation steps reaction; Fluorescent signal in the assaying reaction system also calculates fluorescence change value, then according to fluorescence change value production standard working curve in the concentration of microRNA in the standardized solution and the system.
Silver nitrate concentration is 10～800 μ M in the said step 3, and sodium borohydride solution is a fresh, and its concentration is 10～10mM, and temperature of reaction is 25 ℃～55 ℃, and the reaction times is 20～120 minutes.
Adopt two nicking endonuclease enzymes to cut the design of sequence in the method for the present invention in the DNA cloning template, the single stranded DNA sequence of utilizing amplification to obtain combines with the DNA cloning template again, serves as the primer effect, has greatly improved amplification efficiency.The design of the DNA complementary sequence of synthetic fluorescence nano bunch in the DNA cloning template has realized a kind of application of novel fluorescent nano probe.Compare with traditional fluorescent probe, fluorescent nano probe has good optical characteristics, like high fluorescent, anti-photobleaching property, glow color is adjustable, excitation wavelength and emission wavelength tunable etc.
The invention has the advantages that:
(1) highly sensitive: the design of cutting sequence in the DNA cloning template through two microRNA binding sequences (or pairing becomes the DNA complementary sequence of fluorescence nano bunch) and nicking endonuclease enzyme; Make amplified production the same with target microRNA; Can combine with the DNA cloning template; Start isothermal amplification reactions, reach the effect that exponential signal amplifies, thereby greatly improved detection sensitivity.
(2) selectivity is good: the specificity base complementrity pair principle based on target miRNA and DNA cloning template makes that the disturbing influence of other molecule is very little.
(3) background signal is low: fluorescence nano silver bunch synthetic directly related with the based composition of dna sequence dna, have only silver-colored bunch of the fluorescence nano that the specific dna sequence dna of based composition could the synthesizing stable fluorescent signal.
(4) multiple detection: utilize the difference of the dna sequence dna synthetic nanometer silver aggregate probe optical characteristics of different based compositions,, design different DNA cloning templates, can realize that multiple microRNA detects to different target microRNA.
(5) high throughput testing: adopt 96 or 384 enzyme plates, can realize the detection of while production standard working curve and sample to be tested, reduce and detect error.
(6) operating process is easy fast: whole testing process is simple.
(7) pollution-free: whole testing process need not used organic solvent or toxic reagent.
Description of drawings
Fig. 1 utilizes the principle schematic of the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative miR-141.
The preparation of Fig. 2 working curve.(A) fluorescence spectrum of different concns miR-141 reaction soln; (B) the fluorescence change value (F/F of different concns miR-141 reaction soln
0-1) with the working curve of miR-141 concentration logarithmic value.
The experiment of Fig. 3 specificity.(A) fluorescence spectrum of different target microRNA reaction solns; (B) experimental data compares histogram (F/F
0-1).
Fig. 4 utilizes the principle schematic of the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative miR-21.
Fig. 5 utilizes the principle schematic of the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative let-7a.
Fig. 6 utilizes the principle schematic of the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative miR-646.
Fig. 7 utilizes the principle schematic of the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe multiple detection miR-141 and miR-21.
Nomenclature
In the accompanying drawing 1; MiR-141 is 5 '-UAACACUGUCUGGUAAAGAUGG-3 '; A is target miR-141 binding sequence (5 '-CCATCTTTACCAGACAGTGTTA-3 '); X is that Nt.B stNBI enzyme is cut sequence (5 '-TCTTGACTC-3 '); B is the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '); T produces the dna sequence dna consistent with miR-14 (5 '-TAACACTGTCTGGTAAAGATGG-3 ') for amplification; R is the dna sequence dna of synthetic fluorescence nano silver bunch (5 '-CCCACCCACCCGCCCAA-3 '), and C is the Nt.BstNBI endonuclease, and D is
(exo-) archaeal dna polymerase.
In the accompanying drawing 2, F is the fluorescent value of miR-141 standardized solution at the 644nm place, F
0Be the fluorescent value of dummy (not containing miR-141) at the 644nm place, (F/F
0-1) the fluorescence change value of expression miR-141 standardized solution.
In the accompanying drawing 3, F is the fluorescent value of target microRNA solution at the 644nm place, F
0Be the fluorescent value of dummy (not containing microRNA) at the 644nm place, (F/F
0-1) the fluorescence change value of expression microRNA solution.
