CN105624165A - Cascade amplifying-strategy biomolecule detecting method based on self-locking aptamer probe - Google Patents
Cascade amplifying-strategy biomolecule detecting method based on self-locking aptamer probe Download PDFInfo
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Abstract
The invention discloses a cascade amplifying-strategy biomolecule detecting method based on a self-locking aptamer probe. The probe at least comprises two parts of an aptamer sequence of a 3(minute) terminal having specific recognition on a target and a signal transduction sequence of a 5(minute) terminal, wherein the signal transduction sequence of the 5(minute) terminal is hybridized with a part of the aptamer sequence to form a stem-loop structure of the 5(minute) terminal, and the signal transduction sequence of the 5(minute) terminal comprises a recognition site of Cech endonuclease. The probe combines strand displacement amplification (SDA) with a double-exponential rolling circle amplification (DE-RCA) strategy to achieve ultra-sensitive and high-specific detection on protein-PDGF-BB and micromolecule-adenosine, wherein the detection limit reaches 3.8*10<-16>mol/L and 4.8*10<-8>mol/L respectively.
Description
Technical field
The present invention relates to a kind of nucleic acid detection technique field, be specifically related to a kind of cascade based on the fit probe of self-locking and expand the strategy detection method for highly sensitive, the high specific of protein and small-molecule substance.
Background technology
Fit is a class synthetic, short single strand oligonucleotide acid sequence, the field such as be commonly used for bio-sensing, diagnose and treat. Fit it is generally of specific and compact two grade or tertiary structure, its corresponding object is had high affinity and selectivity. Compared with antibody or antibody fragment, fit have stable, be readily synthesized and modify, object feature widely, its object includes albumen, little molecule, metal ion even cell etc. Therefore, the fit structure having been widely used for molecular probe.
Molecular probe based on fit mainly includes two parts: fit sequence and signal sequence. Two grades or the tertiary structure that fit sequence can be fold into, simultaneously its object of specific recognition. Once fit sequence is combined with object, signal sequence can be released generation signal. But, in actual fit sensing system, fit probe is usually excessive. Theoretical Calculation shows, fit when being not associated with object, also can change by occurred conformation, thus producing interference signal, and the accuracy of impact detection. In order to solve this problem, a kind of method is to be fixed on by fit probe on an out-phase surface, is washed away by unnecessary fit probe by washing. This method can be prevented effectively from interference signal generation, but the range of linearity can be caused limited for the introducing of biphase interface and object, probe joint efficiency reduce defect. In order to avoid these problems, realizing based on fit homogeneous detection, another kind of method is just introduced into retardance DNA molecular one section short so that it is hybridize with the active part of fit sequence, form the fit probe that retardance chain is closed, thus suppressing fit non-specific folding. But, due to this retardance chain shorter (12-15nt), the unstability of duplex structure can cause that retardance chain reveals (coming off from fit probe), and then causes interference with the generation of signal. Therefore, in order to reduce interference signal further, improve detection accuracy, it is highly important for building new, a more stable fit probe.
Summary of the invention
The present invention solves above-mentioned the deficiencies in the prior art, it is provided that a kind of fit probe of self-locking detecting protein and biological micromolecule material and the cascade based on the fit probe of self-locking expand the strategy detection method for highly sensitive, the high specific of protein and small-molecule substance.
The technical solution used in the present invention is as follows:
A kind of fit probe of self-locking being used for detecting protein and biological micromolecule, this probe at least includes two parts: object has the signal transduction sequence that the fit sequence and 5 ' that the 3 ' of specific recognition holds is held, the signal transduction sequence that described 5 ' hold and the fit sequence hybridization of part, form 5 ' end stem-ring structures, the signal transduction sequence that described 5 ' hold comprises the recognition site of nicking restriction endonuclease, and the recognition site of this nicking restriction endonuclease is positioned at 3 ' ends of 5 ' the signal transduction sequences held.
