CN109097446A - A kind of method and kit detecting miRNA - Google Patents
A kind of method and kit detecting miRNA Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a method of detection miRNA, comprising: S1: providing the DNA molecular machine of specific recognition miRNA, and the DNA molecular machine is a nucleotide sequence of the complementary series comprising tetra- serobila sequence of G-；S2: the DNA molecular machine is mutually distinguishable with sample to be tested；S3: archaeal dna polymerase and nickase are introduced, DNA molecular machine is acted on；S4: introducing thioflavine T, and induction generates tetra- stranded structure of G- and in conjunction with tetra- serobila of G-, generates fluorescence；S5: record fluorescence radiation situation.It is a nucleotide sequence that the DNA molecular, which generates machine, including the region being arranged successively from 3 ' ends to 5 ' ends, it may be assumed that miRNA sequence cog region, stable region, nickase cog region and tetra- serobila of G- synthesize template.The present invention is marked without fluorescence probe, and sensitivity is higher, is operated simpler.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of method and kit for detecting miRNA.
Background technique
MicroRNAs (miRNAs) is the endogenic non-coding with adjusting function of one kind found in eucaryote
RNA, size are about 20~25 nucleotide.Mature miRNAs is by longer primary transcript by a series of nucleases
Shearing and generate, be subsequently assembled into RNA induce silencing complex, target is identified by way of base pair complementarity
MRNA, and silencing complex degradation said target mrna is instructed according to the difference of complementarity or checks the translation of said target mrna.Nearest
The study found that miRNA expression is related to kinds cancer, about 50% miRNAs explained is positioned at and swells in the genome
The relevant fragile site of tumor (fragilesite).It is vital that this illustrates that miRNAs serves in tumour generating process, this
A little miRNAs roles are similar to the function of tumor suppressor gene and oncogene, have researcher to be named as miRNA
"oncomirs".There are the miRNA of some unconventionality expressions in primary carcinoma of liver, their liver cancer that target occur, develop correlation more
Gene and signal path, miRNA express spectra also with liver cancer pathologic type, grade malignancy, by stages, classification etc. clinical pathologies process
Closely related, miRNA can be used as the tool of liver cancer judging prognosis.
At present about the detection of miRNA, used isothermal amplification technology needs to carry out fluorescent marker nucleic acid amplification mostly
Detection, fails to break through that fluorescent dye insertion is analyzed in real time or gel electrophoresis carries out traditional nucleic acid amplification such as end point analysis and examines
Survey method fails truly to realize and detects nucleic acid quick, portablely.
Compare patent 1:
Application No. is CN102618664A, patent names are as follows: a kind of MiRNA detection probe and Visual retrieval MiRNA's
A kind of method, wherein disclosing miRNA detection probe, including three parts: being tetra- serobila formation sequence of G- close to the end 3', centre is
Target-complementary sequence to be measured, the end 5' are interference sequence；The tetra- serobila formation sequence of G- contains 4 sections of GGG sequences and participates in tetra- chain of G-
Body is formed；The target-complementary sequence to be measured is loop ring, with corresponding target miRNA complete complementary；The interference sequence has 5-
10 bases complementary with tetra- serobila formation sequence of G-.It wherein, is in sodium potassium particle and ferroheme in specific detection method
Collective effect under, with the aobvious green colour response of Catalyzed Synthesis By Peroxidase, to realize Visual retrieval, and used probe and
Method reduces the link of complicated fluorescent marker.
Compare patent 2:
Application No. is CN106967794A, patent names are as follows: the kit and method of two-way signaling augmentation detection miRNA,
It is mentioned that kit includes: (1) first hairpin structure DNA probe, the first hairpin structure DNA probe can with it is described
MiRNA base pairing, to open hairpin structure；(2) second hairpin structure DNA probes, the second hairpin structure DNA can
With the first hairpin structure DNA probe described in miRNA competitive binding, to discharge miRNA, also, the second hairpin structure DNA
The DNA complex that probe and the first hairpin structure DNA probe are formed has at least one cohesive end；(3) cyclic DNA is visited
Needle, one section of continuous base in the circular DNA probes is complementary with a cohesive end of DNA complex, also, the ring-type
DNA probe and the DNA complex can obtain the G tetrad amplified production containing more G repetitive sequences by rolling circle amplification.Its
Principle is the introducing for designing double hairpin motif and passing through detectable substance target sequence, and rolling ring is carried out under the action of archaeal dna polymerase and dNTPS
The mode of amplification generates a large amount of G tetrad and is mutually distinguishable with THT fluorescent dye, to realize qualitative and quantitative.But the hair
Bright to need to design double hairpin motif, hairpin structure is easy to happen dislocation pairing during rolling circle amplification, reduces the spirit of detection
Sensitivity.
