CN105256037B - The strand replacement reaction of separate type DNA fulcrums mediation is used for the detection method and detection kit of 17 beta estradiols - Google Patents
The strand replacement reaction of separate type DNA fulcrums mediation is used for the detection method and detection kit of 17 beta estradiols Download PDFInfo
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Abstract
Strand replacement reaction the invention discloses a kind of mediation of separate type DNA fulcrums is used for the detection method and detection kit of 17 β estradiol.Kit includes buffer system and nucleic acid sequence A, B, C, D, E and F, and sequence A is the aptamer of 17 β estradiol, can be specifically bound therewith, and the B of A B double-strands is replaced；B and C, D-shaped are into B C D compounds；Using the DNA fulcrums in B C D, reacted with E F, start strand replacement reaction, can the nucleic acid of modification fluorophor from and modification quenching group nucleic acid combination in replace, so as to send out stronger fluorescence.The present invention is easy to operate, economical and practical, washs separation process without fixed, suitable for quickly detecting, suitable for being promoted in base, is with a wide range of applications.The method of the present invention has higher sensitivity, can detect the 17 β estradiol of 0.3 nM, and the range of linearity is 1 nM to 400 nM.
Description
Technical field
The invention belongs to environmental contaminants detection fields, are related to a kind of strand replacement reaction of separate type DNA fulcrums mediation and use
In the detection method and detection kit of 17 beta estradiols.
Background technology
17 beta estradiols are a kind of typical environomental pollution sources, are widely present in river, soil, water source, air and agriculture
In product, belong to environmental estrogens, be it is a kind of act on most strong, most potential hazard incretion interferent, can simulate endogenous
Property estrogen behavior.17 beta estradiols generally exist with trace, and traditional detection method is mainly liquid chromatography, gas chromatography mass spectrometry
Method, Liquid Chromatography/Mass Spectrometry etc..But these mass chromatography technologies need troublesome sample pretreatment process, need expensive analyzer
Device, detection time are longer, it is difficult to realize quick analysis.
Therefore, develop it is a kind of it is easy to operate, of low cost, can quickly detect 17 beta estradiols of environment method and
Coherent detection kit is of great significance.
Invention content
In order to solve the deficiencies in the prior art, it is an object of the invention to establish a kind of chain of separate type DNA fulcrums mediation
Displacement reaction is used for the detection method and detection kit of 17 beta estradiols.
The technical solution used in the present invention is：
A kind of molecular detection kit, including nucleic acid sequence, buffer system；Nucleic acid sequence is by nucleic acid sequence A, B, C, D, E
It is formed with F；Wherein sequence A is the aptamer of testing molecule；Partial sequence and B in A is complementary；Sequence B has a areas and b areas；
Sequence C has a* areas and c areas；Sequence D has d areas and b* areas；Sequence E has c* areas and d* areas；One end of E is modified with fluorescent base
Group or quenching group；Sequence F has c areas；One end of F is modified with quenching group corresponding with E or fluorophor；Wherein above-mentioned a,
B, c, d area respectively with a*, b*, c*, d* region sequence complementary pairing.
Preferably, the length of a, b, c region sequence stands alone as 9~21 bases.
Preferably, the d regions of sequence D can be used as DNA fulcrums, and sequence length is 4-10 base.
Preferably, fluorophor includes FAM, Cy3, Cy5.
Preferably, quenching group includes BHQ, gold nano grain, quantum dot, graphene.
Preferably, buffer system is the buffer system containing salt ionic concentration.
A kind of 17 beta estradiol detection kits, including nucleic acid sequence, buffer system, it is characterised in that：Nucleic acid sequence by
Nucleic acid sequence A, B, C, D, E and F are formed, wherein
Sequence A：5'-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGC
GCTGAAGCGCGGAAGC-3'（SEQ ID NO：1）；
Sequence B:5'-TGCGCAGAGCCGCTA( b )CCCTCTGCGTGTAAT( a )-3'（SEQ ID NO：2）；
Sequence C:5'-ATTACACGCAGAGGG( a* )AACTGCTAGCTTCAG( c )-3'（SEQ ID NO：3）；
Sequence D:5'-AGCTAC( d )TAGCGGCTCTGCGCA( b* )-3'（SEQ ID NO：4）；
Sequence E:5'-FAM-CTGAAGCTAGCAGTT（ c*）GTAGCT（ d*）-3'（SEQ ID NO：5）；
Sequence F:5'-AACTGCTAGCTTCAG（c）-BHQ-3'（SEQ ID NO：6）.