In the accompanying drawing 4; MiR-21 is 5 '-UAGCUUAUCAGACUGAUGUUGA-3 '; A is target miR-21 binding sequence (5 '-TCAACATCAGTCTGATAAGCTA-3 '); X cuts sequence (5 '-ACGTGACTC-3 ') for the Nt.BstNBI enzyme; B is the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '); T produces the dna sequence dna consistent with miR-21 (5 '-TAGCTTATCAGACTGATGTTGA-3 ') for amplification; R is the dna sequence dna of synthetic fluorescence nano silver bunch (5 '-CCCACCCACCCGCCCAA-3 '), and C is the Nt.BstNBI endonuclease, and D is
(exo-) archaeal dna polymerase.
In the accompanying drawing 5; Let-7a is 5 '-UGAGGUAGUAGGUUGUAUAGUU-3 '; A is target let-7a binding sequence (5 '-AACTATACAACCTACTACCTCA-3 '); X cuts sequence (5 '-GCTGAGG-3 ') for the Nb.BbvCI enzyme; B is the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '); T produces the dna sequence dna consistent with let-7a (5 '-TGAGGTAGTAGGTTGTATAGTT-3 ') for amplification; R is the dna sequence dna of synthetic fluorescence nano silver bunch (5 '-CCCACCCACCCGCCCAA-3 '), and C is the Nb.BbvCI endonuclease, and D is
(exo-) archaeal dna polymerase.
In the accompanying drawing 6; MiR-646 is 5 '-AAGCAGCUGCCUCUGAGGC-3 '; A is target miR-646 binding sequence (5 '-GCCTCAGAGGCAGCTGCTT-3 '), and X cuts sequence (5 '-GCTGAGG-3 ') for the Nb.BbvCI enzyme, and B is the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '); T produces the dna sequence dna consistent with miR-646 (5 '-AAGCAGCTGCCTCTGAGGC-3 ') for amplification; R/R ': the dna sequence dna that synthetic fluorescence nano is silver-colored bunch (5 '-CCCACCCACCCGCCCAA-3 '), C is the Nb.BbvCI endonuclease, D is the phi29DNA polysaccharase.
In the accompanying drawing 7; MiR-141 is 5 '-UAACACUGUCUGGUAAAGAUGG-3 '; The miR-21 sequence is 5 '-UAGCUUAUCAGACUGAUGUUGA-3 '; A is target miR-21 binding sequence (5 '-UAGCUUAUCAGACUGAUGUUGA-3 '); X is that Nt.B stNBI enzyme is cut sequence (5 '-TCTTGACTC-3 '); E is target miR-141 binding sequence (5 '-UAACACUGUCUGGUAAAGAUGG-3 '), and B is the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '), and F is the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-ATCGGGGGCGA-3 '); T produces the dna sequence dna consistent with miR-21 (5 '-TAGCTTATCAGACTGATGTTGA-3 ') for amplification; U produces the dna sequence dna consistent with miR-141 (5 '-TAACACTGTCTGGTAAAGATGG-3 ') for amplification, and R is the dna sequence dna of synthetic fluorescence nano silver bunch (5 '-ATCGCCCCCGAT-3 '), and S is the dna sequence dna that synthesizes silver-colored bunch of fluorescence nano (5 '-CCCACCCACCCGCCCAA-3 '); C is the Nt.BstNBI endonuclease, and D is
(exo-) archaeal dna polymerase.
The practical implementation instance:
The present invention will be helped to understand through following embodiment, but content of the present invention can not be limited
Embodiment 1:
Utilize the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative miR-141 (5 '-UAACACUGUCUGGUAAAGAUGG-3 '), detect principle shown in accompanying drawing 1.Employing
is archaeal dna polymerase and Nt.BstNBI endonuclease (exo-).Sequence on the DNA cloning template is arranged and is target miR-141 binding sequence (5 '-CCATCTTTACCAGACAGTGTTA-3 '); The Nt.BstNBI enzyme is cut sequence (5 '-TCTTGACTC-3 '); The miR-141 binding sequence (5 '-CCATCTTTACCAGACAGTGTTA-3 '); The Nt.BstNBI enzyme is cut sequence (5 '-TCTTGACTC-3 '), and the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '), 3 ' end phosphorylation.
