CN105352922A - Mercury ion detection method and detection kit using Y-shaped DNA assembling and bonding signal amplification - Google Patents
Mercury ion detection method and detection kit using Y-shaped DNA assembling and bonding signal amplification Download PDFInfo
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- CN105352922A CN105352922A CN201510675257.1A CN201510675257A CN105352922A CN 105352922 A CN105352922 A CN 105352922A CN 201510675257 A CN201510675257 A CN 201510675257A CN 105352922 A CN105352922 A CN 105352922A
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Abstract
The invention discloses a mercury ion detection method and detection kit using Y-shaped DNA assembling and bonding signal amplification. The kit comprises a nucleic acid sequence, a buffer system, exonuclease II and a double-stranded DNA fluorescent dye, wherein the nucleic acid sequence is composed of a nucleic acid sequence A, a nucleic acid sequence B, a nucleic acid sequence C and a nucleic acid sequence D; nucleic acid sequences A, B and C can form a Y-shaped complex through self assembly and have three universal bulge regions d; and after complementary pairing between the sequence D and the universal bulge regions d, there is at least one unpaired T-T pair and a conservation sequence is highlighted. According to the invention, Y-shaped DNA is used as a sensing and detection platform, reaction can be completed only through simple hybridization, operation is simple, and cost is low; mark, modification, separation and washing are not needed, and detection is convenient and rapid; and exonuclease is used for signal amplification, so high detection sensitivity and good selectivity are obtained. The detection method and detection kit has a mercury detection limit of 5 pM and a large linear range, from 10 pM to 100 nM.
Description
Technical field
The invention belongs to Environmental Analytical Chemistry field, relate to a kind of mercury ion detecting method and detection kit, particularly relate to a kind of Y shape DNA and be assembled amplification of signal for the detection method of mercury ion and detection kit.
Background technology
Mercury ion (Hg
2+) multiple harm is existed to health, can accumulate in vivo, by food chain transport in human body, there is very strong carcinogenic, teratogenesis, mutagenicity.The micro-Hg of the accumulation in human body
2+cannot be drained by own metabolism, heart, liver, nervous system lesion will be caused, even cause canceration.Therefore, to Hg
2+highly sensitive detection significant.Traditional mercury ion detecting method mainly contains atomic absorption spectrography (AAS), atomic fluorescence spectrometry, inductively coupled plasma mass spectrometry etc.But these technology need complicated operation and the instrument of sample pretreatment process and costliness usually, seriously constrain the application of these detection methods.Some biology sensors reported often need to relate to probe mark for the detection of mercury ion, or fixing washing process, and operation steps bothers, and detection sensitivity is inadequate.Therefore, develop a kind of simple to operate, with low cost, without the need to mark, without the need to the highly sensitive mercury ion detecting method of separating, washing and detection kit significant.
Summary of the invention
The object of the invention is to solve the deficiencies in the prior art, set up a kind of Y shape DNA and be assembled amplification of signal for the detection method of mercury ion and detection kit.
The technical solution used in the present invention is:
A kind of super quick mercury or Silver detection kit, comprise nucleotide sequence, buffer system, exonucleaseⅲ and double-stranded DNA fluorescent dyestuff, it is characterized in that: nucleotide sequence is made up of nucleotide sequence A, B, C, D, wherein 5 ' of sequence A be followed successively by a district, b district and general protrusion district d to ' 3 end; 5 ' of sequence B is followed successively by c district, a* district and general protrusion district d to ' 3 end; Sequence C 5 ' to ' 3 end be followed successively by b*, c* and and general protrusion district d; 5 ' of sequence D is followed successively by d* district and protection sequence to ' 3 end; A, b, c district respectively with a*, b*, c* region sequence complementary pairing, at least exist after d* district and general protrusion district d complementary pairing one group of unpaired T-T to or C-C couple; Protection sequence is not containing the random series of 4 or more continuous print T or C.
Further, the length of a, b, c region sequence is independently 9 ~ 21 bases.Preferably, the length of a, b, c region sequence is independently 12 ~ 17 bases.
The length of d region sequence is independently 9 ~ 15 bases.
The protection sequence of sequence D at least has 6 bases.
Exist after d* district and general protrusion district d complementary pairing 2 ~ 6 groups of unpaired T-T to or C-C couple.
Buffer system does not generate with ionic reaction to be measured and precipitates.
Double-stranded DNA fluorescent dyestuff is selected from SYBRGreenI, EB, Genefinder.
