CN105950755A - Method for detecting microRNA based on split-type recognition mode and cascade signal amplification strategy - Google Patents
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Abstract
The invention discloses reagents for detecting miRNA based on a split-type recognition mode and cascade signal amplification strategy design and a detection method. The reagents for detecting miRNA comprise a hairpin recognition probe with a sticky tail end, an auxiliary recognition probe and lock-type DNA with a G tetraploid complementary sequence and a cutting enzyme recognition site. Target miRNA can be accurately distinguished from homologous sequences, the method is good in selectivity and high in sensitivity, and the detection limit of the method is 3.2 pM. Adding standard recovery experiments in serum are conducted, and the recovery rate ranges from 96% to 102%. It is shown through results that the detection reagents and the detection method have larger application potential to miRNA detection in the aspects of clinical diagnosis and disease treatment.
Description
Technical field
The present invention relates to a kind of method combining cascade signal amplification strategy detection microRNA based on Split type recognition mode.
Background technology
MicroRNA (miRNA) is a class endogenous non-coding RNA molecule, has the length of 18-24 base, by right
Target gene mRNA carries out cutting or suppressing the expression of translational control gene.This adjusting function makes miRNA at many physiology
Play an important role during, the propagation of such as cell, migration and apoptosis.Recent research indicate that the exception table of miRNA
Reach the initiation with cancer and develop closely related.MiRNA is used for diagnosis and the treatment of cancer as biomarker.Cause
This, special and sensitive detection miRNA is most important for clinical diagnosis and disease treatment.
Have sequence similarity between miRNA member of the same clan, sequence is short, abundance is low, the feature such as degradable, therefore, for miRNA
Detection method need possess good specificity, sensitivity and stability.The traditional detection method of miRNA has: real time aggregation
Polymerase chain reaction, microarray technology and Northern blotting.In addition, also fluorescence method, electrochemical process, colorimetry and
Biloluminescence method.In these methods, recognition mode is most important for the specific detection of miRNA.The knowledge of recent report
Other pattern is mainly based upon the recognition mode of direct cross reaction and recognition mode based on coupled reaction.Although these recognition modes
Improve the specificity of miRNA detection to a certain extent, but yet suffer from some and limit.Such as: based on the most miscellaneous
Handing in the recognition mode of reaction, the specific recognition of miRNA is to match based on simple Watson Crick.But it is smaller
Thermodynamic energy change makes this recognition mode have poor specificity.In recognition mode based on coupled reaction, miRNA's
Specific recognition is to pass through coupled reaction linking probe based on ligase at the end mated completely.But, when the position of base mispairing
When putting away from connection site, this recognition mode can not show satisfied specificity.
The strand replacement reaction of sticky end mediation is a controlled reaction.In this reaction, target sequence and two hybridization chains
In one sticky end hybridization, cause strand replacement reaction.The part complementary with sticky end when object is complete with sticky end
When entirely mating, object is slower than the speed of strand displacement from the dissociation rate of sticky end.But, when object is mutual with sticky end
When the part mended exists mispairing, object is faster than the speed of strand displacement from the dissociation rate of sticky end, it is impossible to cause strand displacement anti-
Should.Lee seminar constructs the recognition mode of a kind of strand replacement reaction based on sticky end mediation for specific detection
miRNA.Reacting and compared with the recognition mode of coupled reaction with based on direct cross, this recognition mode has an extra competition
Chain, therefore shows higher specificity.But, this recognition mode at specific detection base mismatch site away from sticky end
Homologous sequence time still there is restriction.Therefore, need a kind of base mispairing sequence of can distinguishing of development badly and be positioned at any position
MiRNA specific recognition pattern.
Summary of the invention
For above-mentioned the deficiencies in the prior art, it is an object of the invention to provide one and combine level based on Split type recognition mode
Connection signal amplifies the method for strategy detection miRNA.Wherein, double part identification abilitys of Split type recognition mode make this strategy
Having good selectivity, the high amplification efficiency that cascade signal amplifies makes this strategy have higher sensitivity, and detection limit reaches 3.2
PM, has bigger application potential for clinical diagnosis and disease treatment aspect.