This design makes the fit probe of self-locking be in self-locking state when driftlessness thing, so that fit sequence does not fold when driftlessness thing. Self-locking is fit, and probe molecule passes through folded back on itself, and the base pair making 5 ' the signal transduction sequences held complementary with the fit sequence of part meets, and forms hydrogen bonded, is called hairpin structure (or stem-ring structure).
Described 3 ' the fit sequences held are used to combining target thing, and it is Fas lignand system evolution technology (being called for short SELEX technology) the one section of oligonucleotide sequence being combined with object with high affinity and specificity that in the random oligonucleotide sequences storehouse of synthesis, repeated screening obtains from prosthesis by index concentration.
The fit probe of self-locking of present invention design can be used to detect various bioprotein molecule and biological micromolecule, and described bioprotein molecule is platelet-derived growth factor-BB (PDGF-BB), and biological micromolecule is adenosine. By changing fit sequence and design probe, the method may be alternatively used for the detection of other biomolecule.
Preferably, when the base of fit sequence 5 ' end is not suitable for forming hydrogen bond with signal transduction sequence 3 ' end, this probe also includes for connecting fit sequence and the catenation sequence of signal transduction sequence, described catenation sequence is used for participating in formation 5 ' and holds stem-ring structure, it is ensured that signal transduction sequence and the fit sequence of part can hybridize formation stem-ring structure.
Preferably, this probe is also connected with in 3 ' ends of 3 ' the fit sequences held coordinating sequence, and the base number of described cooperation sequence is 1��5, it is therefore an objective to make the 3 ' hairpin structures holding fit sequence smooth opening 5 ' to hold.
The present invention also provides for the application in detection protein and biological micromolecule content of material of a kind of above-mentioned probe.
Based on the method that the cascade of the fit probe of self-locking expands strategy detection protein or biological micromolecule material, comprise the following steps:
(1) chain replaces amplified reaction: is first combined with the fit probe of self-locking by the thing to be detected containing object, is folded into three-dimensional helical structure, opens stem-ring structure that 5 ' end signal transduction sequence hold fit sequence to be formed with part 3 '; Above-mentioned system is when archaeal dna polymerase, nicking restriction endonuclease and dNTP exist, and the three-dimensional helical structure that 3 ' hold triggers SDA reaction as primer, produces substantial amounts of primer sequence 1;
(2) double indexing type amplification rolling circle amplification: primer sequence in step (1) 1 and template 1 are hybridized, triggers first order exponential type RCA amplified reaction under the effect of archaeal dna polymerase, produces substantial amounts of primer sequence 2; Primer sequence 2 and template 2 are hybridized, under the effect of archaeal dna polymerase, trigger second level exponential type RCA amplified reaction, produce substantial amounts of G-tetraploid sequence, after inserting fluorescence molecule, produce fluorescence signal, by the fluorescence signal detected, protein or biological micromolecule material are carried out quantitative assay.
Specifically comprise the following steps that primer sequence 1 and template 1 are hybridized, under the effect of archaeal dna polymerase, trigger linear RCA reaction, produce a long single-stranded DNA product; This product can the template 1 excessive with system be hybridized, and exposes the recognition site of nicking restriction endonuclease, thus by nicking enzyme restriction endonuclease nicking, producing substantial amounts of primer sequence 2; And free primer 1/ template 1 complex carries out next polymerization, nicking circulation, complete first order exponential type RCA amplified reaction; Finally, primer sequence 2 and template 2 are hybridized, second level exponential type RCA amplified reaction is triggered under the effect of archaeal dna polymerase, produce substantial amounts of G-tetraploid sequence, after inserting fluorescence molecule, produce fluorescence signal, by the fluorescence signal detected, protein or biological micromolecule material are carried out quantitative assay.
If thing to be detected does not contain object, do not occur chain to replace amplified reaction and the amplification rolling circle amplification reaction of double indexing type, after inserting fluorescence molecule, then do not produce fluorescence signal.