A kind of super quick mercury or Silver detection method, comprise the steps:
1) nucleotide sequence A, B, C are mixed in buffer system, make its phase mutual cross form Y shape complex, add double-stranded DNA fluorescent dyestuff;
2) continue to add nucleotide sequence D, exonucleaseⅲ and testing sample in buffer system, continue reaction;
3) according to the concentration of the fluorescent value situation of change determination mercury ion of reaction system;
Wherein, the structure of nucleotide sequence A ~ D is described above.
The invention has the beneficial effects as follows:
1) with the Y shape DNA formed for sensing detection platform, only need single-cross to complete reaction, simple to operate, cheap.
2) modify without the need to mark, without the need to separating, washing process, fast and easy detects.
3) with exonuclease amplified signal, detection sensitivity is high, and selectivity is good.
The detection of the inventive method to mercury is limited to 5pM, and the range of linearity is from 10pM ~ 100nM, and the range of linearity is large.
Accompanying drawing explanation
Fig. 1 is Cleaning Principle schematic diagram of the present invention;
Fig. 2 is the Hg to variable concentrations
2+the result figure detected;
Fig. 3 is specificity experiments result figure.
Embodiment
A kind of super quick mercury or Silver detection kit, comprise nucleotide sequence, buffer system, exonucleaseⅲ and double-stranded DNA fluorescent dyestuff, nucleotide sequence is made up of nucleotide sequence A, B, C, D, wherein 5 ' of sequence A is followed successively by a district, b district and general protrusion district d to ' 3 end; 5 ' of sequence B is followed successively by c district, a* district and general protrusion district d to ' 3 end; Sequence C 5 ' to ' 3 end be followed successively by b*, c* and and general protrusion district d; 5 ' of sequence D is followed successively by d* district and protection sequence to ' 3 end; A, b, c district respectively with a*, b*, c* region sequence complementary pairing, at least exist after d* district and general protrusion district d complementary pairing one group of unpaired T-T to or C-C couple; According to detecting the difference of ion, protection sequence be when not detecting mercury ion containing 4 or more continuous print T() or C(detection silver ion time) random series.
Further, the length of a, b, c region sequence is independently 9 ~ 21 bases.Preferably, the length of a, b, c region sequence is independently 12 ~ 17 bases.The length of d region sequence is independently 9 ~ 15 bases.
This is because the sequence of above-mentioned zone is too short, then the Y type DNA complex formed is stable not, and to react the fluorescence produced more weak with double-stranded DNA fluorescent dyestuff, is unfavorable for detecting; Sequence is long, may form secondary structure, is unfavorable for the cutting of exonucleaseⅲ, has adverse influence to the Detection results of ion.
The protection sequence of sequence D at least has 6 bases.This protection sequence simultaneously not with the complementary in a, b, c district or a*, b*, c* district.Consider from synthesis simplicity, protection sequence can be 6 or more A continuously.
Exist after d* district and general protrusion district d complementary pairing 2 ~ 6 groups of unpaired T-T to or C-C couple.
Buffer system does not generate with ionic reaction to be measured and precipitates.
Double-stranded DNA fluorescent dyestuff is selected from SYBRGreenI, EB, Genefinder.
A kind of super quick mercury or Silver detection method, comprise the steps:
1) nucleotide sequence A, B, C are mixed in buffer system, make its phase mutual cross form Y shape complex, add double-stranded DNA fluorescent dyestuff;
2) continue to add nucleotide sequence D, exonucleaseⅲ and testing sample in buffer system, continue reaction;
3) according to the concentration of the fluorescent value situation of change determination mercury ion of reaction system;
Wherein, the structure of nucleotide sequence A ~ D is described above.
Be detected as example with mercury ion, Cleaning Principle of the present invention as shown in Figure 1.
Nucleotide sequence A, B, C are mixed in buffer system, nucleotide sequence A, B, C mutually match and form A-B-CY shape DNA complex.In Y shape DNA complex, each 3' holds projection; There is double-strand in this Y shape DNA complex, can produce stronger fluorescence with double-stranded DNA fluorescent dyestuff (as SYBRGreen) reaction;
Add nucleotide sequence D, the projection of D and Y shape DNA is complementary, except T-T base is unpaired, as there is mercury ion (Hg in reaction system
2+), under the effect of mercury ion, can by forming T-Hg
2+-T compound, makes D be combined on Y shape DNA, and the 3 ￠ ends of D are protruding; As there is not mercury ion in reaction system, then D can not be mutual with the projection of Y shape DNA;
Add exonuclease III, exonuclease III can from 3 ￠ ends of double-stranded DNA cutting DNA, thus Y shape DNA is cut into single core thuja acid.Exonuclease III can not cut 3 protruding ￠ and hold DNA, and therefore D can not be cut.After cleavage reaction, D and mercury ion can be recycled, and can be combined again with Y shape DNA.Final all Y shape DNA can be cut into single core thuja acid, and now fluorescence intensity is very weak, only has background signal.