For achieving the above object, the present invention uses following technical proposals:
A first aspect of the present invention, it is provided that a kind of hair clip identification probe (HP) for detecting miRNA and assist in identifying probe
(AP)；
Described hair clip identification probe includes: polymerizing template sequence, nicking enzyme recognition sequence and miRNA recognition sequence I；
The described probe that assists in identifying includes: miRNA recognition sequence II and primer sequence；
Described miRNA recognition sequence I and miRNA recognition sequence II is complementary with miRNA sequence to be detected, for simultaneously
Identify miRNA.
Described nicking enzyme recognition sequence is: 5 '-G CTG AGG-3 '.
Described primer sequence is: 5 '-ATG GGA GTT GAG TG-3 '.
In a specific embodiment of the present invention, it is provided that a kind of hair clip identification probe for detecting let-7b and auxiliary are known
Other probe；
The nucleotide sequence of described hair clip identification is as shown in SEQ ID NO.1；Specific as follows:
HP:5 '-GGG AGT TGA GTG CTG AGG TTT TTC ACT CAA CTC CCT ACT ACC TCA-3 '
(SEQ ID NO.1)；(in sequence, normal font part is polymerizing template sequence, and overstriking font component is nicking enzyme identification sequence
Row, italicized item is miRNA recognition sequence I)
The described nucleotide sequence of probe that assists in identifying is as shown in SEQ ID NO.2；Specific as follows:
AP:5 '-AAC CAC ACA ACC ATG GGA GTT GAG TG-3 ' (SEQ ID NO.2)；(in sequence, just
Often font component is primer sequence, and italicized item is miRNA recognition sequence II)
Above-mentioned hair clip identification probe and assist in identifying probe and may be used for identification and the detection of miRNA.
A second aspect of the present invention, it is provided that a kind of reagent detecting miRNA, comprises:
Above-mentioned hair clip identification probe (HP), assist in identifying probe (AP) and there is G tetraploid complementary series and nicking enzyme recognition site
Locking-type DNA；
The nucleotide sequence of described locking-type DNA is as shown in SEQ ID NO.3, specific as follows:
Padlock DNA:p-AGT GCT GAG GAA ACC CAA CCC GCC CTA CCC GCT GAG
GAA ACC CAA CCC GCC CTA CCC GCT GAG GGA GTT G(SEQ ID NO.3).(in sequence,
Overstriking font component is nicking enzyme recognition sequence)
Further, described reagent also comprises: KF polymerase, Nt.BbvCI nicking enzyme, T4DNA ligase and Phi29DNA
Polymerase.
A third aspect of the present invention, it is provided that a kind of method detecting miRNA, step is as follows:
(1) prepare the miRNA solution of variable concentrations, add hair clip identification probe and assist in identifying probe, constant-temperature incubation；Again
Add KF polymerase, Nt.BbvCI nicking enzyme and dNTPs, carry out strand displacement iodine；
(2) in step (1) reacted solution, add locking-type DNA and T4DNA ligase, constant-temperature incubation, carry out
Coupled reaction；Add Nt.BbvCI nicking enzyme, Phi29DNA polymerase and dNTPs, be circulated rolling ring iodine；
(3) in step (2) reacted solution, add NMM and KCl, hatch, fluorescence intensity, build the most glimmering
Linear equation between light intensity and miRNA concentration, to testing sample, can substitute into by measuring the clean fluorescence intensity of sample
Linear equation, calculates the miRNA concentration of sample.
In step (1), the condition of described constant-temperature incubation is: 37 DEG C of constant-temperature incubation 1h.
In step (1), the condition of described strand displacement iodine is: 37 DEG C of constant-temperature incubation 0.5h, then heats 20min in 85 DEG C
Make enzyme inactivate, terminate reaction.
In step (2), the condition of described constant-temperature incubation is: 37 DEG C of constant-temperature incubation 1h.
In step (2), the condition of described circulation rolling ring iodine is: hatch 1.5-4.0h at 37 DEG C, then in 75 DEG C
Heating 20min makes enzyme inactivate, and terminates reaction.
In step (3), the spectral conditions of fluorescence intensity detection is: emission spectrum scope is 550～680nm, and excitation wavelength is
399nm, excites and the slit width launched is 10nm, and excitation voltage is 700V, selects fluorescent emission at 612nm strong
Degree is criterion.
