CN109957607A - A kind of detection probe and its application - Google Patents
A kind of detection probe and its application Download PDFInfo
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- CN109957607A CN109957607A CN201711338526.0A CN201711338526A CN109957607A CN 109957607 A CN109957607 A CN 109957607A CN 201711338526 A CN201711338526 A CN 201711338526A CN 109957607 A CN109957607 A CN 109957607A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
Abstract
The present invention provides a kind of detection probe and its application, the probe includes the probe A for being marked with luciferase and the probe B for being marked with quantum dot；Wherein, there are the sequences of partial complementarity for the other end of one end partial sequence complementarity of one end of the probe A and target nucleic acid, one end of the probe B and the other end partial sequence complementarity of target nucleic acid, the other end of the probe A and probe B；The probe A, probe B and target nucleic acid hybridize to form stable T-type structure, and the luciferase and quantum dot can be mutually paired generation bioluminescence resonance energy transfer；Probe and nucleic acid are dexterously formed stable T structure by design specific probe by the present invention, using the principle of bioluminescence energy resonance transfer, optimize reaction condition, single base mutation can be precisely distinguished, effectively detection target nucleic acid, it is easy to operate, required time is short, convenient and efficient, and cost is relatively low.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of detection probe and its applications.
Background technique
Bioluminescence resonance energy transfer (bioluminescence resonance energy transfer, BRET)
It is a kind of Nonradiative energy transfer occurred between bioluminescent protein such as luciferase (donor) and fluorescent material (receptor).By
It is not required to that outer source excitation, background be low, high sensitivity in BRET, as the optical ruler of slight distance in protein-interacting, living body
The bioanalysis related fieldss such as image, foranalysis of nucleic acids, protease detection and high flux screening are widely used.BRET skill
Art is based on (such as renilla luciferase) the Resonance energy transfer phenomenon in certain marine animal bodies and to lead to energy donor and energy
Measure the energy transfer of on-radiation between receptor.Energy donor is luminous luciferase in BRET, in the presence of corresponding substrate
Emit the spectrum of respective wavelength, the receptor of energy is usually fluorescin or fluorescent material (small molecule dyes or nanometer material
Material), as long as being absorbed into energy in its spectral region that can be excited, optical signal can be emitted in long wave strong point.What BRET was formed
One of key is that donor emission and acceptor absorbance spectrum must be overlapped, and energy transfer could occur.Substrate is different, can quilt
The wave-length coverage of excitation is different, and selected acceptor material is also required to accordingly adjust.
Quantum dot (Quantum dot, QD) is a kind of semiconductor nano material, because it is with high quantum production rate, can be readjusted the distribution
Ejected wave is long, the narrow multicomponent fluorescence detection suitable for biomarker of emission peak, the time needed for can shortening analysis, saves detection
Reagent reduces analysis cost, becomes the important front edge of biological sensor development field.QD can be excited with single light source,
Other fluorescent dyes (such as fluorescein isothiocynate (FITC), rhodamine (Rhodamine)) are different from, it is glimmering greatly to simplify polychrome
Dependence of the light detection to various light sources.QD can also be completely without being bound by external excitation light source.In the detection that the invention is established
In mode, QD obtains energy in a manner of energy transfer of shaking from fixed luciferase (donor) on the surface thereof by the identity of receptor
It measures and shines.Bioluminescence and the important difference of general fluorescence detection method are that it does not need excitation light source.They
The oxidation process to shine dependent on fluorescein, bioluminescence energy is transferred to the quantum dot in a manner of non-radiative resonance makes its hair
Light.Luciferase catalysis substrate in the environment of having oxygen aoxidizes, it is made to shine.Luciferase can be considered the connected egg of two parts
White matter segment composition.This two parts segment has characteristic spectral emission identical with complete luciferase, but its respective is urged
Change luminous intensity and there was only the 1/50 and 1/100 of complete luciferase respectively.The difference of this catalytic luminescence ability is with luciferase
Fragment complementation controls bioluminescence and in the case where keeping low background radiation to the important foundation of quantum dot energy transfer.
