CN105352922B - A kind of Y shape DNA is assembled the detection method and detection kit that amplification of signal is used for mercury ion - Google Patents

Abstract

The invention discloses a kind of Y shape DNA to be assembled detection method and detection kit of the amplification of signal for mercury ion, kit includes nucleotide sequence, buffer system, exonucleaseⅲ and double-stranded DNA fluorescent dyestuff, nucleotide sequence is made up of nucleotide sequence A, B, C, D, and A, B, C can be self-assembly of Y types complex and have 3 general protrusion area d；One group of unpaired T T couple at least be present after sequence D and general protrusion area's d complementary pairings, and protrude protection sequence.The system only needs single-cross to complete to react using Y shape DNA as sensing detection platform, simple to operate, cheap.Without marking modification, without separating, washing process, fast and easy detection.With exonuclease amplified signal, detection sensitivity is high, and selectivity is good.Detection of the inventive method to mercury is limited to 5pM, and the range of linearity is big from 10pM~100nM, the range of linearity.

Description

A kind of Y shape DNA is assembled detection method and the detection that amplification of signal is used for mercury ion Kit

Technical field

The invention belongs to environmental analytical chemistry, is related to a kind of mercury ion detecting method and detection kit, especially relates to And a kind of Y shape DNA is assembled the detection method and detection kit that amplification of signal is used for mercury ion.

Background technology

Mercury ion（Hg²⁺）A variety of harm to health be present, can accumulate in vivo, food chain transport can be passed through To human body, there is very strong carcinogenic, teratogenesis, mutagenicity.The micro Hg of accumulation in human body²⁺It can not be entered by own metabolism Row excretion, will cause heart, liver, nervous system lesion, or even cause canceration.Therefore, to Hg²⁺Highly sensitive detection have it is important Meaning.Traditional mercury ion detecting method mainly has atomic absorption spectrography (AAS), atomic fluorescence spectrometry, inductively coupled plasma Mass spectrography etc..But these technologies usually require operation and the instrument of sample pretreatment process and costliness of complexity, serious system The about application of these detection methods.The detection that some biology sensors reported are used for mercury ion generally requires to be related to probe Mark, or fixed washing process, operating procedure trouble, detection sensitivity are inadequate.Therefore, a kind of simple to operate, cost is developed It is cheap, without mark, without separating, washing highly sensitive mercury ion detecting method and detection kit it is significant.

The content of the invention

It is an object of the invention to solve the deficiencies in the prior art, establish a kind of Y shape DNA and be assembled amplification of signal and be used for The detection method and detection kit of mercury ion.

The technical solution used in the present invention is：

A kind of super quick mercury or Silver detection kit, including nucleotide sequence, buffer system, exonucleaseⅲ and double-strand DNA fluorescent dyes, it is characterised in that：Nucleotide sequence is made up of nucleotide sequence A, B, C, D, and wherein sequence A 5 ' to 3 ' ends are successively For a areas, b areas and general protrusion area d；The 5 ' of sequence B are followed successively by c areas, a* areas and general protrusion area d to 3 ' ends；The 5 ' of sequence C are arrived 3 ' ends are followed successively by b*, c* and general protrusion area d；The 5 ' of sequence D are followed successively by d* areas and protection sequence to 3 ' ends；A, b, c area are distinguished With a*, b*, c* region sequence complementary pairing, at least there is one group unpaired T-T pairs after d* areas and general protrusion area's d complementary pairings Or C-C pairs；Protection sequence is the random sequence without 4 or more continuous T or C.

Further, the length of a, b, c region sequence stands alone as 9~21 bases.Preferably, the length of a, b, c region sequence Stand alone as 12~17 bases.

The length of d region sequences stands alone as 9~15 bases.

The protection sequence of sequence D at least has 6 bases.

There is 2~6 groups unpaired T-T pairs or C-C pairs after d* areas and general protrusion area's d complementary pairings.

Buffer system is not generated with ionic reaction to be measured and precipitated.

Double-stranded DNA fluorescent dyestuff is selected from SYBR Green I, EB, Gene finder.