In a specific embodiment of the present invention, step (3), the clean fluorescence intensity of structure is dense with miRNA (let-7b)
Linear equation between degree is: y=95.4+2.96 × 1011X；Wherein, y is clean fluorescence intensity (i.e. F-F0, F is containing miRNA
The fluorescence intensity of sample, F0Fluorescence intensity for the sample without miRNA)；X is miRNA concentration.
The range of linearity of above-mentioned linear equation is 10pM-10nM.
The present invention based on Split type recognition mode combine cascade signal amplify strategy detection miRNA principle be:
The recognition mode of Split type comprises two species specific identification processes, and both specific identification processes are based on viscous respectively
Property end mediation strand replacement reaction and direct cross reaction.Based on this, the present invention devises two Species specific probes, it may be assumed that tool
Hair clip identification probe (HP) of toughness end and assist in identifying probe (AP).
First, the sticky end of hairpin probe and assist probes identification division miRNA simultaneously, then carrying out strand replacement reaction will send out
Folder probe is unfolded into stable DNA y-type structure.It follows that the assist probes in DNA y-type structure can be as primer
Initiated polymerization in the presence of KF polymerase and dNTPs, generates the double-stranded DNA with Nt.BbvCI nick site.
Then cause polymerization nicking strand displacement iodine, discharge the trigger sequence of many.Finally, these trigger sequences be designed with
The locking-type DNA hybridization of G tetraploid complementary series and nicking enzyme recognition site causes circulation rolling ring iodine, produces many G
Tetraploid sequence.G tetraploid sequence can bind N-methyl porphyrin dipropionic acid IX (NMM), produces the fluorescence signal strengthened,
Realize cascade signal to amplify.On the contrary, when miRNA not in the presence of, HP and AP can not phase mutual cross because HP and AP
The Tm (Tm ≈ 41 DEG C) of the heteroduplex Tm (Tm ≈ 76 DEG C) than HP self is low.It is unable to form DNA
Y-type structure causes iodine, produces relatively low background signal.The principle of its detection is concrete as shown in Figure 1.
The design that it is critical only that the probe to miRNA with identification ability of the present invention, the present invention is by the recognition sequence of miRNA
Design is on two different identification probes.Double part identification abilitys of Split type recognition mode make this strategy have good choosing
Selecting property, in one embodiment of the invention, it is possible to let-7b is distinguished from homology sequence accurately.And, level
The high amplification efficiency that connection signal amplifies makes this strategy have higher sensitivity, and detection is limited to 3.2pM.
Beneficial effects of the present invention:
The miRNA detectable of the present invention and detection method are to combine cascade signal amplification strategy based on Split type recognition mode to enter
Row design, it is possible to achieve target miRNA being distinguished accurately from homology sequence, the selectivity of method is good, spirit
Sensitivity is high, and the detection of method is limited to 3.2pM.The present invention has also carried out the recovery testu in serum, and response rate scope is 96%
-102%.Result shows, the detectable of the present invention and detection method are for clinical diagnosis and the inspection of disease treatment aspect miRNA
Measuring tool has bigger application potential.
Accompanying drawing explanation
Fig. 1: Split type recognition mode combines cascade signal and amplifies strategy for high special, the schematic diagram of Sensitive Detection miRNA；
Fig. 2: the fluorescence emission spectrum of miRNA inspection policies under different situations；
Fig. 3: A is the fluorescence emission spectrum of variable concentrations miRNA；B is variable concentrations miRNA and F-F0Linear relationship；
The relative fluorescence response of Fig. 4: different miRNA.
Detailed description of the invention
The present invention is further illustrated in conjunction with the embodiments, it should explanation, the description below merely to explain the present invention,
Its content is not defined.