The method of detection Microrna mainly has Northern engram analysis, microarray analysis and real time fluorescent quantitative at present
Polymerase chain reaction.Northern engram analysis is the common method based on hybridization check RNA, it is earliest for small
One of several method of RNA analysis, this method is simple and easy, and most of laboratory can be carried out operating, and does not need additional
Capital investment and equipment update.Microarray analysis is also based on the principle of hybridization to detect Microrna, it is specific by measuring
The expression of Microrna in the process understands the expression and regulation mechanism of Microrna and by the base of micro RNA regulation and control to analyze
The expression of cause.Microarray is hybridized using highdensity fluorescence labeling probe with RNA sample, obtains expression figure by fluorescent scanning
Spectrum carries out the expression analysis of miRNA by corresponding software.Due to may include all available miRNA sequences when designing probe,
Therefore microarray can accomplish high-throughput miRNA analysis.Fluorescent quantitative PCR passes through amplification technique, is
Fluorophor is added in polymerase chain reaction system in finger, accumulates the entire reaction process of real-time monitoring using fluorescence signal, most
The method that quantitative analysis is carried out to unknown template by standard curve afterwards.The copy number of initial target nucleic acid is higher, faster sight
Observe dramatically increasing for fluorescence intensity.
CN105807064A provides a kind of luciferase complementation quanta point biological sensor and its construction method and its answers
With the luciferase complementation quanta point biological sensor includes quantum dot, luciferase n terminal fragment, luciferase carboxyl
End fragment, can specific recognition determinand probe and can with the luciferase occur bioluminescence reaction substrate；The invention
The optical advantage of incorporating quantum point sensor, by realizing luciferase n terminal fragment and carboxyl-terminal fragment in quantum dot surface
Induction it is complementary, rebuild catalysis, building obtains new type of high sensitivity biosensor, can be applied to a variety of biomarkers
High targeting detection, and be suitable in homogeneous system to the accurate quantitative analysis of target detection thing, but this method step is complicated, it is time-consuming
It is long.
In conclusion the national concerns to health of the high speed development significant increase of national economy, to obtain high standard
The low cost that health is realized while healthy, need to develop novel detection kit based on bioluminescence resonance energy transfer and
Its detection method, to realize the purpose of efficient detection nucleic acid.
Summary of the invention
In view of the deficiencies of the prior art and actual demand, the present invention provides a kind of detection probe and its application, the present invention
Two probes of the specificity of luciferase and quantum dot are respectively provided with according to target nucleic acid design, dexterously by probe and target
Nucleic acid forms T-type structure, using the principle of bioluminescence energy resonance transfer, optimizes reaction condition, accurate efficient detection target
Nucleic acid saves time and cost, has important application value.
To achieve this purpose, the present invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of detection probe, is designed based on target nucleic acid, and the probe includes being marked with
The probe A of the luciferase and probe B for being marked with quantum dot；
Wherein, one end partial sequence complementarity of one end of the probe A and target nucleic acid, one end of the probe B and mesh
Mark the other end partial sequence complementarity of nucleic acid, there are the sequences of partial complementarity for the other end of the other end of the probe A and probe B
Column；
The probe A, probe B and target nucleic acid hybridize to form stable T-type structure, the luciferase and quantum dot energy
It is mutually paired generation bioluminescence resonance energy transfer.
In the present invention, inventor fully understands bioluminescence energy resonance transfer during long-term experiment is practiced
It is combined with the method for PCR detection nucleic acid, gropes to probe into and verify by many experiments, dexterously set by principle and advantage
Meter is respectively provided with two specific probes of luciferase and quantum dot, and stable T-type structure can be hybridized to target nucleic acid,
So that bioluminescence energy resonance transfer occurs for luciferase and quantum dot, optimize reaction condition, the adjustment reaction temperature time and
Concentration, accurate efficient detection nucleic acid can distinguish single base mutation, save the time, and it is convenient to operate.
