CN103014148A - Isothermal detection method of RNA (Ribonucleic Acid) - Google Patents

Isothermal detection method of RNA (Ribonucleic Acid) Download PDF

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CN103014148A
CN103014148A CN2012104197772A CN201210419777A CN103014148A CN 103014148 A CN103014148 A CN 103014148A CN 2012104197772 A CN2012104197772 A CN 2012104197772A CN 201210419777 A CN201210419777 A CN 201210419777A CN 103014148 A CN103014148 A CN 103014148A
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rna
dnazyme
tetramer
strand displacement
quantitative
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CN103014148B (en
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唐卓
赵永云
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Chengdu Institute of Biology of CAS
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Chengdu Institute of Biology of CAS
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Abstract

The invention provides a qualitative and quantitive isothermal detection method of RNA (Ribonucleic Acid). The method comprises the steps of after cutting RNA at fixed points by dnazyme (DNAzyme), amplifying RNA through a strand displacement isothermal amplification technique (SDA); and carrying out qualitative or quantitive detection by inspecting a reporter group G-tetramer released from an SDA product. The method can measure trace RNA (comprising mRNA and miRNA) quickly, simply, conveniently, highly sensitively and specifically, and meanwhile, the risk of pollution is reduced.

Description

The isothermal detection methods of a kind of RNA
Technical field
The invention belongs to technical field of molecular biology, relate to the RNA Amplification Analysis and detect, more particularly, the present invention relates to a kind of strand displacement isothermal duplication RNA that utilizes, discharge the detection method of reporter.
Background technology
The nucleic acid molecule amplification in vitro is the important means of biotechnology research, not only can increase and separates goal gene, at aspects such as clinical diagnosis, detection in Gene Mutation, forensic identifications important use is arranged also.Polymerase chain reaction (polymerase chain reaction, PCR) be the most widely used nucleic acid amplification technologies, its needs thermally denature repeatedly is to untie the dna double chain, depend on high-quality thermal cycler in application and realize sex change, annealing, three steps of extension, cause temperature control trouble.Different along with scientific development and research purpose, a lot of laboratory attempt development need not the DNA isothermal amplification technique of thermally denature since early 1990s, comprise the isothermal duplication (LAMP) of strand displacement amplification (SDA), rolling circle amplification (RCA), multiple displacement amplification (NASBA), ring mediation etc.And strand displacement amplification reaction has high sensitive, high specific and the characteristic such as easy and simple to handle, is employed gradually.
1992, the Walker in U.S. Becton Dickinson research centre etc. has delivered about the research of strand displacement amplification (Stra nd Displacement Am plification SDA) report, indicates the birth of a kind of new DNA cloning technology SDA.SDA is the external isothermal amplification technique method of a kind of DNA based on enzymatic reaction.By the restriction endonuclease recognition sequence of chemically modified, endonuclease is made a breach double-stranded DNA at its recognition site on the band of target DNA two ends, and next bar DNA chain is held and replaced to 3 ' of the secondary breach that prolongs that archaeal dna polymerase continues.Being replaced the dna single chain that gets off can be combined with primer and extended into two strands by archaeal dna polymerase.This process is constantly carried out repeatedly, and target sequence is efficiently increased.The method is simple to operate, and the low characteristics of high sensitive, high specific and cost are arranged.
Enzyme is the biomacromolecule with catalysis, has Substratspezifitaet and the very high catalytic efficiency of height.The past people think that always enzyme can only be comprised of protein, but the rRNA of the discovery protozoon thermophilass such as T.R.Cech in 1981 has the self splicing function, have disclosed nucleic acid and have also had the ability of catalysis biological reaction, have proposed the concept of ribozyme.1994, Breaker utilized the in-vitro screening scientific discovery, and some single strand dnas have enzymic activity equally, and these dna moleculars with catalysis are called DNAzyme (Deoxyribozyme, DNAzyme).