Preferably, buffer system is 10 mM Tris-HCl buffer solutions, pH=8.
A kind of detection method of molecule, includes the following steps：
1) nucleic acid sequence A and B are dissolved in buffer system, fully reacted, form A-B double-strandednucleic acids；
2) nucleic acid sequence E and F are dissolved in another buffer system, fully reacted, form E-F double-strandednucleic acids；
3) in step 1）Buffer system in continuously add sample to be tested, nucleic acid sequence C, D, fully reaction after, again plus
Enter step 2）The product of acquisition, the reaction was continued；
4) concentration of testing molecule is determined according to the fluorescent value situation of change of reaction system；
Wherein, the structure of nucleic acid sequence A~F is as described above.
A kind of detection method of 17 beta estradiol, includes the following steps：
1) nucleic acid sequence A and B are dissolved in buffer system, fully reacted, form A-B double-strandednucleic acids；
2) nucleic acid sequence E and F are dissolved in another buffer system, fully reacted, form E-F double-strandednucleic acids；
3) in step 1）Buffer system in continuously add sample to be tested, nucleic acid sequence C, D, fully reaction after, again plus
Enter step 2）The product of acquisition, the reaction was continued；
4) concentration of 17 beta estradiols in sample to be tested is determined according to the fluorescent value situation of change of reaction system；
Wherein, the structure of nucleic acid sequence A~F is as described above.
The beneficial effects of the invention are as follows：
（1）Reaction process can once be completed in solution system, without being related to cDNA chip, washing, separation process, operate
Simply, detection is quick, is conveniently used for quickly detecting.
（2）The separate type DNA fulcrums of design are without loop-stem structure DNA, and design is simple, which can be used for detecting a variety of objects
Matter for different aptamers, changes corresponding nucleic acid sequence, you can apply mechanically separate type DNA fulcrums and complete detection.
（3）The method of the present invention has higher sensitivity, can detect 17 beta estradiols of 0.3 nM, the range of linearity 1
NM to 400 nM.
Description of the drawings
Fig. 1 is the schematic diagram of detection method of the present invention；
Fig. 2 is the result figure detected to 17 beta estradiol of various concentration；
Fig. 3 is specificity experiments result figure.
Specific embodiment
A kind of molecular detection kit, including nucleic acid sequence, buffer system；Nucleic acid sequence is by nucleic acid sequence A, B, C, D, E
It is formed with F；Wherein sequence A is the aptamer of testing molecule；Partial sequence and B in A is complementary；Sequence B has a areas and b areas；
Sequence C has a* areas and c areas；Sequence D has d areas and b* areas；Sequence E has c* areas and d* areas；One end of E is modified with fluorescent base
Group or quenching group；Sequence F has c areas；One end of F is modified with quenching group corresponding with E or fluorophor；Wherein above-mentioned a,
B, c, d area respectively with a*, b*, c*, d* region sequence complementary pairing.
Preferably, the length of a, b, c region sequence stands alone as 9~21 bases.
More there is choosing, the length of a, b, c region sequence stands alone as 15 bases.
Preferably, the d regions of sequence D can be used as DNA fulcrums, and sequence length is 4-10 base.
It is furthermore preferred that the length in the d regions of sequence D is 6 bases.
Preferably, fluorophor includes FAM, Cy3, Cy5.
Preferably, quenching group includes BHQ, gold nano grain, quantum dot, graphene.
Preferably, buffer system is the buffer system containing salt ionic concentration.