Therefore, when system does not have mercury ion, the Y shape DNA3 ￠ of formation holds protruding, cannot be cut by exonuclease III, and Y shape DNA keeps complete, after adding SYBRGreen, can send stronger fluorescence.That is, when there is no mercury ion, there is stronger fluorescence; When having mercury ion to exist, fluorescence intensity declines.The fluorescence intensity declined and ion concentration of mercury are proportionate, thus can reach the object detecting mercury ion.
Similar, silver ion (Ag
+) can with C-C (cytimidine) to forming C-Ag
+-C compound, thus realize Ag
+detection.
Below in conjunction with embodiment, further illustrate technical scheme of the present invention.
embodiment 1
Y shape DNA is assembled the detection kit of amplification of signal for mercury ion, comprises following composition:
Self assembly nucleotide sequence, exonucleaseⅲ, SYBRGreenI fluorescent dye, buffer system: 10mMTris-acetate buffer solution (pH7.5, containing 50mM sodium acetate)
The concrete sequence of self assembly nucleotide sequence A, B, C, D is as follows:
A:5-
GCCTACATCGTCACG-
GCGGGCGGGCAACGC -TCCG
TCTAC
TC-3（SEQIDNO：1）
(thickened portion is a district, and italic+underscore part is b district, and remainder is d district)
B:5-
GCAGTTCCGGGGCGC-
CGTGACGATGTAGGC -TCCG
TCTAC
TC-3（SEQIDNO：2）
(thickened portion is c district, and italic+underscore part is a* district, and remainder is d district)
C:5'-
GCGTTGCCCGCCCGC-
GCGCCCCGGAACTGC -TCCG
TCTAC
TC-3（SEQIDNO：3）
(thickened portion is b* district, and italic+underscore part is c* district, and remainder is d district)
D:5'-G
TGTAG
TCGGA-
AAAAAA-3'（SEQIDNO：4）
(thickened portion is protection sequence, and remainder is d* district).
to variable concentrations Hg
2+
detection
Preparation Hg
2+standard solution, concentration is respectively 10pM, 100pM, 1nM, 10nM, 100nM and 500nM, room temperature preservation.
By the Hg of variable concentrations
2+solution is added in the reaction system described in embodiment 1 respectively, fully fluorescence intensity after reaction, as shown in Figure 2, along with Hg
2+the increase of concentration, corresponding fluorescence intensity reduces gradually, works as Hg
2+when concentration is more than 100nM, reach capacity gradually.With Hg
2+logarithm (the lg of concentration
c) be horizontal ordinate, fluorescence intensity is ordinate, drawing standard curve, and the two has good linear relationship, and the range of linearity is from 10pM to 100nM, and linear equation is:
f=58-10lg
c(R
2=0.985) (
ffor fluorescence intensity,
cfor ion concentration of mercury), according to 3 times of signal to noise ratio (S/N ratio) standards (3S/N), detect and be limited to 5pM.
specificity experiments
Compound concentration is the disturbance thing standard solution of 500nM, is Cu respectively
2+, Pb
2+, Fe
3+, Mn
2+, Cr
3+, Co
2+, Cd
2+and Zn
2+.
By the disturbance thing standard solution of 500nM and 100nMHg
2+standard solution is added in the reaction system described in embodiment 1 respectively, fully fluorescence intensity after reaction, as shown in Figure 3, and the Cu of 100nM
2+, Pb
2+, Fe
3+, Mn
2+, Cr
3+, Co
2+, Cd
2+and Zn
2+fluorescence intensity compared with blank sample, change is little, does not have an impact to detection.Only have when adding Hg
2+fluorescence intensity just can be made obviously to decline, and this proves that the method is to Hg
2+detection there is good specificity.