Experiment reagent and instrument:
Experiment reagent used in the embodiment of the present invention and instrument are as follows:
MiRNAs, dNTPs of HPLC purification and pyrocarbonic acid diethyl ester (DEPC) purchased from Sheng Gong biological engineering company limited (in
State, Shanghai).KF polymerase, Nt.BbvCI nicking enzyme, T4DNA ligase are purchased from NEB (China, Beijing).Phi29DNA
Polymerase is purchased from Thermo Fischer Scient Inc. (China, Shanghai).NMM purchased from lark waffle learn a skill company limited (China,
Beijing).In experiment, other chemical reagent used are all analytical pure, and without further purification during use.0.1%DEPC
The deionized water processed is used in whole experimentation.In experiment nucleic acid chains used by Sheng Gong biological engineering limited company (on
Sea, China) synthesize and purification.
In experiment, required buffer composition is as follows:
KCl solution: 2M KCl.
Cut smart buffer: 50mM KAc, 20mM Tris-HAc, 10mM Mg (Ac)2,100μg/mL BSA,pH 7.9。
T4 ligase buffer: 400mM Tris HCl, 100mM MgCl2,100mM DTT,5mM ATP,pH 7.8。
Fluorescence measurement uses Hitachi's F-7000 fluorescence spectrophotometer to be measured (Japan, Hitachi, Ltd).
Embodiment 1: combine cascade signal based on Split type recognition mode and amplify strategy detection miRNA (let-7b)
Concrete grammar is as follows:
(1) design of probe
Separately designing and have hair clip identification probe (HP) of sticky end and assist in identifying probe (AP), its nucleotide sequence is as follows:
HP:5 '-GGG AGT TGA GTG CTG AGG TTT TTC ACT CAA CTC CCT ACT ACC TCA-3 '
(SEQ ID NO.1)；
AP:5 '-AAC CAC ACA ACC ATG GGA GTT GAG TG-3 ' (SEQ ID NO.2)；
Being designed with G tetraploid complementary series and locking-type DNA of nicking enzyme recognition site, its nucleotide sequence is as follows:
p-AGT GCT GAG GAA ACC CAA CCC GCC CTA CCC GCT GAG GAA ACC CAA CCC
GCC CTA CCC GCT GAG GGA GTT G(SEQ ID NO.3)。
(2) the strand displacement iodine of DNA y-type structure mediation
HP, at 95 DEG C of 5min that anneal, is then slowly cooled to room temperature, to form correct structure.To 1 × Cut Smart (50
mM KAc,20mM Tris-HAc,10mM Mg(Ac)2, 100 μ g/mL BSA, pH 7.9) in add 20nM HP,
The AP of 0.50-25nM and the miRNA solution of variable concentrations, hatch 1h at 37 DEG C.Subsequently, to above-mentioned mixed liquor
Middle addition 1U KF polymerase, 2U Nt.BbvCI and the dNTPs of 3 μ L (10mM), hatch 0.5h at 37 DEG C and enter
Row strand displacement iodine.Then make enzyme inactivate in 85 DEG C of heating 20min reaction system, be then slowly cooled to room temperature.
(3) circulation based on locking-type DNA rolling ring iodine
3 μ L 10 × T4DNA ligase buffer (400mM Tris HCl, 100mM MgCl are added in above-mentioned reaction system2,
100mM DTT, 5mM ATP, pH 7.8), locking-type DNA of 50-1200nM and 120U T4DNA ligase, at 37 DEG C
Under hatch 1h be attached reaction.Subsequently, the dNTPs of 3 μ L (10mM), 2 μ L 10 × Cut Smart, 4U are added
Nt.BbvCI and 0.5-5.0U Phi29DNA polymerase, hatches 1.5-4.0h at 37 DEG C and is circulated rolling ring amplification instead
Should.Then make enzyme inactivate in 75 DEG C of heating 20min response system, be then slowly cooled to room temperature.
(4) measurement of fluorescence spectrum
In above-mentioned response system, add the NMM and the KCl of 5 μ L (2M) of 1-7 μM, at 37 DEG C, hatch 1h.
Then the fluorescence spectrum of all samples is measured by Hitachi F-7000 fluorescence spectrophotometer (Hitachi Ltd, Japan, Tokyo).
Emission spectrum scope is 550～680nm, and excitation wavelength is 399nm.Fluorescence intensity at 612nm is used for assessing this sensing system
The performance of system.The slit width excited and launch is 10nm, and excitation voltage is 700V.Build clean fluorescence intensity and miRNA
Linear equation between concentration, to testing sample, can substitute into linear equation by measuring the clean fluorescence intensity of sample, and calculating is treated
The miRNA concentration of test sample product.