Bioluminescence resonance energy transfer technology (BRET) is based on (such as sea pansy fluorescein in certain marine animal bodies
Enzyme) Resonance energy transfer phenomenon and lead to the energy transfer of on-radiation between energy donor and energy acceptor.Energy in BRET
Donor is luminous luciferase, the spectrum of respective wavelength is emitted in the presence of corresponding substrate, the receptor of energy is usually fluorescence
Albumen or fluorescent material (small molecule dyes or nano material), as long as being absorbed into energy in its spectral region that can be excited,
Optical signal can be emitted in long wave strong point.One of the key that BRET is formed, is that donor emission and acceptor absorbance spectrum are necessary
Overlapping, energy transfer could occur.Substrate is different, and the wave-length coverage that can be excited is different, and selected acceptor material is also required to phase
It should adjust.
Preferably, the luciferase includes Gluc, renilla luciferase, extra large firefly luciferase or firefly
Any one in fireworm luciferase, preferably Gluc.
Preferably, the launch wavelength of the luciferase be 400-600nm, such as can be 400nm, 430nm, 460nm,
480nm, 500nm, 530nm or 580nm, preferably 450-550nm.
Gluc is the luciferase found the latest, and molecular weight 19.9kDa is the minimum found so far
Luciferase (Fluc:62kDa；Rluc:36kDa), there is superelevation compared with firefly luciferase and renilla luciferase
Luminous efficiency (1000 times), high thermal stability (95 DEG C of processing are still able to maintain 65% bioluminescence ability for 30 minutes).
Gluc is catalyzed the oxidation of cavity plain (Colenterazine) in the environment of having oxygen, generates its oxidation product
And carbon dioxide and shine, luminescence peak at 480nm (400-600nm wide), be well suited for with 655nm launch wavelength amount
Son point composition BRET pairing.
Preferably, the quantum dot includes CdSe/ZnS quantum dot, CdTe quantum, CdTe/CdS quantum dot, CdTe/
Any one in CdS/ZnS quantum dot, ZnTe quantum dot, InP/ZnS quantum dot or ZnSe quantum dot.
Preferably, the launch wavelength of the quantum dot be 400-750nm, such as can be 400nm, 430nm, 460nm,
500nm, 550nm, 600nm, 655nm, 680nm, 700nm or 750nm, preferably 600-700nm.
Preferably, the distance of the luciferase and quantum dot be 5-10nm, such as can be 5nm, 6nm, 7nm, 8nm,
9nm or 10nm.
Preferably, the luciferase is connected by SMCC activation with the sulfydryl end of the probe A.
The SMCC is succinimide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters.
Preferably, the quantum dot is connected by Streptavidin modification with the biotin labeling end of the probe B.
Preferably, the length of the partial complementarity sequence of the probe A and probe B is 4-9 base, for example, can be 4,
5,6,7,8 or 9, preferably 5-8 base.
In the present invention, when target nucleic acid is not present, probe A and probe B form unstable double-strand, reach 37 in temperature
DEG C when can dissociate, therefore be not present Resonance energy transfer phenomenon, quantum dot will not in the case where no excitation light source
Be excited, but when the nonvolatile memory between probe A and probe B is greater than 9 bases, can in the presence of being not necessarily to object also shape
At double-strand is stablized, experimental result is influenced.
In the present invention, two nucleic acid probes are designed according to target nucleic acid, mark fluorescent element enzyme and 655 quantum dots are (glimmering respectively
Light element enzyme is tagged to above the nucleic acid for having had sulfydryl by SMCC activation, and the quantum dot-labeled to biotin of SA modification is selected
Above the nucleic acid of label), wherein the emission spectrum of luciferase can excite 655nm quantum dot, target RNA there are the case where
Under, can hybridize to form a stable T-type hybrid structure with three, cause the distance between luciferase and quantum dot shorten from
And fluorescence resonance energy transfer can occur, under substrate-function, luciferase catalysis substrate oxidation generate its oxidation product and
Carbon dioxide and luminescence emission wavelength are 480nm, produce the emission peak of quantum dot 655nm under excitation, energy transfer efficiency with
Target concentration is related, is worth in response with the fluorescence signal ratio of QD655/Gluc480, carries out quantitative detection to object.