1997, SANTORO etc. utilized in-vitro screening technology (SELEX) from 10 14Filter out two efficient, general DNAzyme in the individual random dna library: 10-23 type DNAzyme.The function class of this enzyme is similar to a kind of sequence-specific restriction endonuclease, and its active centre district is " 10-23 structure (10-23motif) ", is made of 15 deoxyribonucleotides.The two ends in active centre are respectively the arm of a long 7-9nt, be used for the combination of substrate RNA, be combined pairing with the substrate sequence-specific by the Waston-Crick base pairing, its sequence can change according to the difference of substrate RNA, the phosphodiester bond of cleavage site between the pyrimidine of unpaired purine and pairing on the RNA molecule.The nicking activity of 10-23 structure DNAzyme wherein purine can not form base pairing, and pyrimidine then needs to form base pairing with DNAzyme.Compare with ribozyme, 10-23DNAzyme is in the situation that temperature and ionic strength etc. are identical, and its stability is about 10 of RNA 9Doubly, not only target RNA is had special shear active, and have that relative molecular mass is little, chemical property is relatively stable, be not subject to nuclease degradation, be easy to the advantages such as synthetic and purifying.
Found subsequently various DNAzyme, they have different catalytically active, such as Autophosphorylation, RNA hydrolytic activity, peroxidase activity etc.The concept of G-four serobilas (G-qudruplex) was put forward by scholars such as Gellert in 1962 the earliest.Zalller seminar had reported at K in 1991 +Under the condition that exists, the telomeric dna that is folded into G-four chain body structures can not become the effect substrate of Telomerase, and can suppress Telomerase to the extension of telomere.Dipankar Sen finds again to be folded into the tetrameric single stranded DNA zyme that is rich in guanine of G-subsequently.This DNAzyme has the Peroxidase activity of being similar to after ferrous porphyrin (hemin) or ferrous porphyrin analogue are combined, can catalysis H 2O 2With chromogenic substrate (ABTS 2-) etc. be oxidized to coloring matter, therefore can directly detect nucleic acid by colour-change.
Real-time fluorescence quantitative PCR is to have added fluorescence dye or fluorescent probe on conventional PCR basis, fluorescence dye commonly used for example SYBR Green I can specificity mix the dna double chain, send fluorescent signal, and the dye molecule that does not mix in the two strands does not send fluorescent signal, thereby guarantees that the increase of fluorescent signal and PCR product increase Complete Synchronization.And a large amount of single stranded DNA of the final generation of strand displacement isothermal amplification technique, the chemical structure of triphenylmethane dye is easy to vibration and unstable, and the fluorescence that sends during Individual existence is very weak.When tritane when the tetramer is combined because the tetramer can be stablized the structure of tritane, so fluorescent signal strengthened greatly, so tritane has the potentiality that are developed as G-tetramer fluorescent probe.
How further to expand existing isothermal amplification technique and the range of application of DNAzyme in life science and medical science, how further to improve or develop new Simple fast, but not only can qualitative but also real-time quantitative, and be applicable to simultaneously the detection method of mRNA and miroRNA, remain the key subjects that detect RNA.
Summary of the invention
The invention provides the detection of a kind of RNA and the novel method of RNA isothermal duplication, the method does not need the variation of temperature, and can be rapid, easy and highly sensitive single-minded ground can carry out to RNA the detection method of quantitative and qualitative analysis, is particularly useful for mRNA and miRNA.
The technical scheme that realizes above purpose is as follows:
The isothermal detection methods of a kind of RNA may further comprise the steps:
A) based on the DNAzyme that shears RNA, realize the fixed point of RNA is sheared;
Three strand displacement isothermal duplication circulations occur at polynueleotide kinase in the RNA that b) is sheared under archaeal dna polymerase and the restriction enzyme effect;
Wherein, discharge a series of DNA oligonucleotide small segment trigger through first strand displacement isothermal duplication circulation; Then the oligonucleotide probe that can produce the tetrameric complementation of G-that contains in testing sample is combined and second strand displacement isothermal duplication circulation occurs is discharged the G-tetramer; This G-tetramer with have the DNAzyme that shears the RNA function and be combined and the 3rd strand displacement isothermal duplication circulation occurs discharge again small segment trigger, second strand displacement isothermal duplication of re-initiation thus, discharge the G-tetramer, this G-tetramer causes again the 3rd strand displacement isothermal duplication, then second and the 3 two move in circles and carry out, thereby the lower a large amount of G-tetramer of final displacement;
C) according to the tetrameric character of G-that discharges, can be used as qualitative reporter group or quantitative reporter group, carry out qualitative or the real-time quantitative detection to RNA.