The practical implementation that detects in the face of miR-141 down further specifies the present invention.The experimental technique of unreceipted actual conditions wherein is usually according to the normal condition or the condition of advising according to manufacturer.Room temperature among the present invention is meant 23～28 ℃ of laboratory temperatures that carry out implementation and operation.The concrete operations step is following: in 4 μ L DNA cloning template solution (200nM), add 1 μ L target miR-141 solution, reaction buffered soln is 25mMTris-HNO
3, 50mM SODIUMNITRATE, the 5mM magnesium nitrate, 0.5mM WR 34678, pH are 7.9.In 88 ℃ hatch 10 minutes after, add constant-temperature amplification mixed solution 5 μ L then, comprise
(exo-) archaeal dna polymerase (0.05U μ L
-1), Nt.BstNBI endonuclease (0.4U μ L
-1), RNase suppressor factor (0.8U μ L
-1) and dNTP (250 μ M), reaction buffer is 10mM SODIUMNITRATE, 20mM an ammonium nitrate, 20mM Tris-HNO
3, 2mM magnesium nitrate and 0.1%Triton X-100, pH is 8.8.In 53 ℃ of incubation reaction 50 minutes, hatch 5 minutes termination reactions in 90 ℃ then.In above-mentioned reaction solution, add 8 μ L silver nitrate solutiones (497.5 μ M) and 100 μ L sodium citrate buffer (20mM, pH 7.0), behind the concussion mixing; 12,000 leave the heart gets supernatant solution after 10 minutes, in dark, place after 15 minutes; Add freshly prepared 1 μ L Peng Qinghuana (3.6mM) solution; Mixing was placed 1 hour in dark, measured the fluorescent signal of solution.
The making of working curve: prepare the miR-141 standardized solution of concentration known respectively, concentration is respectively 1aM, 10aM, 100aM; 1fM, 10fM, 100fM, 1pM; 10pM, 100pM, and 1nM, and dummy (does not contain miR-141; Be that miR-141 concentration is 0), operate according to above-mentioned steps, measure the fluorescence spectrum of each reaction solution, shown in Fig. 2 (A).Calculate its fluorescence change value (F/F
0-1), promptly the miR-141 standardized solution at the fluorescent value (F) at 644nm place and the fluorescent value (F of dummy (not containing miR-141) at the 644nm place
0) ratio subtract 1, then according to the concentration of miR-141 standardized solution and its fluorescence change value (F/F
0-1) production standard working curve, shown in Fig. 2 (B), the concentration logarithmic value of miR-141 standardized solution and its fluorescence change value (F/F
0-1) linear in 1aM～1nM scope, linear equation is Y=10.57+1.25lgX.
Shown in Figure 3 is the specificity experiment of this inventive method, chooses miR-429, miR-200b; Let-7d, miR-21 is as miR-141 check sample (detectable level is 1pM), and dummy (does not contain miR-141; Be that miR-141 concentration is 0), the miR-429 sequence is 5 '-UAAUACUGUCUGGUAAAACCGU-3 ', the miR-200b sequence is 5 '-UAAUACUGCCUGGUAAUGAUGA-3 '; The let-7d sequence is 5 '-AGAGGUAGUAGGUUGCAUAGUU-3 '; The miR-21 sequence is 5 '-UAGCUUAUCAGACUGAUGUUGA-3 ', is the fluorescence spectrum of each target reaction solution like Fig. 3 (A), is the fluorescence change value (F/F of each target reaction solution like Fig. 3 (B)
0-1) histogram, experimental result show this method high specificity.
Embodiment 2:
Utilize the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative miR-21 (5 '-UAGCUUAUCAGACUGAUGUUGA-3 '), detect principle shown in accompanying drawing 4.Employing
is archaeal dna polymerase and Nt.BstNBI endonuclease (exo-).Sequence on the DNA cloning template is arranged and is target miR-21 binding sequence (5 '-TCAACATCAGTCTGATAAGCTA-3 '); The Nt.BstNBI enzyme is cut sequence (5 '-ACGTGACTC-3 '); The miR-21 binding sequence (5 '-TCAACATCAGTCTGATAAGCTA-3 '); The Nt.BstNBI enzyme is cut sequence (5 '-ACGTGACTC-3 '), and the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '), 3 ' end phosphorylation.
