CN105385770B - The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method - Google Patents
The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method Download PDFInfo
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Abstract
The invention discloses a kind of Dual-ring hairpin probes of detection bleomycin to mediate label-free strand displacement amplification method, and in the presence of bleomycin, Dual-ring hairpin probe is broken at recognition site, and discharges trigger sequence；Then, trigger sequence is combined with the ring portion of signal probe, and under the action of polymerase and nicking enzyme, and strand displacement amplification reaction occurs, finally generates tetra- serobila sequences of a large amount of G.At the same time, primer chain extension opens signal probe, and the tetra- serobila sequences of G that signal probe neck is sealed up for safekeeping are exposed.Finally, tetra- serobila sequences of G bind NMM molecules, generate fluorescence signal, are quantified to bleomycin by the fluorescence signal detected.The present invention by trigger sequence design by, in the neck of Dual-ring hairpin probe, reducing the background signal of detection architecture；Bleomycin is cut and is combined with strand displacement amplification reaction, realizes that the Sensitive Detection to bleomycin, detection are limited to 0.34nM.This method has easy to operate, label-free and specific good advantage.
Description
Technical field
The present invention relates to a kind of detection method of bleomycin more particularly to a kind of Dual-ring hairpin spies of detection bleomycin
Needle mediates label-free strand displacement amplification method.
Background technology
Bleomycin is a kind of glycopeptide antibiotics generated by the secretion of streptomysin wheel branch bacillus.Clinically, bleomycin
A kind of important anticancer drug, be mainly used for treating a variety of diseases such as squamous cell carcinoma and macrophage tumor (R.H.Blum,
S.K.Carter and K.Agre, Cancer, 1973,31,903；M.Froudarakis, E.Hatzimichael,
L.Kyriazopoulou, K.Lagos, P.Pappas, A.G.Tzakos, V.Karavasilis, D.Daliani,
C.Papandreou and E.Briasoulis, Critical Reviews in Oncology/Hematology, 2013,
87,90；Y.Akiyama, Q.Ma, E.Edgar, A.Laikhter and S.Hecht, Journal of the American
Chemical Society, 2008,130,9650；R.A.Giroux and S.M.Hecht, Journal of the
American Chemical Society, 2010,132,16987；Z.Yu, R.Schmaltz, T.Bozeman, R.Paul,
M.Rishel, K.Tsosie and S.Hecht, Journal of the American Chemical Society, 2013,
135,2883.).But using bleomycin treatment kinds cancer, it is often associated with generation dose-limiting toxicity, such as kidney poison
Property, pulmonary toxicity and pulmonary fibrosis etc. (S.Sleijfer, Chest, 2001,120,617；J.Hay, S.Shahzeidi and
G.Laurent, Archives of Toxicology, 1991,65,81.).Therefore, it in order to monitor bleomycin concentration, reduces
Toxicity obtains best therapeutic effect, and how to build a kind of bleomycin detection method becomes the main of analysis worker research
Content.Currently, researchers establish quantitative analysis of a variety of detection methods for bleomycin, such as high performance liquid chromatography
(R.P.Klett, J.P.Chovana and I.H.Danse, Journal of Chromatography:Biomedical
Sciences and Application, 1984,310,361；G.K.Shiu and T.J.Goehl, Journal of
Chromatography B:Biomedical Sciences and Applications, 1980,181,127.), immune point of enzyme
Analysis method (K.Fujiwara, M.Yasuno and T.Kitagawa, Cancer Research, 1981,41,4121), radiation are exempted from
Epidemic disease analytic approach (J.Teale, J.Clough and V.Marks, British Journal of Cancer, 1977,35,822；
A.Broughton and J.E.Strong, Cancer Research, 1976,36,1418.) and microbiological analysis
(T.Ohnuma, J.F.Holland, H.Masuda, J.A.Waligunda and G.A.Goldberg, Cancer, 33,
1230.) etc..But these methods usually have time-consuming, arduously, are detrimental to health and the shortcomings of cost is higher.Therefore,
It needs to establish a kind of new, sensitive, special detection method, be provided for clinic bleomycin concentration monitor a kind of simpler
Easily analytical plan.