And in the case where target RNA is not present, two nucleic acid probes only have 7 base pair complementarities, unstable double-strand is formed,
Temperature can dissociate when reaching 37 DEG C, therefore Resonance energy transfer phenomenon is not present, and measure in the case where no excitation light source
Son point will not be excited.Wherein, it is selected after being optimized to the buffer solution ion concentration in reaction time and reaction system
Half an hour magnesium ion concentration 10mM is optimum condition.This method can effectively distinguish the similar RNA family of height, even if there are two only
Base changes, and can be used to detect DNA, and can detect the single base mutation of DNA.
Second aspect, the present invention provide a kind of kit, include detection probe as described in relation to the first aspect.
Preferably, the kit further includes luciferase catalysis substrate, magnesium ion solution and Tris-HCl buffer；
The concentration of the Tris-HCl buffer is 50mM, pH=7.4.
The third aspect, it is biological for detecting correlation RNA in blood that the present invention provides one kind kit as described in second aspect
Marker.
Fourth aspect, the present invention provides a kind of method for detecting nucleic acid, using the kit as described in second aspect, including
Following steps:
(1) probe A, probe B and luciferase substrate are added in magnesium ion solution and obtain mixed liquor；
(2) mixed liquor for obtaining determined nucleic acid addition step (1), is hybridized；
(3) fluorescence intensity after hybridization reaction calculates the fluorescence intensity ratio of luciferase and quantum dot.
Preferably, the concentration of the probe A and probe B be 50-200nM, such as can be 50nM, 60nM, 70nM,
80nM, 90nM, 100nM, 120nM, 140nM, 150nM, 160nM, 180nM or 200nM, preferably 80-150nM.
Preferably, the concentration of the luciferase substrate is 0.2-1 μ g/ μ L, such as can be 0.2 μ g/ μ L, 0.3 μ g/ μ
L, 0.5 μ g/ μ L, 0.7 μ g/ μ L, 0.9 μ g/ μ L or 1 μ g/ μ L, preferably 0.5 μ g/ μ L；
Preferably, the concentration of the magnesium ion solution be 5-15mM, such as can be 5mM, 7mM, 9mM, 11mM, 13mM or
15mM, preferably 9-13mM.
Preferably, the concentration of the determined nucleic acid be 5-100nM, such as can be 5nM, 20nM, 40nM, 80nM or
100nM, preferably 20-80nM.
Preferably, the temperature of the hybridization is 30-40 DEG C, such as can be 30 DEG C, 33 DEG C, 35 DEG C, 38 DEG C or 40 DEG C, excellent
It is selected as 35-38 DEG C.
Preferably, the reaction time of the hybridization be 20-40min, such as can be 20min, 23min, 25min,
28min, 30min, 35min or 40min, preferably 25-35min.
As optimal technical scheme, a method of detection nucleic acid specifically comprises the following steps:
(1) by concentration be 50-200nM probe A and probe B and concentration be 0.2-1 μ g/ μ L luciferase substrate be added
Mixed liquor is obtained in the magnesium ion solution that concentration is 5-15nM；
(2) mixed liquor for obtaining determined nucleic acid addition step (1), 30-40 DEG C of progress hybridization reaction 20-40min；
(3) fluorescence intensity after hybridization reaction calculates the fluorescence intensity ratio of luciferase and quantum dot.
Compared with prior art, the invention has the following beneficial effects:
Detection probe provided by the invention utilizes the principle of bioluminescence energy resonance transfer, according to target nucleic acid design point
Not Dai You luciferase and quantum dot specificity probe, probe can hybridize to form stable T-type structure with nucleic acid, pass through inspection
Survey the intensity of luciferase and quantum dot emission fluorescence than detection nucleic acid, precisely efficiently, it is only necessary to step hybrid process operation letter
Single, hybridization only needs half an hour time-consuming short, and convenient and efficient, cost is relatively low, can distinguish single base mutation, to laboratory condition require compared with
It is low.
Detailed description of the invention
Fig. 1 is the primary structure distribution map of the Gluc in specification of the invention；
Fig. 2 is the bioluminescence schematic diagram of the Gluc in specification of the invention；
Fig. 3 is the characteristic spectral emission figure of the Gluc in specification of the invention
Fig. 4 is experimental principle figure of the invention；
Fig. 5 is fluorescence spectrum response diagram of the invention, response diagram of the darker curve for no purpose nucleic acid miR208a, light color
Curve is the response diagram for having purpose nucleic acid miR208a；
Fig. 6 is cognate rna testing result figure of the invention；
Fig. 7 is that DNA single base mutation of the invention distinguishes result figure.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and by specific real
Mode to further illustrate the technical scheme of the present invention is applied, but the present invention is not limited in scope of embodiments.