Summary of the invention
(1) technical problems to be solved
In order to solve the above problem of the prior art, the present invention provides a kind of method for detecting miRNA, will be in sample
MiRNA signal is converted into tetra- serobila of G-, induces richness G genetic fragment to fold to form tetra- serobila of G- by thioflavine T, and thioflavine T
It is embedded in tetra- serobila of G-, forms fluorescence, to realize Visual retrieval, is marked without fluorescence probe, sensitivity is higher, operates simpler
It is single.
The present invention also provides a kind of kits for detecting miRNA.
(2) technical solution
In order to achieve the above object, the main technical schemes that the present invention uses include:
A method of detection miRNA comprising following steps:
S1: providing the DNA molecular machine of specific recognition miRNA, the DNA molecular machine be include that G- tetra- serobila is complementary
The nucleotide sequence of sequence；
S2: the DNA molecular machine is mixed with sample to be tested；
S3: introducing archaeal dna polymerase and nickase, acts on DNA molecular machine, and the new chain synthesis of DNA occurs, forms single-stranded cut
Mouth, constantly removing generate the serial reaction of rich G sequence segment；
S4: introducing thioflavine T, and the rich G sequence segment of induction generates tetra- stranded structure of G- and in conjunction with tetra- serobila of G-, generates glimmering
Light；
S5: record fluorescence radiation situation.
As in a preferred implementation method of the invention, it is successively G- that the DNA molecular machine, which is held from 5 ' ends to 3 ',
Four serobilas synthesize template, nickase cog region, stable region and target sequence complementary region.
As in a preferred implementation method of the invention, the target sequence complementary region and 3 ' termini-complementary of miRNA sequence；
The nickase cog region is the complementary series that nickase can identify sequence；Tetra- serobila of the G- synthesis template is located at core
5 ' ends of nucleotide sequence, tetra- serobila of G- synthesize the complementary series that template is tetra- serobila sequence of G-.
As in a preferred implementation method of the invention, in step S3 by 10~40 μ L of buffer, polymerase 6~
10 μ L, 6~10 μ μ of L and dTP3~10 L of nickase, which are added in the mixed solution in S2, participates in reaction, obtains reaction solution；Step S4
It is middle that 6~10 μ L of thioflavine T, 6~36 μ L of metal cation are added in reaction solution and generate fluorescence reaction.
As in a preferred implementation method of the invention, the temperature of the constant temperature is 45-65 DEG C, proliferation time 15-
30min。
As in a preferred implementation method of the invention, in the S5, first order fluorescence is read every 30s and is recorded, institute
Stating recording mode is, absorption spectrum is taken pictures, records a video or measured to the written description of the shade of comparative solution.
As in a preferred implementation method of the invention, tetra- serobila of the G- synthesis template sequence includes being
5’-…CCCCAAAACCCCAAAACCCCAAAACCCC…-3’。
As in a preferred implementation method of the invention, the base number of the sequence of the target sequence complementary region is 10-
30bp, preferably 12bp, our experiments show that, when the series number of target sequence cog region is 12bp, sensitivity is best.Institute
The miRNA sequence of detection is 5 '-CAATATTACTGTGCTGCTTTA-3 '.
As in a preferred implementation method of the invention, the nickase identification region sequence includes 5 '-GACTC-3 '.
The present invention provides a kind of kit for detecting miRNA, including reagent one, reagent two, reagent three, reagent four and reagent
Five, the reagent one is DNA molecular machine solution, and the reagent two is notch enzyme solutions, and the reagent three is polymerase solution,
The reagent four is thioflavine T solution, and the reagent five is the mixed solution of buffer, dNTP and metal ion.
The principle of the present invention: thioflavin T (ThioflavinT, ThT) is a kind of water-soluble fluorescent dye, due to carbon carbon
The distortion action of key, when individualism, will form vertical dihedral angle structure.This dihedral angle structure does not shine, so ThT is glimmering
Quantum yield is very low.In the presence of metal cation, ThT can induce richness G genetic fragment and fold to form tetra- serobila of G-, while embedding
Enter tetra- serobila of G-, the rotation of intramolecular is suppressed, and the distortion action of carbon-carbon bond is prevented from, and the fluorescence quantum yield of ThT is aobvious
Write enhancing.
(3) beneficial effect
The beneficial effects of the present invention are:
1. tetra- serobila of G- can be realized for converting tetra- serobila of G- for the nucleic acid signal in sample in the present invention at normal temperature
Continuous amplification, draw and amplify the signal height of nucleic acid, stable fluorescence signal is generated by tetra- serobila of thioflavine T combination G-,
To realize highly sensitive nucleic acid fast qualitative analysis utilizing measurement.
2. the comparison patent 1 mentioned in respect to the background art, do not needed in DNA molecular machine provided by the present invention pair
Tetra- serobila of G- synthesizes template sequence and limits, and does not have to design interference sequence, can induce rich G sequence segment by addition thioflavine T
Accurate tetra- serobila of G- is formed, reduces the setting limited tetra- serobila of G- synthesis template and to interference sequence, method is simpler, spirit
Quick property is stronger.