It is reported that in oxygen and in the presence of with the metal ion of redox active, bleomycin can pass through oxygen
The method cutting DNA chain of change deoxynucleotide, primary recognition site 5 '-GT-3 ' and 5 '-GC-3 ' (Q.Ma,
Y.Akiyama, Z.D.Xu, K.Konishi and S.M.Hecht, Journal of the American Chemical
Society, 2009,131,2013；J.Chen, M.K.Ghorai, G.Kenney and J.Stubbe, Nucleic Acid
Research, 2008,36,3781.).Based on this property of bleomycin cutting DNA chain, researchers devise various identifications
The DNA molecular probe of bleomycin, to build a kind of bleomycin detection method of simple and sensitive.Currently, these DNA moleculars are visited
Needle is broadly divided into two classes.The first kind is single-stranded or hair clip DNA (B.C.Yin, the D.Wu and that signaling molecule is combined or marked
B.C.Ye, Analytical Chemistry, 2010,82,8272；Y.Li, C.C.Huang, J.B.Zheng, H.L.Qi,
W.Cao and Y.M.Wei, Biosensors and Bioeletronics, 2013,44,177；Y.F.Qin, Y.F.Ma,
X.Jin, L.L.Zhang, G.J.Ye and S.L.Zhao, Analytica Chimica Acta, 2015,866,84；F.Li,
Y.Feng, C.Zhao, P.Li and B.Tang, Chemical Communications, 2012,48,127.).This kind of probe
After being acted on bleomycin, can release signal molecule, and generate the response of corresponding signal.Second class is with trigger sequence
Function neck-circular DNA structure (F.L.Gao, J.P.Lei and H.X.Ju, Chemical Communications, 2013,
49,7561.).This kind of probe can discharge trigger sequence and trigger signal amplified reaction, by one with after bleomycin effect
Secondary fissure solution event is expressed as repeated signal and generates event.Therefore, this kind of DNA molecular probe, which is more advantageous to, improves detection side
The signal strength of method and sensitivity.Gao et al. devises a neckband there are two 5 '-GC-3 ' recognition sites, and ring portion is one section
The function hairpin probe of trigger sequence, for bleomycin highly sensitive detection (F.L.Gao, J.P.Lei and H.X.Ju,
Chemical Communications, 2013,49,7561.).However, since trigger sequence is located at the ring portion of probe, do not cut
Non-specific hybridization between the DNA molecular probe cut and subsequent reactions chain can cause relatively high background signal and vacation
Positive signal, this limits the application of the probe to a certain extent.
Invention content
The purpose of the present invention is exactly to solve the above-mentioned problems, to provide a kind of Dual-ring hairpin probe Jie of detection bleomycin
Lead label-free strand displacement amplification method.
To achieve the goals above, the present invention adopts the following technical scheme that：
A kind of Dual-ring hairpin probe of identification and detection bleomycin, the Dual-ring hairpin probe include：Two ring portions are known
Other site and triggering site, described two ring portions are：Tool is there are one the end-rings of DNA arms and has the neck bulge loop there are two DNA arms,
The recognition site is located at the neck of Dual-ring hairpin, and the trigger sequence expands iodine, the triggering for triggering following
Base in sequence participates in constituting the DNA arms and neck bulge loop of Dual-ring hairpin probe.
A kind of label-free strand displacement amplification method of Dual-ring hairpin probe mediation of detection bleomycin, when object is rich next mould
In the presence of element, Dual-ring hairpin probe is broken at recognition site, and discharges trigger sequence；Then, trigger sequence and signal
The ring portion of probe is combined, and under the action of polymerase and nicking enzyme, and strand displacement amplification reaction occurs, generates a large amount of fluorescence
Dyestuff binding sequence, at the same time, primer chain extension open signal probe, and the fluorescent dye that signal probe neck is sealed up for safekeeping combines
Sequence is exposed, and finally, fluorescent dye binding sequence binds fluorescent molecular, generates fluorescence signal, passes through the fluorescence detected
Signal quantifies bleomycin；The Dual-ring hairpin probe is as described in the appended claim 1.