A kind of 17 beta estradiol detection kits, including nucleic acid sequence, buffer system, it is characterised in that：Nucleic acid sequence by
Nucleic acid sequence A, B, C, D, E and F are formed, wherein
Sequence A：5'-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGC
GCTGAAGCGCGGAAGC-3'（SEQ ID NO：1）；
Sequence B:5'-TGCGCAGAGCCGCTA( b )CCCTCTGCGTGTAAT( a )-3'（SEQ ID NO：2）；
Sequence C:5'-ATTACACGCAGAGGG( a* )AACTGCTAGCTTCAG( c )-3'（SEQ ID NO：3）；
Sequence D:5'-AGCTAC( d )TAGCGGCTCTGCGCA( b* )-3'（SEQ ID NO：4）；
Sequence E:5'-FAM-CTGAAGCTAGCAGTT（ c*）GTAGCT（ d*）-3'（SEQ ID NO：5）；
Sequence F:5'-AACTGCTAGCTTCAG（c）-BHQ-3'（SEQ ID NO：6）.
Preferably, buffer system is 10 mM Tris-HCl buffer solutions, pH=8.
A kind of detection method of molecule, includes the following steps：
1) nucleic acid sequence A and B are dissolved in buffer system, fully reacted, form A-B double-strandednucleic acids；
2) nucleic acid sequence E and F are dissolved in another buffer system, fully reacted, form E-F double-strandednucleic acids；
3) in step 1）Buffer system in continuously add sample to be tested, nucleic acid sequence C, D, fully reaction after, again plus
Enter step 2）The product of acquisition, the reaction was continued；
4) concentration of testing molecule is determined according to the fluorescent value situation of change of reaction system；
Wherein, the structure of nucleic acid sequence A~F is as described above.
A kind of detection method of 17 beta estradiol, includes the following steps：
1) nucleic acid sequence A and B are dissolved in buffer system, fully reacted, form A-B double-strandednucleic acids；
2) nucleic acid sequence E and F are dissolved in another buffer system, fully reacted, form E-F double-strandednucleic acids；
3) in step 1）Buffer system in continuously add sample to be tested, nucleic acid sequence C, D, fully reaction after, again plus
Enter step 2）The product of acquisition, the reaction was continued；
4) concentration of 17 beta estradiols in sample to be tested is determined according to the fluorescent value situation of change of reaction system；
Wherein, the structure of nucleic acid sequence A~F is as described above.
By taking the detection of 17 beta estradiols as an example, the testing principle that the present invention is further explained（As shown in Figure 1）：
（1）Nucleic acid sequence A is the aptamer of 17 beta estradiols, can be specifically bound with 17 beta estradiols.B can be with A portions
Sub-sequence is complementary.A and B first hybridize, and form A-B double-strandednucleic acids.
（2）17 beta estradiols are added in, A can interact with 17 beta estradiols, so as to which B is replaced.
（3）The B replaced can form B-C-D compounds with a part of C and a part of complementation of D.
（4）A terminal modified fluorophor of E, a terminal modified quenching group of F, E and F are complementary, make fluorophor and are quenched
Group is close, and fluorescent quenching occurs.A terminal modified quenching group of E, a terminal modified fluorophor of F can also be changed to.
（5）In B-C-D compounds, the d regions of D can be used as DNA fulcrums, can with the d* regional complementarities in E-F compounds,
So as to start strand replacement reaction, c regions and the c* regional complementarities in E-F compounds of C, can be after strand replacement reaction starts
F in E-F compounds is replaced, and forms B-C-D-E compounds, and fluorophor and quenching group separation, system are sent out at this time
Stronger fluorescence, and fluorescence intensity is proportionate with 17 beta estradiol concentration, so as to reach 17 beta estradiols of detection
Purpose.
With reference to embodiment, the technical solution further illustrated the present invention, but not limited to this.