Particularly, the inventive method comprises three steps.At first, adopt the DNAzyme that shears RNA that purpose RNA is sheared.Then, the RNA that is sheared is at polynueleotide kinase, cause three strand displacement isothermal duplications circulations under archaeal dna polymerase and the restriction enzyme effect: take the RNA that is sheared as primer, take the DNAzyme that shears RNA as template, first strand displacement isothermal duplication (SDA) occurs, the a series of DNA oligonucleotide small segment trigger of final generation, simultaneously, also contain one in the testing sample and can produce the oligonucleotide probe of the tetrameric complementation of G-, this probe can be combined with DNA oligonucleotide small segment, trigger thus second SDA amplification, discharge a large amount of G-tetramers, this G-tetramer includes one section nucleotide sequence of a bit of non-G of being rich in, this segment nucleotide sequence can be complementary with the sequence at the partially catalyzed center of the DNAzyme that shears RNA, thereby trigger the 3rd SDA amplified reaction, can discharge small segment trigger again, second strand displacement isothermal duplication of re-initiation thus, discharge the G-tetramer, this G-tetramer causes again the 3rd strand displacement isothermal duplication, then second and the 3 two move in circles and carry out, thereby the lower a large amount of G-tetramer of final displacement.With RNA be thus the amplification of primer change into take DNA oligonucleotide small segment as primer amplification, avoided the easily shortcoming of degraded of RNA, realize simultaneously efficient amplification efficiency.Behind three SDA cyclic amplifications, produce a large amount of G-tetramers.At last, has different character according to not the G-tetramer, the present invention has designed two kinds of different probe sequences, finally produce respectively two kinds of different G-tetramers, be respectively to have the DNAzyme of peroxidase activity and can be combined with tritane the D NAzyme that produces fluorescence, carry out qualitative or real-time quantitative detects.
Behind three SDA cyclic amplifications, produce a large amount of G-tetramers.The present invention can produce the different G-tetramers by the different probe sequence of design, is respectively to have the DNAzyme of peroxidase activity and can be combined with tritane the DNAzyme that produces fluorescence.
In the method for the present invention, the terminal bases complete complementary after the DNAzyme of described shearing RNA is sheared with RNA matches, comprise catalytic activity loop section and with brachium conjunctivum two portions of RNA complementary pairing.
The probe that relates in the inventive method by 3 ' to 5 ' comprise with the sequence of small segment trigger complementation, restriction endonuclease recognition sequence, with the sequence of G-tetramer complementation and the sequence identical with the partially catalyzed center of the DNAzyme that shears RNA.
The G-tetramer that relates in the inventive method refers to be rich in the dna fragmentation G base of guanylic acid (dG) by four chain helical dna structures of the interaction of hydrogen bond formation of Hoogsteen pairing.
The G-tetramer of the qualitative reporter group of conduct that relates in the inventive method is the DNAzyme with peroxidase activity, and the result of its acquisition is macroscopic a kind of reporter group; Being to be combined with a kind of dyestuff the DNAzyme that produces fluorescence as the G-tetramer of quantitative reporter group, is a kind of reporter group that can obtain quantitative result.
Preferably, above-mentioned dyestuff is a kind of triphenylmethane organic substance, for example malachite green preferably.
In the SDA reaction system of the present invention, DNAzyme and the concentration and probe concentration of shearing RNA have certain demand in 0.1 μ M-0.5 μ M, sequences Design.The DNAzyme that shears RNA comprise catalytic activity loop section and with brachium conjunctivum two portions of RNA complementary pairing, according to RNA design brachium conjunctivum to be detected, be combined pairing with the substrate sequence-specific by the Waston-Crick base pairing, the catalytic base ring of shearing the DNAzyme of RNA is fixed on the RNA substrate molecule, the cleavage reaction of catalysis RNA privileged site, its sequence can change according to the difference of substrate RNA.It is terminal bases complementary pairing after must being sheared with RNA that the DNAzyme that shears RNA will satisfy condition.
DNAzyme involved in the present invention is the dna sequence dna with catalysis.The DNAzyme that finds at present falls into 5 types: the DNAzyme of cutting RNA, and the DNAzyme of cutting DNA has the DNAzyme of kinase activity, and the DNAzyme with ligase enzyme activity has the DNAzyme of peroxidase activity with ligand binding.