The practical implementation that detects in the face of miR-21 down further specifies the present invention.The experimental technique of unreceipted actual conditions wherein is usually according to the normal condition or the condition of advising according to manufacturer.Room temperature among the present invention is meant 23～28 ℃ of laboratory temperatures that carry out implementation and operation.The concrete operations step is following: in 4 μ LDNA amplification template solution (200nM), add 1 μ L target miR-21 solution, reaction buffered soln is 25mMTris-HNO
3, 50mM SODIUMNITRATE, the 5mM magnesium nitrate, 0.5mM WR 34678, pH are 7.9.In 88 ℃ hatch 10 minutes after, add constant-temperature amplification mixed solution 5 μ L then, comprise
(exo-) archaeal dna polymerase (0.05U μ L
-1), Nt.BstNBI endonuclease (0.4U μ L
-1), RNase suppressor factor (0.8U μ L
-1) and dNTP (250 μ M), reaction buffer is 10mM SODIUMNITRATE, 20mM an ammonium nitrate, 20mMTris-HNO
3, 2mM magnesium nitrate and 0.1%Triton X-100, pH is 8.8.In 53 ℃ of incubation reaction 50 minutes, hatch 5 minutes termination reactions in 90 ℃ then.In above-mentioned reaction solution, add 8 μ L silver nitrate solutiones (497.5 μ M) and 100 μ L sodium citrate buffer (20mM, pH 7.0), behind the concussion mixing; 12,000 leave the heart gets supernatant solution after 10 minutes, in dark, place after 15 minutes; Add freshly prepared 1 μ L Peng Qinghuana (3.6mM) solution; Mixing was placed 1 hour in dark, measured the fluorescent signal of solution.
The making of working curve: prepare the miR-21 standardized solution and the dummy (do not contain miR-21, promptly miR-21 concentration is 0) of concentration known respectively, operate, measure the fluorescence spectrum of each reaction solution, calculate its fluorescence change value (F/F according to above-mentioned steps
0-1), promptly the miR-21 standardized solution subtracts 1 at the fluorescent value and the dummy at 644nm place at the ratio of the fluorescent value at 644nm place, then according to the concentration of miR-21 standardized solution and its fluorescence change value (F/F
0-1) production standard working curve.
Embodiment 3:
Utilize the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative let-7a (5 '-UGAGGUAGUAGGUUGUAUAGUU-3 '), detect principle shown in accompanying drawing 5.Adopt phi29DNA polysaccharase and Nb.BbvCI endonuclease.Sequence on the DNA cloning template is arranged and is target let-7a binding sequence (5 '-AACTATACAACCTACTACCTCA-3 '); The Nb.BbvCI enzyme is cut sequence (5 '-GCTGAGG-3 '); The let-7a binding sequence (5 '-AACTATACAACCTACTACCTCA-3 '); The Nb.BbvCI enzyme is cut sequence (5 '-GCTGAGG-3 '), and the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '), 3 ' end phosphorylation.
The practical implementation that detects in the face of let-7a down further specifies the present invention.The experimental technique of unreceipted actual conditions wherein is usually according to the normal condition or the condition of advising according to manufacturer.Room temperature among the present invention is meant 23～28 ℃ of laboratory temperatures that carry out implementation and operation.The concrete operations step is following: in 4 μ L DNA cloning template solution (200nM), add 1 μ L target let-7a solution, reaction buffered soln is 25mM Tris-HNO
3, 50mM SODIUMNITRATE, the 5mM magnesium nitrate, 0.5mM WR 34678, pH are 7.9.In 88 ℃ hatch 10 minutes after, add constant-temperature amplification mixed solution 5 μ L then, comprise phi29DNA polysaccharase (0.05U μ L
-1), Nb.BbvCI endonuclease (0.4U μ L
-1), RNase suppressor factor (0.8U μ L
-1) and dNTP (250 μ M), reaction buffer is 10mM SODIUMNITRATE, 20mM an ammonium nitrate, 20mM Tris-HNO
3, 2mM magnesium nitrate and 0.1%Triton X-100, pH is 8.8.In 37 ℃ of incubation reaction 50 minutes, hatch 5 minutes termination reactions in 90 ℃ then.In above-mentioned reaction solution, add 8 μ L silver nitrate solutiones (497.5 μ M) and 100 μ L sodium citrate buffer (20mM, pH 7.0), behind the concussion mixing; 12,000 leave the heart gets supernatant solution after 10 minutes, in dark, place after 15 minutes; Add freshly prepared 1 μ L Peng Qinghuana (3.6mM) solution; Mixing was placed 1 hour in dark, measured the fluorescent signal of solution.