Specifically include following steps:
(1) chain of binding induction replaces amplification (SDA)
Thing to be detected containing object and the fit probe of above-mentioned self-locking are carried out first time incubation reaction, hatch afterwards add have the archaeal dna polymerase of strand-displacement activity, nicking restriction endonuclease, dNTPs, buffer and water carry out second time incubation reaction, make SDA reaction terminating finally by heating; Concrete reactions steps is as follows:
Thing to be detected containing object and the fit probe of self-locking (50nM, 5 �� L) are hatched 1h at 37 DEG C; It is subsequently added 0.4 �� LKF polymerase, 0.2 �� LNt.BbvCI, 4 �� L2mMdNTPs, 2 �� LCutsmart and 3.4 �� L water, at 37 DEG C, hatches 2.5h; Heat 10min finally by 80 DEG C and make SDA reaction terminating.
(2) coupled reaction
In the amplified production in step (1), add template 1, T4DNA ligase, T4DNA ligase buffer and water, carry out the reaction of template 1 loop connecting; Concrete reactions steps is as follows:
In above-mentioned 20 �� LSDA amplified productions, add L10 ��M of template of 1.4 �� 1,0.3 �� LT4DNA ligase, 3.0 �� LT4DNA ligase buffer and 5.3 �� L water, at 37 DEG C, react 40min.
(3) double indexing type rolling circle amplification (DE-RCA)
Take the connection product in step (2), add template 2, archaeal dna polymerase, nicking restriction endonuclease, buffer and dNTPs and water carries out amplified reaction, make DE-RCA reaction terminating finally by heating; Concrete reactions steps is as follows:
Take above-mentioned 30 �� L coupled reaction products, add L10 ��M of template of 1.4 �� 2,0.4 �� Lphi29DNA polymerase, 0.4 �� LNt.BbvCI, 5 �� LCutsmart, 10 �� LdNTPs and 22.8 �� L water, at 37 DEG C, react 5h; The reaction of this step is heated 10min by 80 DEG C and is terminated.
(4) fluoroscopic examination
Take the amplified production in step (3), add N-methyl porphyrin dipropionic acid IX (NMM) and react, finally adopt luminoscope to carry out fluoremetry; Concrete reactions steps is as follows:
Take above-mentioned DE-RCA product, add 5 �� L2mMKCl and 5 �� L0.08mMNMM, at 37 DEG C, react 40min. Finally carrying out fluoremetry with HitachiF-7000 luminoscope, excitation wavelength selects 399nm, and the capture range launching wavelength selects 550��680nm, and the final fluorescence intensity selecting 612nm place investigates the sensitivity of method.
Preferably, the signal transduction sequence that described 5 ' hold is such as shown in SEQIDNo:1.
Preferably, the signal transduction sequence that described 5 ' hold is 5 '-GCTGTGGATACTGCTGAGGCCA-3 ', as shown in SEQIDNo:1.
Object according to detection is different, changes corresponding 3 ' and holds fit sequence and catenation sequence. When object is platelet derived growth factor BB (PDGF-BB) relevant to cancer, preferably, the fit sequence of PDGF-BB is 5 '-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3 ', as shown in SEQIDNo:2, catenation sequence is 5 '-CCA-3 ', and cooperation sequence is 5 '-TG-3 '. When object is adenosine, it is preferred that the fit sequence of adenosine is 5 '-ACCTGGGGGAGTATTGCGGAGGAAGGT-3 ', as shown in SEQIDNo:3, catenation sequence is 5 '-CCACAG-3 ', and cooperation sequence is 5 '-CTGT-3 '.
Preferably, described nicking restriction endonuclease is nicking enzyme Et.BbvCI, and the recognition site sequence of corresponding nicking enzyme Et.BbvCI is 5 '-GCTGAGG-3 '.