3. the comparison patent 2 mentioned in respect to the background art, DNA molecular machine provided by the invention, target sequence identification
Afterwards, under the system of nickase, polymerase and dNTP, it can be achieved with amplification and generate a large amount of tetra- serobilas of G-, identify and generate with THT
Fluorescence signal, DNA molecular machine sequencing is simple, and the process of amplification is simpler, reduces the error rate of amplification procedure, cost is more
It is low.
4. the continuous amplification of tetra- serobila of G- can be realized in the present invention under isothermal or room temperature, the signal height of nucleic acid is put
Greatly, stable fluorescence signal is generated by tetra- serobila of thioflavine T combination G-, to realize highly sensitive nucleic acid fast qualitative point
Analysis measurement.The novel sensing that a kind of novel sensor constructs high sensitivity and do not need fluorescence probe label can be constructed
Device.
5. the present invention is developed the color using tetra- serobila of thioflavine T combination G-, fluorescence signal can be in simple handheld device (such as hand-held purple
Outer fluorescent tube/torch) with the help of visually observe determination, suitable for a variety of Rapid nucleic acids measurement occasion.
Detailed description of the invention
Fig. 1 is linear DNA molecular machine construction schematic diagram；
Fig. 2 is circular DNA molecule machine construction schematic diagram；
Fig. 3 is the fluorescent strength determining curve graph of the different DNA molecular machines of embodiment 1；
Fig. 4 is the fluorescent strength determining curve graph of the thioflavine T of various concentration in embodiment 2；
Fig. 5 is the fluorescent strength determining curve graph of different standing times in embodiment 3；
Fig. 6 is the DNA molecular machine of various concentration and the fluorescent strength determining curve of sequence concentration to be measured in embodiment 4
Figure；
Fig. 7 is that the fluorescence intensity for the corresponding different sequence concentrations to be measured of DNA molecular machine that concentration is 50nM in embodiment 4 is surveyed
Determine curve graph.
Specific embodiment
In order to preferably explain the present invention, in order to understand, with reference to the accompanying drawing, by specific embodiment, to this hair
It is bright to be described in detail.
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
A method of miRNA is detected, is included the following steps,
S1: providing the DNA molecular machine of specific recognition miRNA, the DNA molecular machine be include that G- tetra- serobila is complementary
The nucleotide sequence of sequence；
S2: the DNA molecular machine is mutually distinguishable with sample to be tested；It, can be with G- if sample to be tested contains miRNA
MiRNA sequence cog region in four serobila molecule machines is mutually distinguishable；
S3: archaeal dna polymerase and nickase are introduced, DNA molecular machine is acted on, contains miRNA sequence in sample to be tested
Under the premise of, it is expanded using DNA molecular machine as matrix polymerization and generates double-strand, at this point, nickase generates on complementary strand generated
Notch；
S4: introducing thioflavine T, can induce and generates tetra- stranded structure of G- and with tetra- serobila of G- ining conjunction with, generation fluorescence；Thioflavine
T can induce the tetra- serobila sequence of G- that tetra- serobila sequence of G- and other same mode generate and be connected with each other, under notch enzyme effect from
It is peeled off in former double-strand, automatic assembling forms tetra- stranded structure of G-, thioflavine T generation fluorescence chimeric with tetra- stranded structure of G-.If to
Sample is free of miRNA, will not cause the fluorescence reaction of DNA amplification reaction and subsequent G- tetra- serobilas and thioflavine in S3；
S5: record fluorescence radiation situation.
Further, the DNA molecular machine is successively that tetra- serobila of G- synthesizes template, nickase is known from 5 ' ends to 3 ' ends
Other area, stable region and target sequence complementary region.Our experiments show that when the base number of target sequence complementary region is designed as 12bp, detection
Sensitivity highest, effect is best.
As illustrated in fig. 1 and 2, DNA molecular machine can be linear or cyclic annular.
Further, the target sequence complementary region and 3 ' termini-complementary of wheat dwarf virus DNA sequence dna；The nickase
Cog region is the complementary series that nickase can identify sequence；Tetra- serobila of the G- synthesis template is located at the 5 ' of nucleotide sequence
End, tetra- serobila of G- synthesize the complementary series that template is tetra- serobila sequence of G-.
MiRNA sequence of the present invention specifically in conjunction with target sequence identification region after, in polymerization, enzyme effect is downward stretches, and
Nickase cog region forms double-strand, duplex structure energy incision enzyme spcificity identification, and generates notch on a chain wherein,
The extending direction of the notch continues using tetra- serobila of G- synthesis template as tetra- serobila sequence of templated synthesis G-, and by another identical life
It is removed at the tetra- serobila sequence of G- of mode, is self-assembly of tetra- stranded structure of G-, thus by specific binding tetra- stranded structure of G-
Thioflavine T molecule combine, issue fluorescence.When specific detection, sample to be tested is replaced with distilled water, as blank assay, detection
Color contrast is done later, and in obvious green fluorescence, testing result is the positive, and qualitative is to contain miRNA.