Embodiment 2: the feasibility study of the detection method of the present invention
In order to verify the feasibility of the detection method of the present invention, the present invention has investigated the fluorescence emission of reaction system under different situations
Spectrum.Result as in figure 2 it is shown, at negative systems, i.e. when miRNA not in the presence of, it is (bent that system shows the most weak fluorescence signal
Line a), this result show when miRNA not in the presence of, HP and AP can not phase mutual cross, be unable to formed DNA Y
Type structure causes iodine.In contrast, positive systems, i.e. after adding miRNA, it can be observed that significantly fluorescence
(curve d), this sticky end showing HP and AP can form stable DNA by identification division miRNA to intensity enhancing simultaneously
Y-type structure also causes cascade signal amplified reaction.Compared with positive reaction system, it is circulated rolling ring amplification when system lacks
During the Nt.BbvCI nicking enzyme reacted, system shows as fluorescence signal (the curve b) reduced.This result shows, with linear rolling
Ring iodine is compared, and the circulation rolling ring iodine of Nt.BbvCI auxiliary has higher amplification efficiency.
The present invention have also been devised without " AT " assist in identifying probe (AP), its nucleotides sequence is classified as: AAC CAC ACA ACC
GGG AGT TGA GTG。
When AP does not contains " AT " prominent base, system shows as fluorescence signal (the curve c) reduced.This result with to have
Report is consistent, and the most unpaired prominent nucleotide can stablize three nodule structures.More than test result indicate that, the inspection of this present invention
Survey method is feasible.
Embodiment 3: the sensitivity of the detection method of the present invention is investigated
In order to assess the analytical performance of detection method, we measure the glimmering of variable concentrations miRNA in optimal conditions
Optical emission spectroscopy, result is as shown in Figure 3A.Along with miRNA concentration increases to 10nM from 0, fluorescence intensity is also gradually increased.
Build the linear equation between clean fluorescence intensity and miRNA concentration, result as shown in Figure 3 B, net signal F-F0With 10pM-10
The miRNA of nM concentration range is linear, and detection is limited to 3.2pM.The sensitivity that the method is higher is mainly due to chain and puts
The cascade signal changing iodine and circulation rolling ring iodine combination amplifies the high amplification efficiency of strategy.
Embodiment 4: the selectivity of the detection method of the present invention is investigated
Selectivity is the important indicator evaluating detection method, and in order to investigate the selectivity of detection method, we are identical
Have detected several homologous sequences (as shown in table 1) of let-7b in let-7 family under experiment condition, result is as shown in Figure 4.When
In the presence of let-7b, the relative intensity of fluorescence being obviously enhanced can be produced.It is strong that remaining homologous sequence but shows relatively low relative fluorescence
Degree.This result shows which position the base mispairing of no matter homologous sequence is positioned at, and let-7b can be distinguished by the method accurately
Out.The specificity that this strategy is higher is mainly attributed to the identification ability of the double part of Split type recognition mode.
Table 1:
Embodiment 5: the precision of the detection method of the present invention and repeatability are investigated
Precision and repeatability are the important parameters of the weighing apparatus actual application of analysis method, and we in a few days and are in the daytime tested by calculating
Relative standard deviation (RSD) assesses precision and the repeatability (n=3) of detection method.Select is high, medium and low
Three concentration are respectively 8.0nM, 4.0nM and 100pM.In a few days RSD is respectively 1.1%, 2.9% and 1.4%.Same concentrations
Under, RSD is respectively 2.8%, 5.3% and 4.1% in the daytime.These results show: the detection method of the present invention has acceptable
Precision and repeatability.
Embodiment 6: the analysis of complex biological sample
In order to assess the detection method actual application in complex biological sample, we enter in the human serum sample of 10%
Go recovery testu.High, medium and low three concentration selected are respectively 8.0nM, 4.0nM and 100pM.The response rate is divided
It is not 98%, 102% and 96%, and RSD is respectively 2.3%, 4.8% and 3.4%.These results indicate that complex biological sample
Interference effect in product can be ignored, and the method has the biggest application potential for the detection of miRNA in complex biological sample.