Example 1 group installed reagents box
Probe A and probe B is designed according to series of targets miR208a, the 5 ' of middle probe A are terminal modified Gaussia fluorescence
Plain enzyme, the 3 ' of probe B are terminal modified by quantum dot QD655, and the sequence of target nucleic acid, probe A and probe B is as shown in table 1:
Table 1
The primary structure distribution map of Gluc as shown in Figure 1, amino acid sequence 27-97 (hGL-27-97) and
98-168 (hGL-98/168) constitutes two independent enzymatic structures；The bioluminescence schematic diagram of Gluc is such as
Shown in Fig. 2；The characteristic spectral emission figure of Gluc is as shown in Figure 3.
Other conventional reagents include: the Tris-HCl buffer of 50mM pH=7.4.
By concentration be 100nM probe A, probe B and concentration be the luciferase substrate of 0.5 μ g/ μ L, concentration is 10mM's
Magnesium ion solution, other conventional reagents and operation instructions are assembled into kit.
The experiment detection of embodiment 2
(1) by concentration be 100nM probe A and probe B and concentration be 0.5 μ g/ μ L luciferase substrate concentration is added
To obtain mixed liquor in the magnesium ion solution of 10nM；
(2) mixed liquor for obtaining determined nucleic acid addition step (1), 37 DEG C of progress hybridization reaction 30min；
(3) fluorescence intensity after hybridization reaction calculates the fluorescence intensity ratio of luciferase and quantum dot；
The sequence of determined nucleic acid miR208b is as shown in SEQ ID NO:4:
SEQ ID NO:4AUA AGA CGA ACA AAA GGU UUG U
Embodiment 3
Compared with Example 2, in addition to determined nucleic acid is changed to miR-155, other conditions are same as Example 2；
The sequence of MiR-155 is as shown in SEQ ID NO:5:
SEQ ID NO:5UUA AUG CUA AUC GUG AUAGGG GU
Embodiment 4
Compared with Example 2, in addition to determined nucleic acid is changed to miR-183, other conditions are same as Example 2；
The sequence of mi-R183 is as shown in SEQ ID NO:6:
SEQ ID NO:6GUG AAU UAC CGA AGG GCC AUA A
Embodiment 5
Compared with Example 2, in addition to determined nucleic acid is changed to snp1, other conditions are same as Example 2；
The sequence of snp1 is as shown in SEQ ID NO:7:
SEQ ID NO:7ATA ATA CGA GCA AAA AGC TTG T
Embodiment 6
Compared with Example 2, in addition to determined nucleic acid is changed to miR-108a, other conditions are same as Example 2；
Sequence as shown in SEQ ID NO:1:
SEQ ID NO:1AUA AGA CGA GCA AAA AGC UUG U
Embodiment 7
Compared with Example 2, except mixture (including the miR-155, miR-185, miR- that determined nucleic acid is all mRNA
208a, miR-208b) outside, other conditions are same as Example 2.
Embodiment 8
Compared with Example 2, in addition to determined nucleic acid is changed to blank control, other conditions are same as Example 2.
As a result it detects
It is detected using 96 hole microwell plate of black (VICTOR X4, Waltham, MA, PerkinElmer, City, USA) real
The fluorescence of a 2-8 is applied, and for statistical analysis；
Test reaction principle as shown in Figure 4；Target nucleic acid miR208a is not added and adds the glimmering of target nucleic acid miR208a
Light spectral response figure is as shown in Figure 5；MiR208a and miR208b is cognate rna, and miR-155 and miR-183 are cognate rna, together
Source RNA testing result is as shown in fig. 6, single base mutation DNA, the DNA single base mutation that snp1 is miR208a distinguishes result as schemed
Shown in 7.