Tetra- serobila of G- (G-quadruplex) is folded by DNA or RNA rich in tandem sequence repeats guanine (G) in the present invention
The higher structure of formation.G- tetrad (G-quartet) is the structural unit of four serobilas, connects 4 G by Hoogsteen hydrogen bond
Ring plain is formed, two layers or more of tetrad forms four serobilas by pi-pi accumulation.
Thioflavine T (ThioflavinT, ThT) is a kind of water-soluble fluorescent dye, and intrinsic fluorescence signal is very low, but in list
When chain, double-strand or triple strand dna/RNA and tetra- serobila of G- exist simultaneously, thioflavine T (ThioflavinT, ThT) can distinguish difference
Nucleic acid type, to tetra- serobila of G- have very strong structure selectivity, in conjunction with tetra- serobila of G- and generate more stable fluorescence.
In the present invention, nickase is a kind of chain only hydrolyzed in double-stranded DNA, forms " single stranded gaps " on dsDNA
Restrictive slit enzyme, be formed by notch usually at 3 ends with hydroxyl, 5 ends have phosphate radical, can be used as subsequent as replaced
The initiation site of the reactions such as type DNA synthesis, strand displacement amplification.The source nucleic acid nickase Nt.BstNBI that the present invention preferentially selects
In recombinant escherichia coli strain, Vent (exo-) archaeal dna polymerase can be applied to the isothermal duplication of DNA sequence dna.
The base number of the sequence of the target sequence complementary region be 10-30bp, preferably 12bp, our experiments show that, when target sequence
When the series number of column cog region is 12bp, sensitivity is best.The base number of corresponding miRNA sequence to be measured is 10-
30bp, the miRNA sequence less as base number are 5 '-more suitable for the detection in the method for the present invention, such as miRNA sequence to be measured
CCAATATTACTGTGCTGCTTTA-3’。
Preferably, the sequence of tetra- serobila of the G- synthesis template is 5 '-...
CCCCAAAACCCCAAAACCCCAAAACCCC…-3’。
Preferably, the nickase identification region sequence includes 5 '-GACTC-3 ', and stability region sequence is 5 '-TGCATC-3 '
Or 5 '-TTTTTT-3 ' be preferably 5 '-TTTTTT-3 '.
Further, in the S3, dNTP and buffer is added, is placed under constant temperature and expands；In the S4, it is added
Metal cation participates in reaction.Wherein, metal cation is preferably Mg2+。
Further, the temperature of above-mentioned constant temperature is 45-65 DEG C, proliferation time 15-30min.
Further, in the S5, every 30s reads first order fluorescence, and records, and the recording mode is, comparative solution
Absorption spectrum is taken pictures, records a video or measured to the written description of shade.
The present invention also provides a kind of methods for detecting miRNA, include the following steps,
MiRNA sample to be measured is configured to the solution that concentration is 2~8 μM by step 1；
Step 2 takes the DNA molecular machine that 4~8 μ L of solution and 2~4 μ L concentration in step 1 are 0.1~2ug/ml,
It is uniformly mixed, obtains mixed liquor；
Step 3, respectively by the buffer of 10~40 μ L, 6~10 μ L polymerases, 6~10 μ L nickases, 6~10 μ L Sulfurs
The Mg of plain T, the dNTP of 3~10 μ L, 6~36 μ L2+It is added in the mixed liquor of step 2 and is uniformly mixed, obtain reaction solution；
Step 4 observes fluorescence, record detection after standing reaction 20-30 minutes at a temperature of placing reaction liquid into 45-65 DEG C
As a result.Here it can be placed directly in water-bath and keep the constant of temperature.The written description of the shade of comparative solution, take pictures,
Video recording or measurement absorption spectrum.
The dNTP concentration is 0.12~0.50mM；The polymerase concentration is 0.112~0.236U/uL；The notch
The concentration of enzyme is 0.01~0.06U/uL；The concentration of the thioflavine T is 0.1~70uM；The buffer includes that concentration is
Tween20 that 30~70mM and the TrisHCL of PH8.3~9.0, concentration are 0.1%~0.6%, concentration are 130~160mM
KCl, concentration be 10~60mM (NH4)2SO4The NaCL for being 30~70mM with concentration.The concentration of the metal cation is 1
~9mM.
It is 425nm that fluorescence detection, which is in excitation wavelength, and launch wavelength carries out under conditions of being 490nm.