Claims (10)
1. one kind is used for detecting the hair clip identification probe of miRNA and assisting in identifying probe；
Described hair clip identification probe includes: polymerizing template sequence, nicking enzyme recognition sequence and miRNA recognition sequence I；
The described probe that assists in identifying includes: miRNA recognition sequence II and primer sequence；
Described miRNA recognition sequence I and miRNA recognition sequence II is complementary with miRNA sequence to be detected, for simultaneously
Identify miRNA.
2. hair clip identification probe as claimed in claim 1 and assist in identifying probe, it is characterised in that described nicking enzyme identification sequence
It is classified as: 5 '-G CTG AGG-3 '；
Described primer sequence is: 5 '-ATG GGA GTT GAG TG-3 '.
3. one kind is used for detecting the hair clip identification probe of let-7b and assisting in identifying probe, it is characterised in that
The nucleotide sequence of described hair clip identification probe is as shown in SEQ ID NO.1；
The described nucleotide sequence of probe that assists in identifying is as shown in SEQ ID NO.2.
4. the reagent detecting miRNA, it is characterised in that comprise:
Hair clip identification probe described in any one of claim 1-3, assist in identifying probe, and there is G tetraploid complementary series
Locking-type DNA with nicking enzyme recognition site；
The nucleotide sequence of described locking-type DNA is as shown in SEQ ID NO.3.
5. the reagent detecting miRNA as claimed in claim 1, it is characterised in that also comprise in described reagent: KF gathers
Synthase, Nt.BbvCI nicking enzyme, T4 DNA ligase and Phi29 archaeal dna polymerase.
6. the method for the reagent detection miRNA that a kind utilizes described in claim 4 or 5, it is characterised in that step is as follows:
(1) prepare the miRNA solution of variable concentrations, add hair clip identification probe and assist in identifying probe, constant-temperature incubation；Again
Add KF polymerase, Nt.BbvCI nicking enzyme and dNTPs, carry out strand displacement iodine；
(2) in step (1) reacted solution, add locking-type DNA and T4 DNA ligase, constant-temperature incubation, carry out
Coupled reaction；Add Nt.BbvCI nicking enzyme, Phi29 archaeal dna polymerase and dNTPs, be circulated rolling ring iodine；
(3) in step (2) reacted solution, add NMM and KCl, hatch, fluorescence intensity, build the most glimmering
Linear equation between light intensity and miRNA concentration, to testing sample, can substitute into by measuring the clean fluorescence intensity of sample
Linear equation, calculates the miRNA concentration of sample.
7. method as claimed in claim 6, it is characterised in that in step (1), the condition of described strand displacement iodine
For: 37 DEG C of constant-temperature incubation 0.5h, then make enzyme inactivate in 85 DEG C of heating 20min, terminate reaction.
8. method as claimed in claim 6, it is characterised in that in step (2), the condition of described circulation rolling ring iodine
For: hatch 1.5-4.0h at 37 DEG C, then make enzyme inactivate in 75 DEG C of heating 20min, terminate reaction.
9. method as claimed in claim 6, it is characterised in that in step (1) and step (2), described constant-temperature incubation
Condition is: 37 DEG C of constant-temperature incubation 1h.
10. method as claimed in claim 6, it is characterised in that in step (3), the spectral conditions of fluorescence intensity detection is:
Emission spectrum scope is 550～680nm, and excitation wavelength is 399nm, excites and the slit width launched is 10nm, excite
Voltage is 700V, and selecting fluorescent emission intensity at 612nm is criterion.
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| CN106967794B (en) * | 2017-03-14 | 2020-04-14 | 清华大学深圳研究生院 | Kit and method for detecting miRNA (micro ribonucleic acid) by bidirectional signal amplification |
| CN113564229A (en) * | 2021-07-28 | 2021-10-29 | 中国人民解放军陆军军医大学 | Reagent and method for detecting microRNA (microribonucleic acid) based on click chemical ligation and hairpin stacking assembly |
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| CN113564229B (en) * | 2021-07-28 | 2022-04-01 | 中国人民解放军陆军军医大学 | Reagent and method for detecting microRNA (microribonucleic acid) based on click chemical ligation and hairpin stacking assembly |
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