Polynueleotide kinase described in the inventive method is to have hydrolysis 2 ', the nucleoside monophosphate kinase of 3 '-ring-type phosphate group, archaeal dna polymerase be normal temperature have the strand displacement ability and without the polysaccharase of exonuclease activity, restriction enzyme is to identify double-stranded specific site, and shears the restriction endonuclease of strand specific site.Preferably, polynueleotide kinase is T4 polynueleotide kinase (PNK); Archaeal dna polymerase is Klenow exo-DNA polysaccharase or Bsm archaeal dna polymerase; Restriction enzyme is Nb.Bpu10I.
DNAzyme with peroxidase activity of the present invention can with chlorine iron protoheme (Hemin) in conjunction with, can catalysis H 2O 2Oxidation ABTS 2-, make reaction solution by the colourless green that becomes.This moment the colour-change that can directly detect by an unaided eye, thereby reach the purpose of qualitative detection RNA.
Use in the present invention and to be combined DNAzyme with peroxidase activity with Hemin when detecting, SDA will regulate pH to 7.6 after finishing, 94 ℃ of heating 1min, this step is conducive to avoid the probe sealing G-tetramer, adds thermal distortion and makes probe discharge the G-tetramer that is closed.This step increases sensitivity.Then add Hemin and ABTS 2-, room temperature is placed 5min, adds H again 2O 2Observe colour-change.This step fully is combined the G-tetramer with Hemin, make the signal of negative control lower, thereby make detected result sensitiveer.
For improving sensitivity, preferred pH is 7.6 in the color reaction of qualitative experiment of the present invention.
Detection by quantitative of the present invention refers to add certain density triphenylmethane dye in reaction system, the DNAzyme that utilizes another probe to produce through the SDA amplification can produce in conjunction with triphenylmethane dye the character of fluorescence, make the carrying out along with amplified reaction, signal is progressively accumulated the whole amplification process of Real-Time Monitoring that realizes, by typical curve the RNA of the unknown is carried out quantitative analysis at last.The present invention realizes the purpose of detection by quantitative RNA by detecting single stranded DNA, avoided because the false positive signal that the existence of primer dimer causes.Triphenylmethane dye and the exploitation of the G-tetramer are applied in the real-time fluorescence detection as reporter group, have stronger novelty, and reduced the cost of real-time detection, easy to operate, sensitive high.
For further strengthening sensitivity, the preferred triphenylmethane dye of the present invention are malachite green (Malachite green, MG).
The RNA that method of the present invention is applicable to detect is the RNA that contains AU or GU, and AU and the frequency of occurrences of GU in RNA be that approximately per 8 bases will occur once, and the scope that therefore detects RNA is wider.RNA can synthesize, in-vitro transcription or from sample, extract.Sample can be any source, comprise virus, cell extraction, environmental sample is from sample, microbiological specimens, tissue or the humoral sample etc. of commercial run.
In addition, owing to shearing the characteristic of the DNAzyme of RNA, detect different RNA and will design corresponding DNAzyme, the present invention has very high specificity.
To be reaction solution can carry out under 37 ℃ that optimize or 39 ℃ of constant temperatures of the present invention one large feature.Because adopt strand displacement isothermal duplication RNA under constant temperature, to carry out, thus temperature needn't periodically be changed up and down, thus can adopt easy device to realize the detection of the quantitative and qualitative analysis of RNA.
In addition, detection by quantitative of the present invention can adopt single stage method to implement to finish in a reaction vessel, so that operate further easy, rapidization reduced contaminated danger simultaneously.
Description of drawings
Fig. 1 is the RNA schematic diagram that strand displacement amplification is sheared;
As shown in the figure, at first be that the DNAzyme that shears RNA shears RNA at specific site, then be to carry out strand displacement isothermal duplication RNA to produce a series of DNA oligonucleotide small segment trigger, cause a series of SDA amplification, finally produce a large amount of reporter groups.Character by reporter group reaches the purpose that detects RNA.