A kind of kit detecting miRNA, including reagent one, reagent two, reagent three, reagent four and reagent five,
Reagent one is that DNA molecular generates machine solution, and reagent two is notch enzyme solutions, and reagent three is polymerase solution, institute
Stating reagent four is thioflavine T solution, and reagent five is the mixed solution of buffer, dNTP, metal cation.Reagent one, reagent two,
Reagent three, the solution concentration of reagent four and reagent five and volume with it is above-mentioned it is a kind of detect miRNA method in corresponding numerical value phase
Together.
Kit is used as sample sets in use, only need to be uniformly mixed reagent one with sample to be tested, sequentially adds reagent
Three, reagent two and reagent five stand reaction 20-30 minutes, while distilled water are substituted sample to be tested at a temperature of being placed in 45-63 DEG C
It is mixed with reagent one as blank control group, the same sample sets of subsequent step.After standing reaction terminates, by sample sets and blank pair
According to group, control color, if sample sets are in apparent green fluorescence relative to blank control group, the result judgement of sample to be tested is
The positive, i.e., qualitative is to contain miRNA.
The embodiment of the present invention:
1. experimental instruments used in the embodiment of the present invention
Testing the instrument used mainly has: Thermo centrifuge, PCR instrument (Thermo), and the macro thermostat water bath of essence is pure
Water instrument (ELGA-purelab option), liquid-transfering gun, superclean bench, pH meter (MettlerToledo) ultraviolet specrophotometer
(Thermo ND-1000V1.6) etc..
2 test reagents
Thioflavine T (ThT), magnesium sulfate (MgSO4), distilled water (DD H2O)、Isothermal Amplification
Buffer II and NEB buffering.
3. nickase, polymerase
DNTP is purchased from Lai Feng Biotechnology Co., Ltd (Shanghai).
NBstNBI nickase is purchased from NBI company
BstPol polymerase is purchased from Sheng Gong company
Embodiment 1:
The method for detecting liver cancer miRNA,
Step 1: synthetic dna molecule machine, and it is configured to the solution that concentration is 20nM,
Step 2: crushing rat liver cancer tissue at room temperature, and juice is extracted in filtering, and it is molten to be configured to the sample that concentration is 2 μM
Liquid,
Step 3: taking the sample solution of 4 μ L and the DNA molecular machine solution of 2 μ L, and micro oscillation is uniformly mixed, is mixed
Solution,
Step 4: successively respectively by the buffer of 10 μ L, 6 μ L polymerases, 6 μ L nickases, 6 μ L thioflavine Ts, 6 μ LdNTP,
6μLMg2+It is added into the obtained mixed solution of step 3, obtains reaction solution.
Wherein, above-mentioned dNTP concentration is 0.12mM；Polymerase concentration is 0.112U/uL；The concentration of nickase is 0.01U/
uL；The concentration of thioflavine T is 0.1uM；Buffer includes that concentration is 30mM, and the TrisHCL of PH8.3, concentration are 0.1%
(NH4) that KCl that tween20, concentration are 130mM, concentration are 10mM2SO4, concentration be 30mM NaCL；Mg2+Concentration be
1mM。
Step 5: expanding 15min under placing reaction liquid into the constant temperature that temperature is 45 DEG C.
Step 6: absorption spectrum is taken pictures, records a video or is measured in the shade of comparative solution, clerking.
When being detected using the present embodiment method, the solution in step 1 is replaced with into distilled water, other conditions are identical
Blank detection comparison is done, sample detection result is that sample is in obvious green fluorescence, detection knot with distilled water control test result
Fruit is the positive, i.e., it is to contain liver cancer miRNA that testing result is qualitative.
Experimental result:
This experiment is that validating DNA molecule machine measures the feasibility of nucleic acid and the selection of optimal sequence, preferably to test
Demonstrate,prove DNA molecular machine, this experiment using simple miRNA sequence CAATATTACTGTGCTGCTTTA as determined nucleic acid sequence, and
The synthesis of student on commission's work company.
The three DNA molecular machine sequencings verified are respectively
G4M-1:5'-CCCCAAAACCCCAAAACCCCAAAACCCCTGCATTCGACTCTAAAGCAGCACA-3'；
G4M-2:5'-CCCCAAAACCCCAAAACCCCAAAACCCCTTTTTTGACTCTAAAGCAG-3'；
G4M-3:5'-CCCCAAAACCCCAAAACCCCAAAACCCCTTTTTTGACTCTAAAGCAGCACA-3'。
Under the premise of other reaction conditions and all identical reagent, while three groups of detection reaction experiments are carried out, testing result,
So that it is determined that most suitably DNA molecular life device, and blank test is done respectively, blank test is designed as sequence to be measured and replaces with
Pure water.