A kind of super quick mercury or Silver detection method, comprise the following steps：

1) nucleotide sequence A, B, C are mixed in buffer system, its phase mutual cross is formed Y shape complex, add double-stranded DNA Fluorescent dye；

2) nucleotide sequence D, exonucleaseⅲ and testing sample are continuously added in buffer system, continues to react；

3) concentration of mercury ion is determined according to the fluorescent value situation of change of reaction system；

Wherein, nucleotide sequence A~D structure is as described above.

The beneficial effects of the invention are as follows：

1) using the Y shape DNA of formation as sensing detection platform, single-cross is only needed to complete to react, simple to operate, price It is cheap.

2) without marking modification, without separating, washing process, fast and easy detection.

3) with exonuclease amplified signal, detection sensitivity is high, and selectivity is good.

Detection of the inventive method to mercury is limited to 5pM, and the range of linearity is big from 10pM~100nM, the range of linearity.

Brief description of the drawings

Fig. 1 is the Cleaning Principle schematic diagram of the present invention；

Fig. 2 is the Hg to various concentrations²⁺The result figure of detection；

Fig. 3 is specificity experiments result figure.

Embodiment

A kind of super quick mercury or Silver detection kit, including nucleotide sequence, buffer system, exonucleaseⅲ and double-strand DNA fluorescent dyes, nucleotide sequence are made up of nucleotide sequence A, B, C, D, wherein sequence A 5 ' to 3 ' end be followed successively by a areas, b areas and General protrusion area d；The 5 ' of sequence B are followed successively by c areas, a* areas and general protrusion area d to 3 ' ends；The 5 ' of sequence C are followed successively by 3 ' ends B*, c* and general protrusion area d；The 5 ' of sequence D are followed successively by d* areas and protection sequence to 3 ' ends；A, b, c area respectively with a*, b*, c* At least there is one group unpaired T-T pairs or C-C pairs after region sequence complementary pairing, d* areas and general protrusion area's d complementary pairings；Root According to the difference of detection ion, protection sequence is without 4 or more continuous T（When detecting mercury ion）Or C（Detect silver ion When）Random sequence.

Further, the length of a, b, c region sequence stands alone as 9~21 bases.Preferably, the length of a, b, c region sequence Stand alone as 12~17 bases.The length of d region sequences stands alone as 9~15 bases.

Because the sequence of above-mentioned zone is too short, then the Y type DNA complexs formed are not sufficiently stable, and glimmering with double-stranded DNA Fluorescence caused by photoinitiator dye reaction is weaker, is unfavorable for detecting；And sequence is long, secondary structure is likely to form, is unfavorable for nucleic acid The cutting of excision enzyme III, there is detrimental effect to the Detection results of ion.

The protection sequence of sequence D at least has 6 bases.This protect sequence and meanwhile not with a, b, c area or a*, b*, c* area Sequence it is complementary.Consider from convieniently synthesized property, protection sequence can be 6 or more continuous A.

There is 2~6 groups unpaired T-T pairs or C-C pairs after d* areas and general protrusion area's d complementary pairings.

Buffer system is not generated with ionic reaction to be measured and precipitated.

Double-stranded DNA fluorescent dyestuff is selected from SYBR Green I, EB, Gene finder.

A kind of super quick mercury or Silver detection method, comprise the following steps：

1) nucleotide sequence A, B, C are mixed in buffer system, its phase mutual cross is formed Y shape complex, add double-stranded DNA Fluorescent dye；

2) nucleotide sequence D, exonucleaseⅲ and testing sample are continuously added in buffer system, continues to react；

3) concentration of mercury ion is determined according to the fluorescent value situation of change of reaction system；

Wherein, nucleotide sequence A~D structure is as described above.

By taking the detection of mercury ion as an example, Cleaning Principle of the invention is as shown in Figure 1.