Embodiment 1
The strand replacement reaction of DNA fulcrums mediation is used for the detection kit of 17 beta estradiols, including following component：
（1）Nucleic acid sequence A, B, C, D, E, F；Particular sequence is as follows：
Sequence A：5'-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGC
GCTGAAGCGCGGAAGC-3'（SEQ ID NO：1）；
Sequence B:5'-TGCGCAGAGCCGCTA( b )CCCTCTGCGTGTAAT( a )-3'（SEQ ID NO：2）；
Sequence C:5'-ATTACACGCAGAGGG( a* )AACTGCTAGCTTCAG( c )-3'（SEQ ID NO：3）；
Sequence D:5'-AGCTAC( d )TAGCGGCTCTGCGCA( b* )-3'（SEQ ID NO：4）；
Sequence E:5'-FAM-CTGAAGCTAGCAGTT（ c*）GTAGCT（ d*）-3'（SEQ ID NO：5）；
Sequence F:5'-AACTGCTAGCTTCAG（c）-BHQ-3'（SEQ ID NO：6）.
（2）17 beta estradiol standard solution.
（3）10 mM Tris-HCl buffer solutions（100 mM NaCl, 20 mM KCl, 5 mM MgCl are contained in pH=82）.
The strand replacement reaction of DNA fulcrums mediation is used for the detection method of 17 beta estradiols, carries out in accordance with the following steps：
10 mM Tris-HCl buffer solutions of the B of the A and 200 nM of (1) 200 nM（PH=8, containing 100 mM NaCl, 20
MM KCl, 5 mM MgCl2）Dissolving, abundant mixing react at room temperature 30 minutes.
(2) 17 beta estradiols are added in, are reacted at room temperature 45 minutes.
(3) C and D of 200 nM is added in, is reacted at room temperature 30 minutes.
The E and F of (4) 200 nM is first dissolved in 10 mM Tris-HCl buffer solutions（PH=8, containing 100 mM NaCl, 20
MM KCl, 5 mM MgCl2）In, abundant mixing reacts at room temperature 30 minutes, forms E-F.
(5) E-F is added to step（3）Reaction product in, abundant mixing, react at room temperature 60 minutes.Reaction solution is 495
Under nm excitations, 525 nm emitted luminescence intensities are recorded.
Embodiment 2：Detection to 17 beta estradiol of various concentration：
Prepare 17 beta estradiol standard solution, concentration be respectively 1 nM, 10 nM, 100 nM, 200nM, 400 nM and
800nM room temperature preservations.
17 beta estradiol solution of various concentration are added separately in the reaction system described in embodiment 1, are fully reacted
Fluorescence intensity afterwards, as shown in Fig. 2, with the increase of 17 beta estradiol concentration, corresponding fluorescence intensity gradually increases, when 17
When beta estradiol concentration is more than 400 nM, saturation is progressivelyed reach.With a concentration of abscissa of 17 beta estradiols, fluorescence intensity is vertical
Coordinate draws standard curve, and the two has good linear relationship, and the range of linearity is from 1 nM to 400 nM, and linear equation is：
F = 138.75+1.1C（R2=0.952）（F is fluorescence intensity, and C is 17 beta estradiol concentration）, according to 3 times of signal-to-noise ratio standards
（3S/N）, detect and be limited to 0.3 nM.
Embodiment 3：Specificity experiments：
Compound concentration be 500 nM disturbance object standard solution, be respectively bisphenol-A, progesterone, sevin, lincomycinum,
Mitomycin, estriol.
The disturbance object standard solution of 500 nM and 100 nM, 17 beta estradiol standard solution are added separately to embodiment
In reaction system described in 1, fully reaction after record fluorescence intensity change, as shown in figure 3, the bisphenol-A of 500 nM, progesterone,
Sevin, lincomycinum, mitomycin, estriol do not generate apparent fluorescence intensity change, and detection is not had an impact.Only
Fluorescence intensity significantly increases after 17 beta estradiol is added in, and it is special well to show that this method has the detection of 17 beta estradiols
Property.