Experimental result as shown in figure 3,
Trigger0: DNA molecular machine be added is represented as G4M-1, G4M-2, G4M-3, the sample of detection is pure water
Three curves, since three curve difference are small, three are essentially coincided, and are shown as a curve in Fig. 3, unified to mark
Trigger0 is denoted as,
Trigger1: representing DNA molecular machine be added as the fluorescence curve of G4M-1,
Trigger2: representing DNA molecular machine be added as the fluorescence curve of G4M-2,
Trigger3: DNA molecular machine be added is represented as the fluorescence curve of G4M-3.
As can be seen from Figure 3 plus G4M-3 group wave crest highest at 490nm wavelength, other each group wave crests are close.The above results
Show that target sequence can generate a large amount of DNA structures with G4M-3, generates notch, the nucleic acid sequence rich in G, which falls off, generates tetra- serobila of G-
Structure, tetra- serobila of G- significantly increase fluorescence intensity, thus prove in conjunction with ThT, and G4M-3 detects core as DNA molecular machine
Acid, feasibility are strong.
Embodiment 2:
Step 1: synthetic dna molecule machine, and it is configured to the solution that concentration is 60nM,
Step 2: crushing rat liver cancer tissue at room temperature, and juice is extracted in filtering, and it is molten to be configured to the sample that concentration is 8 μM
Liquid,
Step 3: taking the sample solution of 8 μ L and the DNA molecular machine solution of 4 μ L, and micro oscillation is uniformly mixed, is mixed
Solution,
Step 4: successively respectively by the buffer of 40 μ L, 10 μ L polymerases, 10 μ L nickases, 10 μ L thioflavine Ts, 10 μ
LdNTP、10μLMg2+It is added into the obtained mixed solution of step 3, obtains reaction solution.
Wherein, above-mentioned dNTP concentration is 0.50mM；Polymerase concentration is 0.236U/uL；The concentration of nickase is 0.06U/
uL；Buffer includes that concentration is 70mM, and tween20 that the TrisHCL of PH9.0, concentration are 0.6%, concentration are 160mM's
KCl, (NH4) that concentration is 60mM2SO4, concentration be 70mM NaCL；Mg2+Concentration be 6mM.
Step 5: expanding 30min under placing reaction liquid into the constant temperature that temperature is 55 DEG C.
Step 6: absorption spectrum is taken pictures, records a video or is measured in the shade of comparative solution, clerking.
When being detected using the present embodiment method, the solution in step 1 is replaced with into distilled water, other conditions are identical
Blank detection comparison is done, sample detection result is that sample is in obvious green fluorescence, detection knot with distilled water control test result
Fruit is the positive, i.e., it is to contain miRNA that testing result is qualitative.
Experimental result:
This experiment is the optimum addition of preferred key substance thioflavine T hT of the present invention, and verifies other reaction systems and exist
Feasibility under additive amount and concentration levels described in the present embodiment, as the method for the present invention detection nucleic acid.
Wherein, DNA molecular machine is designed as G4M-3 (particular sequence is as described in Example 1) in this experiment, other reaction items
Part is as described in Example 2, this experiment using simple miRNA sequence CAATATTACTGTGCTGCTTTA as determined nucleic acid sequence,
And student on commission's work company synthesizes.
Thioflavine T is single factor test, and concentration separately designs as 0.1uM, 1uM, 5uM, 10uM, 20uM, 40uM.Every group of detection it
Afterwards, fluorescent strength determining is carried out after placing 3 hours.
As shown in figure 4, occurring wave crest at 490nm, fluorescence intensity is most strong at this time.It can be concluded that when ThT concentration is 20uM
Fluorescence intensity is maximum at wave crest, and intensity is up to 25000a.u or more very much, so the optimal addition concentration of ThT is 20uM,
Also the method for provable the present embodiment has the stronger feasibility of measurement nucleic acid sequence.
Embodiment 3:
Step 1: synthetic dna molecule machine, and it is configured to the solution that concentration is 100nM,
Step 2: crushing rat liver cancer tissue at room temperature, and juice is extracted in filtering, and it is molten to be configured to the sample that concentration is 2 μM
Liquid,
Step 3: taking the sample solution of 4 μ L and the DNA molecular machine solution of 4 μ L, and micro oscillation is uniformly mixed, is mixed
Solution,
Step 4: successively respectively by the buffer of 20 μ L, 6 μ L polymerases, 7 μ L nickases, 10 μ L thioflavine Ts, 3 μ
LdNTP、36μLMg2+It is added into the obtained mixed solution of step 3, obtains reaction solution.
Wherein, above-mentioned dNTP concentration is 0.3mM；Polymerase concentration is 0.212U/uL；The concentration of nickase is 0.03U/
uL；The concentration of thioflavine T is 0.1uM；Buffer includes that concentration is 30mM, and the TrisHCL of PH8.5, concentration are 0.4%
(NH4) that KCl that tween20, concentration are 145mM, concentration are 45mM2SO4, concentration be 46mM NaCL；Mg2+Concentration be
9mM。
Step 5: expanding 30min under placing reaction liquid into the constant temperature that temperature is 56 DEG C.