The making of working curve: prepare the let-7a standardized solution and the dummy (do not contain let-7a, promptly let-7a concentration is 0) of concentration known respectively, operate, measure the fluorescence spectrum of each reaction solution, calculate its fluorescence change value (F/F according to above-mentioned steps
0-1), promptly the let-7a standardized solution subtracts 1 at the fluorescent value and the dummy at 644nm place at the ratio of the fluorescent value at 644nm place, then according to the concentration of let-7a standardized solution and its fluorescence change value (F/F
0-1) production standard working curve.
Embodiment 4:
Utilize the analytical procedure of the synthetic fluorescence nano silver of isothermal amplification reactions aggregate probe detection by quantitative miR-646 (5 '-AAGCAGCUGCCUCUGAGGC-3 '), detect principle shown in accompanying drawing 6.Adopt phi29DNA polysaccharase and Nb.BbvCI endonuclease.Sequence on the DNA cloning template is arranged and is target miR-646 binding sequence (5 '-GCCTCAGAGGCAGCTGCTT-3 '); The Nb.BbvCI enzyme is cut sequence (5 '-GCTGAGG-3 '); The DNA complementary sequence that synthetic fluorescence nano is silver-colored bunch (5 '-GGGTGGGTGGGCGGGTT-3 '); The Nb.BbvCI enzyme is cut sequence (5 '-GCTGAGG-3 '), and the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-GGGTGGGTGGGCGGGTT-3 '), 3 ' end phosphorylation.
The practical implementation that detects in the face of miR-646 down further specifies the present invention.The experimental technique of unreceipted actual conditions wherein is usually according to the normal condition or the condition of advising according to manufacturer.Room temperature among the present invention is meant 23～28 ℃ of laboratory temperatures that carry out implementation and operation.The concrete operations step is following: in 4 μ LDNA amplification template solution (200nM), add 1 μ L target miR-646 solution, reaction buffered soln is 25mMTris-HNO
3, 50mM SODIUMNITRATE, the 5mM magnesium nitrate, 0.5mM WR 34678, pH are 7.9.In 88 ℃ hatch 10 minutes after, add constant-temperature amplification mixed solution 5 μ L then, comprise phi29DNA polysaccharase (0.05U μ L
-1), Nb.BbvCI endonuclease (0.4U μ L
-1), RNase suppressor factor (0.8U μ L
-1) and dNTP (250 μ M), reaction buffer is 10mM SODIUMNITRATE, 20mM an ammonium nitrate, 20mMTris-HNO
3, 2mM magnesium nitrate and 0.1%Triton X-100, pH is 8.8.In 37 ℃ of incubation reaction 50 minutes, hatch 5 minutes termination reactions in 90 ℃ then.In above-mentioned reaction solution, add 8 μ L silver nitrate solutiones (497.5 μ M) and 100 μ L sodium citrate buffer (20mM, pH 7.0), behind the concussion mixing; 12,000 leave the heart gets supernatant solution after 10 minutes, in dark, place after 15 minutes; Add freshly prepared 1 μ L Peng Qinghuana (3.6mM) solution; Mixing was placed 1 hour in dark, measured the fluorescent signal of solution.
The making of working curve: prepare the miR-646 standardized solution and the dummy (do not contain miR-646, promptly miR-646 concentration is 0) of concentration known respectively, operate, measure the fluorescence spectrum of each reaction solution, calculate its fluorescence change value (F/F according to above-mentioned steps
0-1), promptly the miR-646 standardized solution subtracts 1 at the fluorescent value and the dummy at 644nm place at the ratio of the fluorescent value at 644nm place, then according to the concentration of miR-646 standardized solution and its fluorescence change value (F/F
0-1) production standard working curve.