It is preferred that：The fluorescent dye binding sequence of the signal probe neck is tetra- serobila sequences of G-, and the fluorescent molecular is
NMM。
It is preferred that：The recognition site is 5 '-GTGC-3 '.
It is preferred that：The trigger sequence is 5 '-GAGGAAGAAGAGAGGGAAGGA-3 ' (such as SEQ ID No：Shown in 1).
It is preferred that：The sequence of the Dual-ring hairpin probe is TCC TTC CCT CTC AAA ACC TCG CAC CAA AAG
GTG CGA GGA AGA AGA GAG GGA AGG A(such as SEQ ID No：Shown in 2).
The sequence of fluorescence probe is CCC AAC CCG CCC TAC CCT TTT TGA TCC TTC CCT CTC TTC
TTC CTC CCT CAA AAA GGG TAG GGC GGG TTG GG (such as SEQ ID No：Shown in 3).
It is preferred that：Strand displacement amplification reaction the specific steps are：Take 1 μ L 2mM dNTPs, 1U KF polymerases, 4U
2 μ 10 × NEB of L buffer2 (10mM Tris-HCl, 10mM MgCl of Nt.AlwI, 3 0.75 μM of μ L SP and2, 50mM
NaCl, 1.0mM dithiothreitol, pH 7.9) above-mentioned reaction system is added, it is reacted 40 minutes at 37 DEG C, you can.
It is preferred that：In the amplification method, Dual-ring hairpin probe be 100nM, signal probe 75nM, archaeal dna polymerase 1U,
Nicking restriction endonuclease is 4U, a concentration of 6 μM of NMM.
A kind of kit of detection bleomycin, including Dual-ring hairpin probe and signal probe, the Dual-ring hairpin probe
As described in the appended claim 1；The signal probe is hairpin structure, and the neck of the signal probe has fluorescent dye combination sequence
Row；The trigger sequence of the Dual-ring hairpin probe and the ring portion sequence of signal probe are matched, fluorescent molecular, archaeal dna polymerase, nicking
Restriction endonuclease, 10 × NEB buffer2 (10mM Tris-HCl, 10mM MgCl2, 50mM NaCl, 1.0mM
Dithiothreitol, pH 7.9).
Beneficial effects of the present invention：
The present invention constructs a kind of Dual-ring hairpin probe, is used for the identification and detection of bleomycin.Dual-ring hairpin probe master
To include two regions：Bleomycin 5 '-GTGC-3 ' recognition sites and trigger sequence.In addition, there are two Dual-ring hairpin probe tools
Ring portion, wherein there are two the rings of DNA arms to be known as neck bulge loop for tool.It is different from function hairpin probe in the prior art, Dual-ring hairpin
The trigger sequence of probe seals the neck in probe up for safekeeping with semi-enclosed pattern, effectively prevents non-specific hybridization.Semiclosed mould
Formula, i.e., most of base participates in constituting the DNA arms of neck bulge loop both sides in trigger sequence, and only sub-fraction base constitutes neck bulge loop
(such as the trigger sequence for the Dual-ring hairpin probe used in the embodiment of the present invention is：5’-GAGGAAGAAGAGAGGGAAGGA-
3 ', the only subparticipation neck bulge loop of underscore, remaining constitutes the DNA arms of both sides).There are two protrusion is excellent for this design pattern tool
Gesture.On the one hand, Dual-ring hairpin probe by intramolecular hybridize in the way of silence trigger sequence, effectively avoid non-specific hybridization.