Fig. 2 is the polyreaction figure after RNA is sheared;
Wherein A represents that 10-23DNAzyme (Gerald, 1997) shears RNA and produces 2 ', 3 ' ring-type phosphate group, but since terminal bases be unworthy of (A-A) to the appearance without extension; B represents that extension appears in the DNAzyme (namely shearing terminal pairing A-T) that improves.
Fig. 3 is that the RNA of present method detects schematic diagram.
A represents that malachite green is in conjunction with the schematic diagram of the G-tetramer as reporter among Fig. 4; B represents that real-time quantitative detects the Flag-RNA result schematic diagram of gradient dilution; C represents the canonical plotting of real-time quantitative detected result.
Fig. 5 is as a result figure of single-minded detection by quantitative Let-7a.
Embodiment
Below with specific embodiment technical scheme of the present invention is described; but be not to be restriction to technical scheme of the present invention; those skilled in the art should understand that; still can make amendment or be equal to replacement invention; and not breaking away from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed among protection scope of the present invention.
Embodiment 1: the strand displacement amplification experiment take RNA as primer
(1) extension behind the DNAzyme shearing RNA of employing shearing RNA
At first use in this experiment a kind of DNAzyme, in reaction solution 10 μ L systems, comprise 50nm Flag-RNA, 2 μ M DNAzyme, 10mM Tris-HCl (pH8.5), 100mM KCl, 10mM MgCl 2, and 0.1mg/ml BSA, 0.5unit/ μ l PNK and 0.4unit/ μ l Bsm archaeal dna polymerase behind 37 ℃ of lower reaction 1h, are analyzed with the polyacrylamide gel (PAGE) of sex change, occur without extending band.Adopt the DNAzyme of improvement, DNAzyme (fCat) complementary pairing of the terminal bases after namely RNA is sheared and improvement at this.Through above same reaction conditions, band appears extending.Wherein the effect of PNK is 2 ', 3 ' the ring-type phosphate group of shearing end of hydrolysis RNA, carries out extension so that RNA can be used as primer after being sheared.As shown in Figure 2.
In reaction solution, add certain density Flag-RNA, 0.5 μ M fCat, 0.25unit/ μ l PNK, 10mM Tris-HCl (pH8.5), 100mM KCl, 10mMMgCl 2, and 0.1mg/ml BSA, add ddH 2O adds to 10 μ L.
1 negative control pipe does not only add Flag-RNA, and all the other are identical.All place under 37 ° of C and reacted 2 hours 30 minutes.
Flag-RNA(SEQ?ID?NO1):
5’-GGAGGACGAAAUGGACUACAAGGACGAUGACGAUAAGCAGCUGCGUAACUCUAAAAA-3’
10-23(SEQ?ID?NO2):
5’-CTT?ATC?GTC?A?GGC?TAG?CTA?CAA?CGA?C?GTC?CTT?GTAGTC?CAT-3’
fCat(SEQ?ID?NO3):
5’-CAACAGCGACCCTCAGCCTTATCGTCAGGCTAGCTACAACGTCGTCCTTGTAGTCCAT-3’
(2) take the RNA that is sheared of (1) preparation as primer, carry out strand displacement amplification reaction
Because the different G-tetramers has different character, so when quantitative and qualitative detection, the present invention adopts the probe that produces different G-tetramer sequences.The probe Ta of qualitative detection comprises and the sequence of shearing lower first paragraph oligonucleotide sequence (trigger) complementation take RNA as primer amplification by 3 ' to 5 ', restriction endonuclease recognition sequence is with the sequence of the PW-17 complementary pairing with peroxidase activity and the sequence identical with the partially catalyzed center of fCat.As shown in Figure 3.
Ta(SEQ?ID?NO4):
5’-CTTATCGTCAAGCTAACTATTTCCCAACCCGCCCTACCCTCAGCCAACAGCGACCCTCA-3’
In (1) reaction solution, add 0.25 μ M probe Ta (qualitative probe), dNTPs (250 μ m), Nb.Bpu10I (0.125UmL/ μ l), Klenow exo-polymerase (0.125U m L/1) adds dd H 2O adds to 20 μ L, places 37 ℃ of lower reaction 1h.
(3) qualitative detection of amplified production
1 of the negative control pipe of a qualitative detection only contains ddH 2O.