Embodiment 5:
Utilize the analytical procedure of the multiple detection of isothermal amplification reactions synthetic fluorescence nano silver aggregate probe miR-141 (5 '-UAACACUGUCUGGUAAAGAUGG-3 ') and miR-21 (5 '-UAGCUUAUCAGACUGAUGUUGA-3 '), the detection principle is shown in accompanying drawing 7.Employing
is archaeal dna polymerase and Nt.BstNBI endonuclease (exo-).Sequence on the DNA cloning template 1 is arranged and is target miR-141 binding sequence (5 '-CCATCTTTACCAGACAGTGTTA-3 '); The Nt.BstNBI enzyme is cut sequence (5 '-TCTTGACTC-3 '); The miR-141 binding sequence (5 '-CCATCTTTACCAGACAGTGTTA-3 '); The Nt.BstNBI enzyme is cut sequence (5 '-TCTTGACTC-3 '), and the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '), 3 ' end phosphorylation.Sequence on the DNA cloning template 2 is arranged and is target miR-21 binding sequence (5 '-TCAACATCAGTCTGATAAGCTA-3 '); The Nt.BstNBI enzyme is cut sequence (5 '-ACGTGACTC-3 '); The miR-21 binding sequence (5 '-TCAACATCAGTCTGATAAGCTA-3 '); Nt.B stNBI enzyme is cut sequence (5 '-ACGTGACTC-3 '), and the DNA complementary sequence of synthetic fluorescence nano silver bunch (5 '-TTGGGCGGGTGGGTGGG-3 '), 3 ' end phosphorylation.
The practical implementation that detects in the face of miR-141, miR-21 down further specifies the present invention.The experimental technique of unreceipted actual conditions wherein is usually according to the normal condition or the condition of advising according to manufacturer.Room temperature among the present invention is meant 23～28 ℃ of laboratory temperatures that carry out implementation and operation.The concrete operations step is following: in the mixing solutions (each 200nM) of 4 μ LDNA amplification templates 1 and DNA cloning template 2, add 1 μ L target miR-141 or miR-21 or the mixing solutions of the two, reaction buffered soln is 25mM Tris-HNO
3, 50mM SODIUMNITRATE, the 5mM magnesium nitrate, 0.5mM WR 34678, pH are 7.9.In 88 ℃ hatch 10 minutes after, add constant-temperature amplification mixed solution 5 μ L then, comprise
(exo-) archaeal dna polymerase (0.05U μ L
-1), Nt.BstNBI endonuclease (0.4U μ L
-1), RNase suppressor factor (0.8U μ L
-1) and dNTP (250 μ M), reaction buffer is 10mM SODIUMNITRATE, 20mM an ammonium nitrate, 20mMTris-HNO
3, 2mM magnesium nitrate and 0.1%Triton X-100, pH is 8.8.In 53 ℃ of incubation reaction 50 minutes, hatch 5 minutes termination reactions in 90 ℃ then.Add 8 μ L silver nitrate solutiones (497.5 μ M) and 100 μ L sodium citrate buffer (20mM, pH 7.0) in the above-mentioned reaction solution, behind the concussion mixing; 12,000 leave the heart gets supernatant solution after 10 minutes, in dark, place after 15 minutes; Add freshly prepared 1 μ L Peng Qinghuana (3.6mM) solution; Mixing was placed 1 hour in dark, measured the fluorescent signal of solution.
The making of working curve: prepare miR-141 and the miR-21 standardized solution and the dummy (not containing miR-141 and miR-21) of concentration known respectively, operate, measure the fluorescence spectrum of each reaction solution, calculate its fluorescence change value (F/F according to above-mentioned steps
0-1); Be that fluorescent value and the dummy of miR-141 standardized solution at its characteristic emission peak 644nm place subtracts 1 at the ratio of the fluorescent value at 644nm place; The miR-21 standardized solution subtracts 1 at the fluorescent value and the dummy at its characteristic emission peak 625nm place at the ratio of the fluorescent value at 625nm place, then according to the concentration of miR-141 and miR-21 standardized solution and its fluorescence change value (F/F
0-1) difference production standard working curve.