Preferably, the sequence of described template 1 include after forming ring-type one section of primer sequence 1 complementary series and and the complementary series of two primer sequences 2, between the complementary series of each primer sequence 2, be connected each through nicking endonuclease recognized site sequence between complementary series and the complementary series of primer sequence 1 of primer sequence 2. It is preferably 5 '-TACTGCTGAGGGAGTTGAGTGCTGAGGGAGTTGAGTGCTGAGGCTGTGGA-3 ', as shown in SEQIDNo:4, wherein underlined sequence is the recognition site of nicking restriction endonuclease. Wherein, described primer sequence 1 is the product of above-mentioned SDA reaction, and described primer sequence 2 is the product of above-mentioned first order exponential type RCA amplified reaction.
Preferably, the sequence of described template 2 includes the complementary series of one section of primer sequence 2 and the complementary series of two G-tetraploid sequences after forming ring-type, between each G-tetraploid complement thereof, is connected each through nicking endonuclease recognized site sequence between complementary series and the complementary series of primer sequence 2 of G-tetraploid sequence. It is preferably 5 '-AGTGCTGAGGAAACCCAACCCGCCCTACCCGCTGAGGAAACCCAACCCGCCCTACCCGCT GAGGGAGTTG-3 ', as shown in SEQIDNo:5, wherein drawing single line sequence is the tetraploid complementary series of G-, draws the recognition site that two-wire sequence is nicking restriction endonuclease.
Preferably, described fluorescence molecule is NMM (N-methyl porphyrin dipropionic acid IX).
Preferably, the archaeal dna polymerase adopted in described SDA reaction is the archaeal dna polymerase with strand-displacement activity, such as KlenowFragment polymerase.
Preferably, the archaeal dna polymerase adopted in rolling circle amplification reaction is phi29DNA polymerase.
The present invention also provides for a kind of test kit detecting protein and biological micromolecule material, including:
(1) the fit probe of self-locking according to any one of claims 1 to 3;
(2) archaeal dna polymerase, nicking restriction endonuclease, T4DNA ligase;
(3) dNTPs, fluorescence molecule and KCl solution;
(4) buffer of Cutsmart buffer, T4DNA ligase;
(5) above-mentioned template 1 and template 2.
Preferably, described archaeal dna polymerase includes KlenowFragment polymerase and phi29DNA polymerase.
The invention has the beneficial effects as follows:
The present invention constructs a fit probe of self-locking. This probe has triple functions: one is based on its 3 ' molecular recognition function holding fit sequence; Two signal transduction functionalities being based on its 5 ' terminal signal sequence; Three auto-lock functions being based on signal sequence and fit sequence hybridization. Owing to the hybridization of signal sequence Yu fit sequence is to hybridize in molecule, so the fit probe of this self-locking is than the fit probe steady of retardance chain retardance, therefore advantageously in reducing fit non-specific folding and interference signal. Additionally, for the sensitivity of ensuring method, the present invention have also been devised the cascade amplification strategy based on the fit probe of this self-locking, it is achieved that the highly sensitive and high specific detection of platelet derived growth factor BB (PDGF-BB), detection limit reaches 3.8 �� 10-16Mol/L, the range of linearity is more than 6 orders of magnitude. By changing fit sequence, this strategy is also employed successfully in the Sensitive Detection of little molecule-adenosine, illustrates that the fit probe of this self-locking has good versatility, biomolecule actually detected in there is very big application potential.
The present invention adopts the strategy that the fit probe of self-locking reacts and DE-RCA reacting phase is combined with SDA, it is achieved the high sensitivity of bioprotein molecule and biological micromolecule material and high specific detection.
Accompanying drawing explanation
Fig. 1: the cascade amplification strategy of self-locking is fit probe mediation is for the schematic diagram of sensitive, the specific detection of protein.
The fluorogram spectrogram of Fig. 2 (A): variable concentrations PDGF-BB; Wherein, a �� l be followed successively by blank, 2 �� 10-15M��5��10-15M��1��10-14M��1��10-13M��1��10-12M��1��10-11M��1��10-10M��1��10-9M��1��10-8M��2.0��10-8M��5��10-8M��
Fig. 2 (B): the linear relationship chart of fluorescence intensity and PDGF-BB concentration.
Fig. 3: the specificity of method investigates schematic diagram.
Fig. 4 (A): adenosine Cleaning Principle figure.