Nucleotide sequence A, B, C are mixed in buffer system, nucleotide sequence A, B, C, which are mutually paired, to be formed A-B-C Y shapes DNA and answer It is fit.In Y shape DNA complex, each 3 ' all ends are raised；There is double-strand in the Y shape DNA complex, with double-stranded DNA fluorescent dyestuff （Such as SYBR Green）Reaction can produce stronger fluorescence；

It is complementary to add nucleotide sequence D, D and Y shape DNA projection, except T-T bases are unpaired, as existed in reaction system Mercury ion（Hg²⁺）, can be by forming T-Hg in the presence of mercury ion²⁺- T compounds, D is set to be incorporated on Y shape DNA, the 3 ' of D End is raised；As mercury ion is not present in reaction system, then D can not be mutual with Y shape DNA projection；

Exonuclease III is added, exonuclease III can start cutting DNA since the 3 ' of double-stranded DNA ends, so that Y shape DNA cuts into single nucleotide acid.Exonuclease III can not cut 3 ' end DNA of projection, therefore D will not be cut.Cutting is anti- Ying Hou, D and mercury ion can be recycled, and can be combined again with Y shape DNA.Final whole Y shape DNA can be cut into single nucleotide acid, Now fluorescence intensity is very weak, only background signal.

Therefore, when system does not have mercury ion, the ends of Y shape DNA 3 ' of formation are raised, can not be cut by exonuclease III Cut, Y shape DNA keeps complete, after adding SYBR Green, can send stronger fluorescence.That is, in the presence of without mercury ion, have Stronger fluorescence；In the presence of having mercury ion, fluorescence intensity declines.The fluorescence intensity of decline is proportionate with ion concentration of mercury, from And the purpose of detection mercury ion can be reached.

Similar, silver ion（Ag⁺）Can be with C-C (cytimidine) to forming C- Ag⁺- C compounds, so as to realize Ag⁺Inspection Survey.

With reference to embodiment, technical scheme is further illustrated.

Embodiment 1

A kind of Y shape DNA is assembled the detection kit that amplification of signal is used for mercury ion, including following component：

Self assembly nucleotide sequence, exonucleaseⅲ, SYBR Green I fluorescent dyes, buffer system：10 mM Tris- Acetate buffer solution（PH 7.5, contain 50 mM sodium acetates）

Self assembly nucleotide sequence A, B, C, D particular sequence are as follows：

A: 5-GCCTACATCGTCACG- GCGGGCGGGCAACGC -TCCGTCTACTC-3（SEQ ID NO：1）

（Thickened portion is a areas, and italic+underscore part is b areas, and remainder is d areas）

B: 5-GCAGTTCCGGGGCGC- CGTGACGATGTAGGC -TCCGTCTACTC-3（SEQ ID NO：2）

（Thickened portion is c areas, and italic+underscore part is a* areas, and remainder is d areas）

C: 5’-GCGTTGCCCGCCCGC- GCGCCCCGGAACTGC -TCCGTCTACTC-3（SEQ ID NO：3）

（Thickened portion is b* areas, and italic+underscore part is c* areas, and remainder is d areas）

D: 5’-GTGTAGTCGGA-AAAAAA-3’（SEQ ID NO：4）

（Thickened portion is protection sequence, and remainder is d* areas）.

To various concentrations Hg²⁺Detection

Prepare Hg²⁺Standard liquid, concentration are respectively 10 pM, 100 pM, 1 nM, 10 nM, 100 nM and 500 nM, room temperature Preserve.

By the Hg of various concentrations²⁺Solution is added separately in the reaction system described in embodiment 1, is fully detected after reaction glimmering Luminous intensity, as shown in Fig. 2 with Hg²⁺The increase of concentration, corresponding fluorescence intensity are gradually reduced, and work as Hg²⁺Concentration is more than 100 During nM, saturation is progressivelyed reach.With Hg²⁺The logarithm of concentration（lgC）For abscissa, fluorescence intensity is ordinate, draws standard curve, The two has good linear relationship, and the range of linearity is to be from 10 pM to 100 nM, linear equation：F = 58 -10lgC（R²= 0.985）（FFor fluorescence intensity,CFor ion concentration of mercury）, according to 3 times of signal to noise ratio standards（3S/N）, detect and be limited to 5 pM.