<110> Guangdong Prov. Inst. of Ecological Environment & Soil Science
<120> Y shape DNA is assembled amplification of signal for the detection method of mercury ion and detection kit
<130>
<160>4
<170>PatentInversion3.5
<210>1
<211>41
<212>DNA
<213> artificial sequence
<400>1
gcctacatcgtcacggcgggcgggcaacgctccgtctactc41
<210>2
<211>41
<212>DNA
<213> artificial sequence
<400>2
gcagttccggggcgccgtgacgatgtaggctccgtctactc41
<210>3
<211>41
<212>DNA
<213> artificial sequence
<400>3
gcgttgcccgcccgcgcgccccggaactgctccgtctactc41
<210>4
<211>17
<212>DNA
<213> artificial sequence
<400>4
gtgtagtcggaaaaaaa17
Claims (9)
1. a super quick mercury or Silver detection kit, comprise nucleotide sequence, buffer system, exonucleaseⅲ and double-stranded DNA fluorescent dyestuff, it is characterized in that: nucleotide sequence is made up of nucleotide sequence A, B, C, D, wherein 5 ' of sequence A be followed successively by a district, b district and general protrusion district d to ' 3 end; 5 ' of sequence B is followed successively by c district, a* district and general protrusion district d to ' 3 end; Sequence C 5 ' to ' 3 end be followed successively by b*, c* and and general protrusion district d; 5 ' of sequence D is followed successively by d* district and protection sequence to ' 3 end; A, b, c district respectively with a*, b*, c* region sequence complementary pairing, at least exist after d* district and general protrusion district d complementary pairing one group of unpaired T-T to or C-C couple; Protection sequence is not containing the random series of 4 or more continuous print T or C.
2. super quick mercury according to claim 1 or Silver detection kit, is characterized in that: the length of a, b, c region sequence is independently 9 ~ 21 bases.
3. super quick mercury according to claim 1 or Silver detection kit, is characterized in that: the length of a, b, c region sequence is independently 12 ~ 17 bases.
4. super quick mercury according to claim 1 or Silver detection kit, is characterized in that: the length of d region sequence is independently 9 ~ 15 bases.
5. super quick mercury according to claim 1 or Silver detection kit, is characterized in that: the protection sequence of sequence D at least has 6 bases.
6. super quick mercury according to claim 1 or Silver detection kit, is characterized in that: exist after d* district and general protrusion district d complementary pairing 2 ~ 6 groups of unpaired T-T to or C-C couple.
7. super quick mercury according to claim 1 or Silver detection kit, is characterized in that: buffer system does not generate with ionic reaction to be measured and precipitates.
8. super quick mercury according to claim 1 or Silver detection kit, is characterized in that: double-stranded DNA fluorescent dyestuff is selected from SYBRGreenI, EB, Genefinder.
9. super quick mercury or a Silver detection method, comprise the steps:
1) nucleotide sequence A, B, C are mixed in buffer system, make its phase mutual cross form Y shape complex, add double-stranded DNA fluorescent dyestuff;
2) continue to add nucleotide sequence D, exonucleaseⅲ and testing sample in buffer system, continue reaction;
3) according to the concentration of the fluorescent value situation of change determination mercury ion of reaction system;
Wherein, the structure of nucleotide sequence A ~ D is as described in claim 1 ~ 6 any one.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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|---|---|---|---|
| CN201510675257.1A CN105352922B (en) | 2016-01-04 | 2016-01-04 | A kind of Y shape DNA is assembled the detection method and detection kit that amplification of signal is used for mercury ion |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109136334A (en) * | 2018-09-21 | 2019-01-04 | 中国人民解放军陆军军医大学第附属医院 | MicroRNA rapid detection method based on Y type DNA structure |
| CN113252619A (en) * | 2020-02-12 | 2021-08-13 | 青岛科技大学 | Hg can be detected simultaneously2+And Ag+The nanocapsule-nucleic acid biomolecule compound and the preparation method thereof |
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| US20080241866A1 (en) * | 2007-03-30 | 2008-10-02 | Pacific Biosciences Of California, Inc. | Systems and methods for enhancing fluorescent signals |
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| US20080241866A1 (en) * | 2007-03-30 | 2008-10-02 | Pacific Biosciences Of California, Inc. | Systems and methods for enhancing fluorescent signals |
| CN103773856A (en) * | 2014-01-02 | 2014-05-07 | 广东省生态环境与土壤研究所 | Ultra-sensitive detection method of mercury ions and detection kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136334A (en) * | 2018-09-21 | 2019-01-04 | 中国人民解放军陆军军医大学第附属医院 | MicroRNA rapid detection method based on Y type DNA structure |
| CN109136334B (en) * | 2018-09-21 | 2020-06-02 | 中国人民解放军陆军军医大学第一附属医院 | Rapid microRNA detection method based on Y-type DNA structure |
| CN113252619A (en) * | 2020-02-12 | 2021-08-13 | 青岛科技大学 | Hg can be detected simultaneously2+And Ag+The nanocapsule-nucleic acid biomolecule compound and the preparation method thereof |
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Address after: Tianhe District Tianyuan road Guangzhou City, Guangdong province 510650 No. 808 Patentee after: Guangdong Institute of eco environmental technology Address before: Guangzhou City, Guangdong province 510650 Tianyuan Road No. 808 Patentee before: Guangdong Prov. Inst. of Ecological Environment & Soil Science |