Step 6: absorption spectrum is taken pictures, records a video or is measured in the shade of comparative solution, clerking.Using this reality
When applying a method and being detected, the solution in step 1 is replaced with into distilled water, other conditions are identical to do blank detection comparison, sample
Product testing result is that for sample in obvious green fluorescence, testing result is the positive, i.e. detection knot with distilled water control test result
Qualitative fruit is to contain virus.If a period of time is placed after taking-up at room temperature, is swept with quantitatively come qualitative by fluorescence spectrometry
The fluorescence spectrum under 425nm excitation wavelength is retouched, launch wavelength range is 455nm-695nm.
Experimental result:
This experiment is to verify the feasibility of the present embodiment, and after the reaction, it is long to explore the time being placed at room temperature for
The short influence to testing result.
Wherein, DNA molecular machine is designed as G4M-3 (particular sequence is as described in Example 1) in this experiment, other reaction items
Part is as described in Example 3, this experiment using simple miRNA sequence CAATATTACTGTGCTGCTTTA as determined nucleic acid sequence,
And student on commission's work company synthesizes, and when testing, determined nucleic acid sequence is configured to the solution that concentration is 0.1uM and replaces with this reality
The sample solution in example is applied, this is the estimation carried out according to 2 μM in the present embodiment of sample solution.When after reaction, take out
Different time (0h, 1h, 2h, 18h) is placed at room temperature and does four groups of experiment of single factor, is scanned under 425nm excitation wavelength respectively
Fluorescence spectrum, launch wavelength range is 455nm-695nm.
Experimental result as shown in figure 5, comparison different standing times do the fluorescence curve of detection response sample, in 1h, 2h and
Response sample fluorescence curve wave crest is significantly higher than the wave crest of 0h when 18h, therefore, after reaction, when being placed at room temperature for certain
Between the sensitivity of testing result can be improved.The micro- crest height greater than 1h and 2h of the crest height of 18h, the crest height of 1h and 2h
Difference is negligible, and the overlong time of placement will not generate more effective influence for the sensitivity of testing result, to sum up, inspection
It surveys after reaction, is placed at room temperature for the sensitivity that 1h is remarkably improved result, but the time placed is not because too long.
Embodiment 4:
Step 1: synthetic dna molecule machine,
Step 2: crushing rat liver cancer tissue at room temperature, and juice is extracted in filtering, is configured to the sample that concentration is 2~8 μM
Solution,
Step 3: taking the sample solution of 4~8 μ L and the DNA molecular machine solution of 2~4 μ L, and micro oscillation is uniformly mixed, obtains
To mixed solution,
Step 4: successively respectively by the buffer of 25 μ L, 7 μ L polymerases, 9 μ L nickases, 6 μ L thioflavine Ts, 8 μ LdNTP,
30μLMg2+It is added into the obtained mixed solution of step 3, obtains reaction solution.
Wherein, above-mentioned dNTP concentration is 0.26mM；Polymerase concentration is 0.120U/uL；The concentration of nickase is 0.02U/
uL；The concentration of thioflavine T is 0.1uM；Buffer includes that concentration is 60mM, and the TrisHCL of PH8.4, concentration are 0.3%
(NH4) that KCl that tween20, concentration are 150mM, concentration are 45mM2SO4, concentration be 66mM NaCL；Mg2+Concentration be
5mM。
Step 5: expanding 18min under placing reaction liquid into the constant temperature that temperature is 48 DEG C.
Step 6: absorption spectrum is taken pictures, records a video or is measured in the shade of comparative solution, clerking.
When being detected using the present embodiment method, the solution in step 1 is replaced with into distilled water, other conditions are identical
Blank detection comparison is done, sample detection result is that sample is in obvious green fluorescence, detection knot with distilled water control test result
Fruit is the positive, i.e., it is to contain virus that testing result is qualitative.
This experiment is to verify the feasibility of the present embodiment, and verify sensitivity of the invention.
DNA molecular machine is designed as G4M-3 (particular sequence is as described in Example 1) in this experiment, other reaction conditions are such as
Described in embodiment 4, this experiment is entrusted using simple miRNA sequence CAATATTACTGTGCTGCTTTA as determined nucleic acid sequence
The synthesis of reincarnation work company.
The present embodiment tests (one), in the case where other parameters are constant, with the concentration of DNA molecular machine and sequence to be measured
Concentration do dual factors experiment, the concentration of DNA molecular machine is respectively set to 10nM, 50nM, 100nM, sequence to be measured it is dense
Degree is respectively set to 10nM, 1nM, 100pM, 10pM, 1pM, 100fM, 10fM, 0,24 groups of experiments is designed altogether, at wavelength 490nm
The fluorescence intensity of every group of measurement result is measured respectively, experimental result is as shown in Figure 6, wherein horizontal axis coordinate indicates sequence to be measured
Various concentration, ordinate indicate fluorescence intensity, and each line chart respectively indicates different DNA molecular machine concentration.