Specificity experiments

Compound concentration is 500 nM disturbance thing standard liquid, is Cu respectively²⁺ 、Pb²⁺ 、Fe³⁺、Mn²⁺、Cr³⁺、Co^{2 +}、Cd²⁺And Zn²⁺。

By 500 nM disturbance thing standard liquid and 100 nM Hg²⁺Standard liquid is added separately to described in embodiment 1 Reaction system in, fully reaction after fluorescence intensity, as shown in figure 3,100 nM Cu²⁺ 、Pb²⁺ 、Fe³⁺、Mn²⁺、Cr^{3 +}、Co²⁺、Cd²⁺And Zn²⁺Fluorescence intensity compared with blank sample, change is little, and detection is not had an impact.Only work as addition Hg²⁺Can just fluorescence intensity be decreased obviously, this proves this method to Hg²⁺Detection have well specificity.

<110>Guangdong Prov. Inst. of Ecological Environment ＆ Soil Science

<120>A kind of Y shape DNA is assembled the detection method and detection kit that amplification of signal is used for mercury ion

<130>

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 41

<212> DNA

<213>Artificial sequence

<400> 1

gcctacatcg tcacggcggg cgggcaacgc tccgtctact c 41

<210> 2

<211> 41

<212> DNA

<213>Artificial sequence

<400> 2

gcagttccgg ggcgccgtga cgatgtaggc tccgtctact c 41

<210> 3

<211> 41

<212> DNA

<213>Artificial sequence

<400> 3

gcgttgcccg cccgcgcgcc ccggaactgc tccgtctact c 41

<210> 4

<211> 17

<212> DNA

<213>Artificial sequence

<400> 4

gtgtagtcgg aaaaaaa 17

Claims (9)

1. a kind of super quick mercury or Silver detection kit, including nucleotide sequence, buffer system, exonucleaseⅲ and double-strand DNA fluorescent dyes, it is characterised in that：Nucleotide sequence is made up of nucleotide sequence A, B, C, D, and wherein sequence A 5 ' to 3 ' ends are successively For a areas, b areas and general protrusion area d；The 5 ' of sequence B are followed successively by c areas, a* areas and general protrusion area d to 3 ' ends；The 5 ' of sequence C are arrived 3 ' ends are followed successively by b*, c* and general protrusion area d；The 5 ' of sequence D are followed successively by d* areas and protection sequence to 3 ' ends；A, b, c area are distinguished With a*, b*, c* region sequence complementary pairing, at least there is one group unpaired T-T pairs after d* areas and general protrusion area's d complementary pairings Or C-C pairs；Protection sequence is the random sequence without 4 or more continuous T or C.

2. super quick mercury according to claim 1 or Silver detection kit, it is characterised in that：A, the length of b, c region sequence Degree stands alone as 9~21 bases.

3. super quick mercury according to claim 1 or Silver detection kit, it is characterised in that：A, the length of b, c region sequence Degree stands alone as 12~17 bases.

4. super quick mercury according to claim 1 or Silver detection kit, it is characterised in that：The length of d region sequences is only Stand as 9~15 bases.

5. super quick mercury according to claim 1 or Silver detection kit, it is characterised in that：The protection sequence of sequence D At least there are 6 bases.

6. super quick mercury according to claim 1 or Silver detection kit, it is characterised in that：D* areas and general protrusion area There is 2~6 groups unpaired T-T pairs or C-C pairs after d complementary pairings.

7. super quick mercury according to claim 1 or Silver detection kit, it is characterised in that：Buffer system not with it is to be measured Ionic reaction generation precipitation.

8. super quick mercury according to claim 1 or Silver detection kit, it is characterised in that：Double-stranded DNA fluorescent dyestuff Selected from SYBR Green I, EB, Gene finder.

9. a kind of super quick mercury or Silver detection method, comprise the following steps：

1) nucleotide sequence A, B, C are mixed in buffer system, its phase mutual cross is formed Y shape complex, add double-stranded DNA fluorescent Dyestuff；

2) nucleotide sequence D, exonucleaseⅲ and testing sample are continuously added in buffer system, continues to react；

3) concentration of mercury ion is determined according to the fluorescent value situation of change of reaction system；

Wherein, nucleotide sequence A~D structure is as described in claim 1~6 any one.