On the other hand, after cleavage reaction occurs for Dual-ring hairpin probe, the double-spiral structure that a centre carries neck bulge loop is generated；Due to
Neck bulge loop reduces double-stranded stability, and trigger sequence is untwisted with its not fully complementary chain, then with enough
Base number and subsequent reactions chain generate stable double helix, finally generate apparent positive signal.Based on this Dual-ring hairpin
Probe, and strand displacement amplification (SDA) is combined to react, the present invention constructs a novel fluorescence sensing platform and is examined for bleomycin
It surveys.This method generates that a large amount of DNA are single-stranded and tetra- serobilas of G-/NMM compounds emit fluorescence by strand displacement amplification reaction, for the first time
Realize the label-free detection of bleomycin based on amplification technique.It is bound by single-stranded DNA product and the signal probe of opening
NMM dye molecules generate the fluorescence signal of enhancing, and this method shows superior sensitivity.The detection of method is limited to 0.34nM,
This method has easy to operate, label-free and specific good advantage.In addition, experiment has carried out the experiment of the human serum rate of recovery,
The result shows that the detection method has larger clinical application potential.
Description of the drawings
Fig. 1 is the BLM detection architecture experimental principle figures based on bicyclic identification probe and strand displacement amplification reaction structure, A.
Bleomycin cuts Dual-ring hairpin probe, discharges trigger sequence, and open signal probe under the action of polymerase, exposes
One section of tetra- serobila sequence of G-, after B. trigger sequences are combined with signal probe, then triggers chain under the action of polymerase and nicking enzyme
Replace amplified reaction；
Fig. 2 (A) is that polyacrylamide gel electrophoresis verifies bleomycin cracking and strand displacement amplification iodine；M:
DNA ladder；(1)BHP；(2)SP；(3)BHP+BLM；(4)BHP+BLM+SP；(5)BHP+BLM+SP+KF polymerase；
(6)BHP+BLM+SP+KF polymerase+Nt.AlwI；(7)BHP+SP+KF polymerase+Nt.AlwI；(B) body is detected
It is fluorescence emission spectrum：Curve a, BHP+SP+KF polymerase+Nt.AlwI+NMM；Curve b, BHP+BLM+SP+KF
polymerase+Nt.AlwI+NMM；
Fluorescence spectrum under Fig. 3 (A) difference bleomycin concentration：Curve a → h is followed successively by 0nM, 2nM, 5nM, 20nM,
100nM, 150nM, 200nM, 220nM；(B) linear relationship between fluorescence response and bleomycin concentration；
Fig. 4 is influence of the different target object to detection architecture fluorescence response.
Specific implementation mode
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Material and instrument Dual-ring hairpin probe (BHP) and signal probe (SP) oligonucleotide sequence are by Sangon
(Shanghai, the China) synthesis of Biotechnology Co., Ltd.s and purifying.Pingyangmycin, mitomycin and daunorubicin purchase
From National Institute for Food and Drugs Control.Actinomycin D purchased from Melone Pharmaceutical Co., Ltd.s (Dalian, in
State).Frerrous chloride (FeCl2 ●H2O) it is purchased from Tianjin recovery fine chemistry industry research institute.Klenow fragment (3 ' -5 ' exo-) are poly-
Synthase and Nt.AlwI are purchased from New England Biolabs Ltd. (Beijing, China).DNTPs is purchased from Thermo Fisher
Scientific Ltd. (China).NMM is purchased from Frontier Scientific Inc. (Utah, USA).Clinical serum sample
It is derived from affiliated hospital of Shandong University.Every other chemical reagent is that analysis is pure.Aqueous solution used uses ultra-pure water in experiment
Configure (resistivity>18.25MΩ●cm)。
15mM phosphate buffers：13.4mM Na2HPO4, 1.6mM NaH2PO4With 50mM NaCl (pH=8.0).
Fluorescence spectrum is to scan acquisition at ambient temperature using luminoscope Hitachi F-2500 (Japan).Excitation wave
Long 399nM, scanning range 550nM-680nM.Excitation and transmite slit width are 10nM.Real-time voltage 700V.