Reaction tubes is at 94 ℃ of sex change 1min, and negative control pipe and reaction tubes all add 1.7 μ lTris-HCl (pH7.3), hemin (1.8 μ M), ABTS 2-(2.1mM), room temperature is placed 5min, H 2O 2(2.1mM).With digital camera record colour developing result.Wherein 94 ℃ of heating 1min are conducive to avoid the probe sealing G-tetramer, to increase sensitivity.Then add Hemin and ABTS 2-, room temperature is placed 5min, and the G-tetramer fully is combined with Hemin, adds H again 2O 2Observe colour-change.Be conducive to make the signal of 2 negative control pipes lower, thereby make detected result sensitiveer.
The Chinese and English full name of the colouring reagents that wherein occurs is as shown in table 1 below.
Table 1 colouring reagents Chinese and English full name
Figure DEST_PATH_GDA00002797315900131
(4) qualitative detection result
Under the Flag-RNA of gradient dilution concentration, concentration from high to low presents the colour-change trend that weakens gradually.When concentration is 1fmol, still with the blank pipe macroscopic difference is arranged.Illustrate that the inventive method has higher sensitivity.
Embodiment 2: malachite green is developed as quantitative reporter experiment in conjunction with the Hum21 DNAzyme with G-tetramer structure of fluorescence (can be combined with dyestuff produce)
Quantitative probe Tb of the present invention is by 3 ' to the 5 ' sequence that comprises with the trgger complementation, and restriction endonuclease recognition sequence and can be combined with malachite green the sequence of the hum21 complementary pairing that produces fluorescence and the sequence identical with the partially catalyzed center of fCat.When primer is combined with Tb, the displacement amplification occurs to connect, thereby produces a large amount of Hum21, utilize this G-tetramer to be combined the character of generation fluorescence with malachite green, along with the accumulation of signal reaches real-time detection.Therefore, illustrate that malachite green can develop as quantitative reporter in conjunction with Hum21.
Reaction solution 20 μ L comprise 10mM Tris-HCl, pH8.5,100mM KCl, 10mM MgCl 2, 0.01mg/ml BSA, dNTPs (250 μ M), restriction enzyme Nb.Bpu10I (0.17unit/ μ l), Bsm DAN polysaccharase (0.17unit/ μ l), malachite green (15 μ Mol), 0.25 μ Mol Tb, 0.125 μ Mol trigger adds ddH 2O to 20 μ L system.
1 negative control pipe: the negative control pipe that does not add the malachite green of trigger
Do respectively two repetitions, place real-time fluorescence quantitative PCR instrument (4 passages excite at 620-650nm, are transmitted in 675-690nm), response procedures is: 39 ℃, 70 circulations are read number one time every 1min.The results are shown in accompanying drawing 4.
Embodiment 3 utilizes DNAzyme and the quantitatively experiment of probe quantitative detection RNA
For detection by quantitative RNA, the present invention adopts single stage method to implement in a reaction vessel, so that operate further easy, rapidization reduced contaminated danger simultaneously.In single stage method reaction, at first the DNAzyme (fCat) of improvement shears RNA, is 2 ', 3 ' ring-type phosphate group of the shearing end of hydrolysis RNA with the effect of PNK, and then cloning RNA occurs even to replace.Reached the purpose that detects RNA by quantitative reporter.
Reaction solution 20 μ L, 10mM Tris-HCl, pH7.0,100mM KCl, 10mMMgCl 20.1mg/ml BSA, dNTPs (250 μ M), Nb.Bpu10I (0.17unit/ μ l), Bsm polymerase (0.17unit/ μ l), PNK (0.08unit/ μ l), malachite green (15 μ mol), 0.25 μ M fCat, 0.25 μ M Tb, certain density Flag-RNA.
1 negative control pipe: just do not add Flag-RNA, all the other are identical.
Do respectively two repetitions, place real-time fluorescence quantitative PCR instrument (Chanel4, Excitation600-640nm, Emission666-740nm.; PikoREAL, Thermo, USA) in.Response procedures is: 39 ℃, 120 circulations are read number one time every 1min.The results are shown in, detected result shows that this method has good discrimination to the Flag-RNA of gradient dilution, and becomes good linear relationship, R 2=0.969.