It can be obtained from Fig. 6, when DNA molecular machine concentration is 50nM, sensitivity big with sequence curve amplitude of variation to be measured
It is high.
The present embodiment tests (two) in the case where other parameters are constant, and concentration is the DNA molecular machine of 50nM, with to be measured
Sequence concentration does the experiment of single factor of fluorescence spectrum, concentration be respectively set to 10nM, 1nM, 100pM, 10pM, 1pM, 100fM,
10fM, 0, experimental result as shown in fig. 7, with sequence concentration to be measured continuous increase, the fluorescence intensity at 485nm constantly increases
Greatly, therefore sequence concentration to be measured is higher, and sensitivity is higher, and fluorescence intensity change at low concentrations is small, and sensitivity is not as good as height
The height of concentration.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (10)
1. it is a kind of detect miRNA method, which is characterized in that itself the following steps are included:
S1: providing the DNA molecular machine of specific recognition miRNA, and the DNA molecular machine is to include the mutual of tetra- serobila sequence of G-
One nucleotide sequence of complementary series；
S2: the DNA molecular machine is mixed with sample to be tested；
S3: introducing archaeal dna polymerase and nickase, acts on DNA molecular machine, occur the new chain synthesis of DNA, formed single-stranded nick,
The constantly serial reaction of the rich G sequence segment of removing generation；
S4: introducing thioflavine T, and the rich G sequence segment of induction generates tetra- stranded structure of G- and in conjunction with tetra- serobila of G-, generates fluorescence；
S5: record fluorescence radiation situation.
2. the method for detection miRNA as described in claim 1, it is characterised in that: the DNA molecular machine is held from 5 ' to 3 '
End is successively tetra- serobila of G- synthesis template, nickase cog region, stable region and target sequence complementary region.
3. the method for detection miRNA as claimed in claim 2, it is characterised in that: the target sequence complementary region and miRNA sequence
3 ' termini-complementaries；The nickase cog region is the complementary series that nickase can identify sequence；Tetra- serobila of G- closes
It is located at 5 ' ends of nucleotide sequence at template, and tetra- serobila of G- synthesis template is the complementary series of tetra- serobila sequence of G-.
4. the method for detection miRNA as described in claim 1, it is characterised in that: in the S3, dNTP and buffer is added,
It is placed under constant temperature and expands；In the S4, metal cation is added and participates in reaction.
5. the method for detection miRNA as claimed in claim 4, it is characterised in that: the temperature of the constant temperature is 45-65 DEG C, is expanded
The increasing time is 15-30min.
6. the method for detection miRNA as described in claim 1, it is characterised in that: in the S5, read every 30s primary glimmering
Light simultaneously records；The recording mode is that absorption light is taken pictures, records a video or measured to the written description of the shade of comparative solution
Spectrum.
7. the method for detection miRNA as described in claim 1, it is characterised in that: tetra- serobila of G- synthesizes template sequence packet
The CCCCAAAACCCCAAAACCCCAAAACCCC ... -3 ' that includes 5 '-....
8. the method for detection miRNA as claimed in claim 2, it is characterised in that: the base number of the target sequence complementary region is
10-30bp。
9. the method for detection miRNA as claimed in claim 2, it is characterised in that: the sequence of the nickase cog region includes
5’-GACTC-3’。
10. a kind of kit for detecting miRNA, it is characterised in that: including reagent one, reagent two, reagent three, reagent four and reagent
Five, the reagent one is DNA molecular machine solution, and the reagent two is notch enzyme solutions, and the reagent three is polymerase solution,
The reagent four is thioflavine T solution, and the reagent five is the mixed solution of buffer, dNTP and metal ion.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110229872A (en) * | 2019-06-14 | 2019-09-13 | 中国科学院化学研究所 | A kind of detection method of the visualization identification microRNA to be untwisted based on tetra- serobila probe structure of G- |
| CN112080552A (en) * | 2020-09-16 | 2020-12-15 | 清华大学深圳国际研究生院 | Method for detecting target miRNA based on G quadruplex molecular beacon double-enzyme cascade isothermal amplification |
| CN112080552B (en) * | 2020-09-16 | 2023-04-07 | 清华大学深圳国际研究生院 | Method for detecting target miRNA based on G quadruplex molecular beacon double-enzyme cascade isothermal amplification |
| CN113337627A (en) * | 2021-06-03 | 2021-09-03 | 浙江省农业科学院 | Label-free visual detection method for vibrio parahaemolyticus gene based on CRISPR/Cas12a |
| CN113549678A (en) * | 2021-08-09 | 2021-10-26 | 四川大学 | Method and kit for detecting biological molecules based on split G-quadruplex nuclease by fluorescence or naked eyes |
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