Bleomycin activates and the reaction of Dual-ring hairpin probe cleavage：First, Dual-ring hairpin probe and signal probe difference
It anneals 5 minutes, is slowly cooled to room temperature in 95 DEG C, it is spare；Then, bleomycin mixes with frerrous chloride isoconcentration and in room temperature
Under the conditions of be incubated 30 minutes, the bleomycin activated；Finally, above-mentioned bleomycin and Dual-ring hairpin probe is fully mixed
It closes, is incubated 30 minutes at 37 DEG C so that bleomycin fully cracks Dual-ring hairpin probe.
Strand displacement amplification reaction：After cracking reaction completion, 1 μ L 2mM dNTPs, 1U KF polymerases, 4U are taken
2 μ 10 × NEB of L buffer2 (10mM Tris-HCl, 10mM MgCl of Nt.AlwI, 3 0.75 μM of μ L SP and2, 50mM
NaCl, 1.0mM dithiothreitol, pH 7.9) above-mentioned reaction system is added, it is reacted 40 minutes at 37 DEG C, you can.
Fluorescence measurement：After strand displacement amplification reaction completion, 1.2 μ L, 150 μM of NMM, 4.8 μ L 1M KCl is taken to be added
Reaction system is incubated 30 minutes at 37 DEG C.30 μ L of end reaction acquired solution are taken to be placed in fluorescence pond, according to material and instrument portion
The sweep parameter carries out spectral scan.
Gel electrophoresis：10% polyacrylamide gel electrophoresis verifies the reaction of Dual-ring hairpin probe cleavage and strand displacement amplification
Reaction.In order to clearly observe DNA bands, we use the bleomycin of high concentration, Dual-ring hairpin probe and letter
Number probe is tested.The 10 μ L of solution that end reaction obtains are taken, are sufficiently mixed with 6 × loading buffer, 2 μ L, loading,
Run glue.Finally, obtained DNA gel ethidium bromide (EB) is dyed 5 minutes, after clear water rinses, is placed in ultraviolet imagery system
Lower imaging.
Results and discussion
Experimental principle
As shown in Figure 1, first, bleomycin cracks Dual-ring hairpin probe at recognition site, a new bob is generated
Clamping structure and the intermediate double helix for carrying neck bulge loop.Since neck bulge loop reduces double-stranded stability, double helix solution
Section of DNA sequence is revolved and releases, which is cutting primer strand (Cut-primer, abbreviation CP).Then, primer is cut
Chain is combined with the ring portion of signal probe, and under the action of KF polymerases, is extended by template strand of signal probe, forms one
A double-spiral structure with nicking enzyme Nt.AlwI recognition sites.Then, nicking enzyme nicking and generation one at recognition site
New polymerase binding site point.Then, polymerase extends, and replicates chain release and nicking digestion is carved process cycle and carried out, finally
Produce tetra- serobila sequences of a large amount of single-stranded G-；At the same time, primer chain extension opening signal probe exposes envelope presence signal and visits
The tetra- serobila sequences of G- of needle neck.Finally, tetra- serobila sequences of G- bind NMM, generate detectable fluorescence signal.