<110>The strand replacement reaction of separate type DNA fulcrums mediation is used for 17 β
The detection method and detection kit of estradiol
<120>Guangdong Prov. Inst. of Ecological Environment ＆ Soil Science
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 76
<212> DNA
<213>Artificial combination sequence
<400> 1
gcttccagct tattgaatta cacgcagagg gtagcggctc tgcgcattca attgctgcgc 60
gctgaagcgc ggaagc 76
<210> 2
<211> 30
<212> DNA
<213>Artificial combination sequence
<400> 2
tgcgcagagc cgctaccctc tgcgtgtaat 30
<210> 3
<211> 30
<212> DNA
<213>Artificial combination sequence
<400> 3
attacacgca gagggaactg ctagcttcag 30
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<400> 4
agctactagc ggctctgcgc a 21
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ctgaagctag cagttgtagc t 21
<210> 6
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aactgctagc ttcag 15
Claims (8)
1. a kind of molecular detection kit, including nucleic acid sequence, buffer system, it is characterised in that：Nucleic acid sequence is by nucleic acid sequence
A, B, C, D, E and F are formed；Wherein sequence A is the aptamer of testing molecule；Partial sequence and B in A is complementary；
Sequence B has a areas and b areas；Sequence C has a* areas and c areas；Sequence D has d areas and b* areas；Sequence E has c* areas and d*
Area；One end of E is modified with fluorophor or quenching group；Sequence F has c areas；One end of F, which is modified with, corresponding with E is quenched base
Group or fluorophor；Wherein above-mentioned a, b, c, d area respectively with a*, b*, c*, d* region sequence complementary pairing.
2. kit according to claim 1, it is characterised in that：A, the length of b, c region sequence stands alone as 9~21 alkali
Base.
3. kit according to claim 1, it is characterised in that：The d regions of sequence D can be used as DNA fulcrums, and sequence is long
It spends for 4-10 base.
4. kit according to claim 1, it is characterised in that：Fluorophor includes FAM, Cy3, Cy5.
5. kit according to claim 1, it is characterised in that：Quenching group include BHQ, gold nano grain, quantum dot,
Graphene.
6. kit according to claim 1, it is characterised in that：Buffer system is the buffer system containing salt ionic concentration.
7. a kind of 17 beta estradiol detection kits, including nucleic acid sequence, buffer system, it is characterised in that：Nucleic acid sequence is by core
Acid sequence A, B, C, D, E and F are formed, wherein
Sequence A：5'-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTG
AAGCGCGGAAGC-3'（SEQ ID NO：1）；
Sequence B:5'-TGCGCAGAGCCGCTA( b )CCCTCTGCGTGTAAT( a )-3'（SEQ ID NO：2）；
Sequence C:5'-ATTACACGCAGAGGG( a* )AACTGCTAGCTTCAG( c )-3'（SEQ ID NO：3）；
Sequence D:5'-AGCTAC( d )TAGCGGCTCTGCGCA( b* )-3'（SEQ ID NO：4）；
Sequence E:5'-FAM-CTGAAGCTAGCAGTT（ c*）GTAGCT（ d*）-3'（SEQ ID NO：5）；
Sequence F:5'-AACTGCTAGCTTCAG（c）-BHQ-3'（SEQ ID NO：6）.
8. kit according to claim 7, it is characterised in that：Buffer system is 10 mM Tris-HCl buffer solutions, pH=
8。
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| CN106520927A (en) * | 2016-09-23 | 2017-03-22 | 广东省生态环境技术研究所 | Nucleic acid detection method and kit |
| CN107462705B (en) * | 2017-07-26 | 2019-10-18 | 广东省生态环境技术研究所 | A kind of sensitivity is strong, decomposable quantum dot nano talent scout needle and preparation method thereof |
| CN107727623B (en) * | 2017-10-12 | 2020-06-05 | 广东省生态环境技术研究所 | Mercury ion fluorescence detection kit |
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| CN104975079A (en) * | 2015-05-28 | 2015-10-14 | 广东省生态环境与土壤研究所 | 17beta-estradiol visualization detection method based on DNA nano-structure, and 17beta-estradiol visualization detection kit based on DNA nano-structure |
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| CN104975079A (en) * | 2015-05-28 | 2015-10-14 | 广东省生态环境与土壤研究所 | 17beta-estradiol visualization detection method based on DNA nano-structure, and 17beta-estradiol visualization detection kit based on DNA nano-structure |
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