In conclusion the present invention provides a kind of detection probe and its application, pass through the band respectively designed according to target nucleic acid
There is the probe of the specificity of luciferase and quantum dot, probe and nucleic acid are dexterously formed into stable T structure, utilize bioluminescence
The principle of resonance energy transfer optimizes reaction condition, can precisely distinguish single base mutation, effectively detection target nucleic acid, operation letter
Single, required time is short, and convenient and efficient, cost is relatively low.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Sequence table
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Claims (10)
1. a kind of detection probe, is designed based on target nucleic acid, which is characterized in that the probe includes being marked with luciferase
Probe A and be marked with the probe B of quantum dot；
Wherein, one end partial sequence complementarity of one end of the probe A and target nucleic acid, one end of the probe B and target core
There are the sequences of partial complementarity for the other end of the other end partial sequence complementarity of acid, the other end of the probe A and probe B；
The probe A, probe B and target nucleic acid hybridize to form stable T-type structure, and the luciferase and quantum dot can be mutually
Bioluminescence resonance energy transfer occurs for pairing.
2. probe according to claim 1, which is characterized in that the luciferase includes Gluc, sea pansy
Any one in luciferase, extra large firefly luciferase or firefly luciferase, preferably Gluc；
Preferably, the launch wavelength of the luciferase is 400-600nm, preferably 450-550nm；
Preferably, the quantum dot includes CdSe/ZnS quantum dot, CdTe quantum, CdTe/CdS quantum dot, CdTe/CdS/
Any one in ZnS quantum dot, ZnTe quantum dot, InP/ZnS quantum dot or ZnSe quantum dot；
Preferably, the launch wavelength of the quantum dot is 400-750nm, preferably 600-700nm；
Preferably, the distance of the luciferase and quantum dot is 5-10nm.
3. probe according to claim 1 or 2, which is characterized in that the luciferase is by SMCC activation and the spy
The sulfydryl end of needle A is connected；
Preferably, the quantum dot is connected by Streptavidin modification with the biotin labeling end of the probe B.
4. probe according to any one of claim 1-3, which is characterized in that the partial complementarity of the probe A and probe B
The length of sequence is 4-9 base, preferably 5-8 base.
5. a kind of kit, which is characterized in that comprising such as detection probe of any of claims 1-4；
Preferably, the kit further includes luciferase catalysis substrate, magnesium ion solution and Tris-HCl buffer.
6. a kind of kit as claimed in claim is for detecting correlation RNA biomarker in blood.
7. a kind of method for detecting nucleic acid, which is characterized in that use kit as claimed in claim 5, include the following steps:
(1) probe A, probe B and luciferase substrate are added in magnesium ion solution and obtain mixed liquor；
(2) mixed liquor for obtaining determined nucleic acid addition step (1), is hybridized；
(3) fluorescence intensity after hybridization reaction calculates the fluorescence intensity ratio of luciferase and quantum dot.
8. the method according to the description of claim 7 is characterized in that the concentration of the probe A and probe B be 50-200nM, it is excellent
It is selected as 80-150nM；
Preferably, the concentration of the luciferase substrate is 0.2-1 μ g/ μ L, preferably 0.5 μ g/ μ L；
Preferably, the concentration of the magnesium ion solution is 5-15mM, preferably 9-13mM；
Preferably, the concentration of the determined nucleic acid is 5-100nM, preferably 20-80nM.
9. method according to claim 7 or 8, which is characterized in that the temperature of the hybridization is 30-40 DEG C, preferably 35-
38℃；
Preferably, the reaction time of the hybridization is 20-40min, preferably 25-35min.
10. the method according to any one of claim 7-9, which is characterized in that specifically comprise the following steps:
(1) by concentration be 50-200nM probe A and probe B and concentration be 0.2-1 μ g/ μ L luciferase substrate concentration is added
To obtain mixed liquor in the magnesium ion solution of 5-15nM；
(2) mixed liquor for obtaining determined nucleic acid addition step (1), 30-40 DEG C of progress hybridization reaction 20-40min；
(3) fluorescence intensity after hybridization reaction calculates the fluorescence intensity ratio of luciferase and quantum dot.
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| CN111321205A (en) * | 2020-03-11 | 2020-06-23 | 昆明理工大学 | MiRNA detection method |
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