Fig. 4 (B): the fluorescence response figure of variable concentrations adenosine; Wherein, a �� i be followed successively by blank, 1 �� 10-7M��5��10-7M��1��10-6M��5��10-6M��1��10-5M��5��10-5M��8��10-5M��1��10-4M��
Fig. 4 (C): the linear relationship chart of variable concentrations adenosine and fluorescence intensity.
Detailed description of the invention
Embodiment 1
1. experimental section
1.1 reagent and material
In experiment, DNA (sequence is table 1 such as) and dNTPs used is by Sheng Gong biological engineering company limited's (China, Shanghai) synthesis and purification; Platelet derived growth factor (PDGF-BB), immunoglobulin G while (IgG), huamn tumor necrosis factory alpha (TNF-��), human interferon gamma (IFN-��) and thrombin are purchased from ProSpec-Tany company (Nai Siciaona, Israel); Adenosine is purchased from Sigma-Aldrich company (St. Louis, the U.S.); KlenowFragment polymerase, nicking enzyme Et.BbvCI, T4DNA ligase and phi29DNA polymerase are purchased from NewEnglandBiolabs company (U.S.); NMM is purchased from J&KScientific company (Beijing, China); Other reagent (analytical pure) is purchased from standard suppliers.
In experimentation, HEPES buffer used comprises 50mMNaCl and 25mMHEPES (pH7.0); PBS comprises 0.15MNaCl, 2.4mMNaH2PO4And 7.6mMNa2HPO4(PH7.4); TE buffer comprises 10mMTris and 1.0mMNa2EDTA(PH8.0)��
DNA sequence used in table 1 experiment
Note: double; two underlined sequence are the fit sequence difference of PDGF-BB, the fit sequence of chain-dotted line sequence adenosine; Single line sequence nicking enzyme Et.BbvCI recognition site, wave sequence is the tetraploid complementary series of G-.
The chain of 1.2 binding inductions replaces amplification (SDA)
PDGD-BB (10nM, 5 �� L) and the fit probe of self-locking (50nM, 5 �� L) are hatched 1h at 37 DEG C. It is subsequently added 0.4 �� LKF polymerase, 0.2 �� LNt.BbvCI, 4 �� L2mMdNTPs, 2 �� LCutsmart and 3.4 �� L water, at 37 DEG C, hatches 2.5h. Heat 10min finally by 80 DEG C and make SDA reaction terminating.
1.3 coupled reactions
In above-mentioned 20 �� LSDA products, add L10 ��M of template of 1.4 �� 1,0.3 �� LT4DNA ligase, 3.0 �� LT4DNA ligase buffer and 5.3 �� L water, at 37 DEG C, react 40min.
1.4 double indexings type rolling circle amplification (DE-RCA)
Take above-mentioned 30 �� L coupled reaction products, add L10 ��M of template of 1.4 �� 2,0.4 �� Lphi29 polymerase, 0.4 �� LNt.BbvCI, 5 �� LCutsmart, 10 �� LdNTPs and 22.8 �� L water, at 37 DEG C, react 5h. The reaction of this step is heated 10min by 80 DEG C and is terminated.
1.5 fluoroscopic examinations
Take above-mentioned DE-RCA product, add 5 �� L2mMKCl and 5 �� L0.08mMNMM, at 37 DEG C, react 40min. Finally carrying out fluoremetry with HitachiF-7000 luminoscope, excitation wavelength selects 399nm, and the capture range launching wavelength selects 550��680nm, and the final fluorescence intensity selecting 612nm place investigates the sensitivity of method.