Claims (10)
1. one kind is utilized isothermal amplification reactions to synthesize the analytical procedure that fluorescence nano silver aggregate probe detects microRNA; It is characterized in that; After utilizing target microRNA and the DNA cloning template combining; Induce isothermal amplification reactions and the specific endonuclease reaction of nicking endonuclease to obtain a large amount of single stranded DNA products, the silver nitrate solution of this single stranded DNA product and adding prepares fluorescence nano silver aggregate probe under the sodium borohydride reduction effect, and the fluorescent signal in the assaying reaction system also calculates fluorescent signal change value; With the standard working curve comparison, extrapolate the concentration of target microRNA.
2. the method for claim 1 is characterized in that, said fluorescence nano silver aggregate probe particle diameter is 1～10nm, and excitation wavelength is 500～680nm, and emission wavelength is 510～900nm.
3. the method for claim 1 is characterized in that, described target microRNA detects and comprises following operation steps:
Step 1 joins target microRNA in the DNA cloning template solution, carries out incubation reaction;
Add the constant-temperature amplification mixed solution in the reaction solution of step 2 gained in step 1, after the incubation reaction, heat inactivation enzyme, termination reaction;
Add silver nitrate solution in the reaction solution of step 3 gained in step 2, behind the mixing, the centrifuging and taking supernatant adds silver nitrate solution and freshly prepared sodium borohydride solution then, and the synthetic fluorescence nano of reaction is silver-colored bunch in the dark;
The fluorescent signal of the fluorescence nano of gained silver bunch solution in step 4 determination step 3.
4. method as claimed in claim 3 is characterized in that target microRNA comes from the biological specimen of tissue, blood or cell in the said step 1.
5. method as claimed in claim 3; It is characterized in that; Incubation reaction in the said step 1 37 ℃～55 ℃ with the reaction buffered soln in; DNA cloning template reaction liquid in the step 1 and the volume ratio of target microRNA are 0.1～10, and the reaction times is 5～50 minutes, and described DNA cloning template concentrations is 10～800nM; Described target microRNA concentration is 1aM～100nM; The described DNA cloning template of described DNA cloning template and target microRNA contains three kinds of function sequences: with target microRNA binding sequence; Nicking endonuclease enzyme is cut the DNA complementary sequence of sequence and synthetic fluorescence nano bunch, and described reaction buffered soln is 25mM Tris-HNO
3, 50mM SODIUMNITRATE, 5mM magnesium nitrate, 0.5mM WR 34678 and pH are 6.0～8.9.
6. method as claimed in claim 3; It is characterized in that; The incubation reaction of said step 2 be 35 ℃～55 ℃ with reaction buffer in; The reaction solution of step 1 gained and constant-temperature amplification mixeding liquid volume ratio are 0.1～10, react 20～200 minutes, contain constant temperature polysaccharase, nicking endonuclease, RNase suppressor factor and dNTP in the constant-temperature amplification mixed solution; Described constant temperature polymerase concentration is 0.01U μ L
-1～1.0U μ L
-1, nicking endonuclease enzyme concn is 0.1U μ L
-1～1.0U μ L
-1, the RNase inhibitor concentration is 0.1U μ L
-1～1.0U μ L
-1, the dNTP strength of solution is 80～500 μ M; Described reaction buffer is 10mM SODIUMNITRATE, 20mM an ammonium nitrate, 20mM Tris-HN0
3, 2mM magnesium nitrate and 0.1%TritonX-100 and pH be 6.0～8.9.
7. method as claimed in claim 6; It is characterized in that said constant temperature polysaccharase is commercial
(exo-) archaeal dna polymerase, phi29DNA polysaccharase or Klenow Fragment polysaccharase.
8. method as claimed in claim 6 is characterized in that, said nicking endonuclease is commercial Nt.CviPII, Nb.BbvCI, Nb.BtsI, Nt.BsmAI, Nt.BbvCI, Nt.BspQI, Nt.AlwI or Nt.BstNBI.
9. method as claimed in claim 3 is characterized in that, silver nitrate concentration is 10～800 μ M in the said step 3, and sodium borohydride solution is a fresh, and its concentration is 10 μ M～10mM, and temperature of reaction is 25 ℃～55 ℃, and the reaction times is 20～120 minutes.
10. the method for claim 1; It is characterized in that; Described standard working curve is the standardized solution by the target microRNA of concentration known, after said operation steps reaction; Fluorescent signal in the assaying reaction system also calculates fluorescence change value, then according to fluorescence change value production standard working curve in the concentration of microRNA in the standardized solution and the system.
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