Feasibility is verified
In order to verify Dual-ring hairpin probe whether can play a role according to above-mentioned design process and analytical plan can
Row, We conducted the experiments of 10% polyacrylamide gel electrophoresis.As shown in Figure 2 A, compared with swimming lane 1, swimming lane 3 is identical
Position there are dark DNA bands, and there is a new DNA band close to the position of 20bp.This illustrates Dual-ring hairpin
Cracking reaction has occurred in probe.By in the complete solution of signal probe addition bleomycin and Dual-ring hairpin probe reaction, swimming lane 4 goes out
The new DNA bands that a migration velocity is considerably slower than signal probe are showed.This new DNA bands explanation, cracking reaction occur with
Afterwards, CP chains are released, and are combined to form a hybridization double-spiral structure with the ring portion of SP.KF polymerases are added, swimming lane 5 is in 60bp
Position there is a bright DNA band, it is anti-to illustrate that chain extension occurs using SP chains as template under the action of polymerase for CP chains
It answers, generates a more stable DNA double spiral.When reaction system is added in KF polymerases and nicking enzyme simultaneously, swimming lane 6 occurs one
Migration velocity is faster than the DNA bands of 20bp, illustrates that SDA reactions produce a large amount of single strand dna.Swimming lane 7 only has bicyclic hair
The DNA bands for pressing from both sides probe and signal probe illustrate that there is Dual-ring hairpin probe duplexes structure enough stability, system not to send out
Raw SDA reactions.In order to further prove the feasibility of the design of Dual-ring hairpin probe and analytical plan, We conducted corresponding
Fluorescence spectrum scanning experiment.Such as Fig. 2 B, in the absence of object, detection architecture shows low background signal (Curve a).This says
Bright when not having cracking reaction, the CP sequences in Dual-ring hairpin probe are consequently not used for strand displacement amplification reaction and generate signal.
When reaction system is added in object bleomycin, detection architecture shows the response signal (Curve b) significantly improved.This says
After bright cracking reaction occurs, CP sequences are released and are used for the fluorescence signal that subsequent strand displacement amplification reaction generates enhancing.
Result above show the detection scheme be it is feasible, Dual-ring hairpin probe can successfully silence trigger chain, to lower or
Eliminate non-specific hybridization caused background signal in homogeneous analysis.
Experimental condition optimization
In order to obtain best sensing efficiency, test respectively to Dual-ring hairpin concentration and probe concentration, signal probe concentration, enzyme concentration
And the parameters such as NMM concentration are optimized.Final choice Dual-ring hairpin probe is 100nM, signal probe 75nM, KF polymerization
Enzyme is 1U, a concentration of 6 μM of Nt.AlwI 4U, NMM.
Linear and range
Under optimal experiment condition, we have investigated sensitivity and the range of linearity of detection architecture.As shown in figure 3, working as
When bleomycin concentration is gradually increased from 0nM to 220nM, fluorescence response intensity also increases as.Fluorescence intensity and bleomycin
Concentration is linear within the scope of 0nM-220nM (R=0.998).Detection limit 0.34nM.
Specificity
In order to investigate the specificity of method, bleomycin, mitomycin, daunorubicin and D actinomycin D are chosen in experiment
D is investigated respectively as object.The results are shown in Figure 4, and under identical experiment condition, detection architecture is only to bleomycin
Stronger response signal is shown, shows that the detection method has preferable specificity.
The rate of recovery is investigated
In order to investigate the actual application ability of this method, We conducted the experiments of the human serum rate of recovery.Experimental result exists
Between 95%-100%, illustrate that this method has potential clinical application ability.
It summarizes
Based on the strand displacement amplification reaction that Dual-ring hairpin probe mediates, this work constructs a kind of label-free fluorescence strategy
Sensitive Detection for bleomycin.As far as we know, this method realizes the bleomycin based on DNA cloning technology and exempts from for the first time
Label detection.Detection is limited to 0.34nM.The investigation of the human serum rate of recovery meets the requirements, and illustrates this method in Pharmaceutical Analysis and clinic
There is potential actual application ability in.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention
The limitation enclosed, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not
Need to make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (9)
1. a kind of Dual-ring hairpin probe of detection bleomycin mediates label-free strand displacement amplification method, it is characterized in that：Work as target
In the presence of object bleomycin, Dual-ring hairpin probe is broken at recognition site, and discharges trigger sequence；Then, sequence is triggered
Row are combined with the ring portion of signal probe, and under the action of polymerase and nicking enzyme, and strand displacement amplification reaction occurs, and are generated big
The fluorescent dye binding sequence of amount, at the same time, primer chain extension opens signal probe, the fluorescence that signal probe neck is sealed up for safekeeping
Dyestuff binding sequence is exposed, and finally, fluorescent dye binding sequence binds fluorescent molecular, generates fluorescence signal, passes through detection
To fluorescence signal bleomycin is quantified；The Dual-ring hairpin probe includes：Two ring portions, recognition site and trigger bit
Point, described two ring portions are：Tool is there are one the end-rings of DNA arms and has the neck bulge loop there are two DNA arms, the recognition site position
Base in the neck of Dual-ring hairpin, the trigger sequence participates in constituting the neck and neck bulge loop of Dual-ring hairpin probe.