2. result and discussion
2.1 principles
Such as Fig. 1, first design and construct a fit probe of self-locking. This probe includes two parts: the signal transduction sequence that 3 ' the fit sequences and 5 ' held are held. Additionally, this signal transduction sequence can be hybridized with fit Sequence, probe is made to be in self-locking state when driftlessness thing, thus not folding, it is ensured that the low background of method. In this method, select the PDGF-BB that cancer is relevant as model analysis thing. When there being PDGF-BB to exist in system, fit probe is combined with PDGF-BB, is folded into three-dimensional helical structure, opens the 5 ' hairpin structures held. It follows that when KF polymerase, nicking enzyme Et.BbvCI and dNTP exist, the three-dimensional helical structure that 3 ' hold triggers SDA reaction as primer, produces substantial amounts of primer 1. Then, primer 1 and template 1 Eclectics, trigger linear RCA reaction under the effect of phi29 polymerase, produce a long single-stranded DNA product. This product can the template 1 excessive with system be hybridized, and exposes the recognition site of nicking enzyme, thus being carved by nicking enzyme action, produces substantial amounts of primer 2. And free primer 1/ template 1 complex has and can carry out next polymerization, nicking circulation, complete first order exponential type RCA amplification. Finally, primer 2 and template 2 are hybridized, and trigger second level exponential amplification reaction, produce substantial amounts of G-tetraploid sequence, after inserting NMM, produce the fluorescence signal strengthened.
2.2 condition optimizings
Respectively SDA response time, DE-RCA response time, phi29 polymerase consumption, Et.BbvCI consumption and NMM concentration are optimized. It is final that to select the SDA time be 2.5h, DE-RCA response time be 5h, phi29 polymerase consumption be 4U, Et.BbvCI consumption is final concentration of 5 ��Ms of 4U, NMM.
2.3 sensitivity are investigated
In optimal conditions, the sensitivity of method is investigated, such as Fig. 2,2.0 �� 10-15Mol/L��1.0 �� 10-8Within the scope of mol/L, fluorescence intensity is good with the linear relationship of target concentration, and linear equation is �� F=2548.4+713.0lgC, and detection is limited to 3.8 �� 10-16mol/L��
2.4 specificitys are investigated
With IgG, TNF-��, IFN-�� and thrombin for jamming target thing, the specificity of method is investigated. Such as Fig. 3, PDGF-BB is only had strong fluorescence response by this method, and the fluorescence response of other albumen is only small, illustrates that this method has good specificity.
2.5 versatilities are investigated
In order to investigate the versatility of method, the fit part of probe is replaced with the fit sequence of adenosine, it is achieved that the Sensitive Detection to adenosine. Such as Fig. 4, method is 1.0 �� 10-7Mol/L��1.0 �� 10-4Within the scope of mol/L, fluorescence intensity is good with the linear relationship of adenosine concentration, and detection is limited to 4.8 �� 10-8mol/L��
It addition, the fit sequence of probe to be replaced by the fit sequence of other biomolecule, also it can be carried out high sensitivity and detect with high specificity.
3. sum up
In this work, the present invention constructs a fit probe of self-locking, and the cascade in conjunction with SDA and DE-RCA expands, it is achieved that protein and micromolecular highly sensitive, high specific detection, detection limit reaches 3.8 �� 10 respectively-16Mol/L and 4.8 �� 10-8mol/L��
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Claims (10)
1. the fit probe of self-locking being used for detecting protein and biological micromolecule, it is characterized in that: this probe at least includes two parts: object is had the signal transduction sequence that the fit sequence and 5 ' that the 3 ' of specific recognition holds is held, the signal transduction sequence that described 5 ' hold and the fit sequence hybridization of part, forming 5 ' end stem-ring structures, the signal transduction sequence that described 5 ' hold comprises the recognition site of nicking restriction endonuclease.
2. probe as claimed in claim 1, is characterized in that: this probe also includes for connecting fit sequence and the catenation sequence of signal transduction sequence, and described catenation sequence is used for participating in formation 5 ' and holds stem-ring structure.
3. probe as claimed in claim 1, is characterized in that: this probe is also connected with coordinating sequence in 3 ' the fit sequences held.