Embodiment 4 quantitative and qualitative analysis detect the RNA of Let-7 family
The RNA (sequence is as shown in table 2) of chemosynthesis Let-7 family, take Let-7 as example, design can be sheared the DNAzyme (Let-7a-Cat) of the improvement of Let-7, and qualitative checking method is with embodiment 1, and quantitative detecting method is with embodiment 3.Detected result shows that this method can well detect the Let-7a analogue, even there are very fine distinction in analogue and Let-7a, even the difference of one to two base all can differentiate with this method.The result as shown in Figure 5.
The RNA of table 2Let-7 family
Figure DEST_PATH_GDA00002797315900151
Embodiment 5 investigates the specificity experiment
Get single stranded DNA, the full RNA in double-stranded DNA and the HepG2 cell, detection method is with embodiment 1.
2 positive control pipes: contain Flag-RNA, contain the full RNA in Flag-RNA and the HepG2 cell.
The result shows single stranded DNA, and the full RNA in double-stranded DNA and the HepG2 cell all shows colourless, only in the situation that Flag-RNA exists, the generation of color reaction is arranged.Illustrate that the method has very high specificity.

Claims (10)

1. the isothermal detection methods of a RNA comprises following step:
A) based on the DNAzyme that shears RNA, realize the fixed point of RNA is sheared;
Three strand displacement isothermal duplication circulations occur at polynueleotide kinase in the RNA that b) is sheared under D NA polysaccharase and the restriction enzyme effect;
Wherein, discharge a series of D NA oligonucleotide small segment trigger through first strand displacement isothermal duplication circulation; Then the oligonucleotide probe that can produce the tetrameric complementation of G-that contains in testing sample is combined and second strand displacement isothermal duplication circulation occurs is discharged the G-tetramer; This G-tetramer with have the DNAzyme that shears the RNA function and be combined and the 3rd strand displacement isothermal duplication circulation occurs discharge again small segment trigger, second strand displacement isothermal duplication of re-initiation thus, discharge the G-tetramer, this G-tetramer causes again the 3rd strand displacement isothermal duplication, then second and the 3 two move in circles and carry out, thereby the lower a large amount of G-tetramer of final displacement;
C) according to the tetrameric character of G-that discharges, can be used as qualitative reporter group or quantitative reporter group, carry out qualitative or the real-time quantitative detection to RNA.
2. according to claim 1 method, wherein said RNA comprises mRNA and miRNA.
3. according to claim 1 method, the DNAzyme of wherein said shearing RNA and RNA
The pairing of terminal bases complete complementary after being sheared, comprise catalytic activity loop section and with brachium conjunctivum two portions of RNA complementary pairing.
4. according to claim 1 method, wherein said polynueleotide kinase is to have hydrolysis 2 ', the nucleoside monophosphate kinase of 3 '-ring-type phosphate group, archaeal dna polymerase be normal temperature have the strand displacement ability and without the polysaccharase of exonuclease activity, restriction enzyme is to identify double-stranded specific site, and shears the restriction endonuclease of strand specific site.
5. according to claim 4 method, wherein said polynueleotide kinase can be T4 polynueleotide kinase (PNK), and archaeal dna polymerase can be the Bsm archaeal dna polymerase, and restriction enzyme can be Nb.Bpu10I.
6. according to claim 1 method, wherein said probe by 3 ' to 5 ' comprise with the sequence of small segment trigger complementation, restriction endonuclease recognition sequence, with the sequence of G-tetramer complementation and the sequence identical with the partially catalyzed center of the DNAzyme that shears RNA.
7. the four chain helical dna structures that the interaction of hydrogen bond of the dna fragmentation G base that according to claim 1 method, the wherein said G-tetramer refer to be rich in guanylic acid (dG) by the Hoogsteen pairing forms.
8. according to claim 1 method, the wherein said G-tetramer as qualitative reporter group is the DNAzyme with peroxidase activity, the result of its acquisition is macroscopic a kind of reporter group.
9. according to claim 1 method, the G-tetramer of wherein said quantitative reporter group are to be combined with a kind of dyestuff the DNAzyme that produces fluorescence, are a kind of reporter groups that can obtain quantitative result.
10. according to claim 8 method, wherein said dyestuff is a kind of triphenylmethane organic substance-malachite green.
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