2. the method as described in claim 1, it is characterized in that：The fluorescence binding sequence of the signal probe neck is tetra- serobilas of G-
Sequence, the fluorescent molecular are NMM.
3. the method as described in claim 1, it is characterized in that：The recognition site is 5 '-GTGC-3 '.
4. the method as described in claim 1, it is characterized in that：The trigger sequence such as SEQ ID No：Shown in 1.
5. the method as described in claim 1, it is characterized in that：The sequence of the Dual-ring hairpin probe such as SEQ ID No：Shown in 2.
6. the method as described in claim 1, it is characterized in that：The sequence of fluorescence probe such as SEQ ID No：Shown in 3.
7. the method as described in claim 1, it is characterized in that：Strand displacement amplification reaction the specific steps are：Take 1 μ L, 2 mM
DNTPs, 1 U KF polymerases, 4 U Nt.AlwI, 3 0.75 μM of μ L SP and 2 μ 10 × NEB of L buffer2, described 10
× NEB buffer2：10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1.0 mM dithiothreitol,
pH 7.9；Above-mentioned reaction system is added, is reacted 40 minutes at 37 DEG C, you can.
8. the method as described in claim 1, it is characterized in that：In the amplification method, Dual-ring hairpin probe is 100 nM, signal
Probe is 75 nM, and archaeal dna polymerase 1U, nicking restriction endonuclease is 4U, a concentration of 6 μM of NMM.
9. a kind of kit of detection bleomycin, it is characterized in that：Including Dual-ring hairpin probe and signal probe, the bicyclic hair
Pressing from both sides probe includes：Two ring portions, recognition site and triggering site, described two ring portions are：Tool there are one DNA arms end-rings and
There are two the neck bulge loop of DNA arms, the recognition sites to be located at the neck of Dual-ring hairpin for tool, and the base in the trigger sequence participates in
Constitute the neck and neck bulge loop of Dual-ring hairpin probe；The signal probe is hairpin structure, and the neck of the signal probe has
Fluorescent dye binding sequence；The trigger sequence of the Dual-ring hairpin probe and the ring portion sequence of signal probe are matched, fluorescent molecular,
Archaeal dna polymerase, nicking restriction endonuclease, 10 × NEB buffer2,10 × NEB buffer2：10 mM Tris-HCl, 10
mM MgCl2, 50 mM NaCl, 1.0 mM dithiothreitol, pH 7.9.
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| CN105950757B (en) * | 2016-06-17 | 2019-11-22 | 山东大学 | Method based on the cascade amplification strategy detection bleomycin that Dual-ring hairpin probe and enzyme mediate |
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| CN107151694B (en) * | 2016-09-30 | 2020-05-12 | 山东大学 | Loop-mediated cascade amplification strategy for high-sensitivity detection of DNA methyltransferase activity |
| CN106520764B (en) * | 2016-12-16 | 2019-02-22 | 福州大学 | A kind of nanometer-double-ring aptamers probe and its application |
| CN108342456A (en) * | 2018-02-08 | 2018-07-31 | 中国农业大学 | A kind of visualization of dual heavy metal ion quantifies new detecting method |
| CN117571982B (en) * | 2024-01-09 | 2024-04-09 | 德州学院 | Marker-free fluorescent aptamer sensor for detecting kanamycin with low background and application thereof |
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