4. the method expanding strategy detection protein or biological micromolecule based on the cascade of the fit probe of self-locking, is characterized in that, comprise the following steps:
(1) chain replaces amplified reaction: be combined with the probe according to any one of claims 1 to 3 by the thing to be detected containing object, it is folded into three-dimensional helical structure, opens stem-ring structure that 5 ' end signal transduction sequence hold fit sequence to be formed with part 3 '; Above-mentioned system, when archaeal dna polymerase, nicking restriction endonuclease and dNTP exist, occurs chain to replace amplified reaction, produces substantial amounts of primer sequence 1;
(2) double indexing type amplification rolling circle amplification: primer sequence in step (1) 1 and template 1 are hybridized, triggers first order exponential type RCA amplified reaction, produce substantial amounts of primer sequence 2; Primer sequence 2 and template 2 are hybridized, and trigger second level exponential type RCA amplified reaction, produce substantial amounts of G-tetraploid sequence, after inserting fluorescence molecule, produce fluorescence signal, by the fluorescence signal detected, protein or biological micromolecule are carried out quantitative assay;
If thing to be detected does not contain object, do not occur chain to replace amplified reaction and the amplification rolling circle amplification reaction of double indexing type, then there is no fluorescence signal.
5. method as claimed in claim 4, is characterized in that: described protein is platelet derived growth factor BB, and described biological micromolecule is adenosine, and the signal transduction sequence that described 5 ' hold is such as shown in SEQIDNo:1.
6. method as claimed in claim 4, it is characterized in that, in step (1), the step of SDA reaction is: the thing to be detected containing object and the fit probe of self-locking carry out first time incubation reaction, hatch afterwards add have the archaeal dna polymerase of strand-displacement activity, nicking restriction endonuclease, dNTPs, buffer and water carry out second time incubation reaction, make SDA reaction terminating finally by heating.
7. method as claimed in claim 4, is characterized in that, in step (2), concrete reactions steps is:
Primer sequence 1 and template 1 are hybridized, and trigger linear RCA reaction, produce a long single-stranded DNA product under the effect of archaeal dna polymerase; This product and template 1 are hybridized, and expose the recognition site of nicking restriction endonuclease, thus by nicking restriction endonuclease nicking, producing substantial amounts of primer sequence 2; And free primer 1/ template 1 complex carries out next polymerization, nicking circulation, complete first order exponential type RCA amplification; Finally, primer sequence 2 and template 2 are hybridized, and trigger second level exponential amplification reaction, produce substantial amounts of G-tetraploid sequence, after inserting fluorescence molecule, produce fluorescence signal, by the fluorescence signal detected, protein or biological micromolecule are carried out quantitative assay.
8. the method as described in claim 4 or 7, it is characterized in that, before there is RCA amplified reaction, the coupled reaction step of template 1 is: add template 1 in the amplified production in step (1), T4DNA ligase, T4DNA ligase buffer and water, be attached reaction.
9. method as claimed in claim 4, is characterized in that: described nicking restriction endonuclease is nicking enzyme Et.BbvCI, and the recognition site sequence of described nicking enzyme Et.BbvCI is 5 '-GCTGAGG-3 ', the sequence of described template 1 include after forming ring-type the primer sequence 1 described in one section of claim 4 complementary series and and the complementary series of primer sequence 2 described in two claim 4, between the complementary series of each primer sequence 2, it is connected each through nicking endonuclease recognized site sequence between the complementary series of primer sequence 2 and the complementary series of primer sequence 1, the sequence of described template 2 includes the complementary series of one section of primer sequence 2 and the complementary series of two G-tetraploid sequences after forming ring-type, between the complementation of each G-tetraploid sequence, it is connected each through nicking endonuclease recognized site sequence between complementary series and the complementary series of primer sequence 2 of G-tetraploid sequence.
10. detect a test kit for protein or biological micromolecule, it is characterized in that: the fit probe of self-locking according to any one of (1) claims 1 to 3;
(2) archaeal dna polymerase, nicking restriction endonuclease, T4DNA ligase;
(3) dNTPs, fluorescence molecule and KCl solution;
(4) buffer of Cutsmart buffer, T4DNA ligase;
(5) template 1 described in claim 4 and template 2.
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