CN104946770A - Novel exonuclease-III-based mercury ion detection method - Google Patents

Novel exonuclease-III-based mercury ion detection method Download PDF

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Publication number
CN104946770A
CN104946770A CN201510382389.5A CN201510382389A CN104946770A CN 104946770 A CN104946770 A CN 104946770A CN 201510382389 A CN201510382389 A CN 201510382389A CN 104946770 A CN104946770 A CN 104946770A
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mercury ion
mercury
dna
concentration
exonucleaseiii
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朱颖越
邓大庆
朱敏
李海霞
蔡义林
袁爱梦
王立梅
齐斌
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Changshu Institute of Technology
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

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Abstract

The invention relates to a novel exonuclease-III-based mercury ion detection method which comprises the following steps: (1) mixing two DNA (deoxyribonucleic acid) segments, which can specifically recognize mercury ions and contain rich T bases, with a sample to be detected so that the two DNA segments are combined into a 3'-terminal closed double-chain DNA under the action of Hg<2+> in the sample to be detected; (2) carrying out enzyme digestion on the double-chain DNA by using exonuclease III; (3) carrying out fluorescent quantitative PCR (polymerase chain reaction) amplification on the rest DNA segments which are not subjected to enzyme digestion; and (4) determining the Hg<2+> concentration according to the fluorescent detection result. The invention designs a mercury ion biosensor which is used for implementing simple quick high-sensitivity detection on the mercury ions in water.

Description

A kind of mercury ion detecting novel method based on exonucleaseⅲ
Technical field
The invention belongs to technical field of analytical chemistry and technical field of food safety, relate to a kind of mercury ion detecting novel method based on exonucleaseⅲ.
Background technology
Mercury is mainly present in occurring in nature with the form such as inorganic mercury and organic mercury, and inorganic mercury can be converted into organic mercury by methylated effect at occurring in nature.The mercury of different states is different to the harm of human body, and wherein the body harm of organic mercury to people is maximum.Mercury can act on people's cylinder accumulation by food chain enrichment, brings harm to human body health.Such as: the brain of grievous injury human body and neural system, cause to organ dysfunctions such as immunity system, Digestive tract, kidney and lungs and have a strong impact on, the novel method therefore developing a kind of simple and quick detection mercury ion remains very necessary.
The concentration determination traditional method of mercury ion mainly contains atomic absorption spectrometry, inductively coupled plasma mass spectrometry, spectrophotometry, voltammetry etc.Although each tool advantage of these traditional methods, has been still many limitation in actual applications, as plant and instrument is expensive, pre-treatment is complicated, also need skilled skilled worker etc.
From about T-Hg 2+since-T Idiotype structure is in the news, some researchers start to pay close attention to T-Hg 2+the Idiotype structure that-T is such, T-Hg 2+-T specific binding reactive force is greater than " A " " T " base pair complementarity reactive force, and reports some and utilize T-Hg 2+this kind of sensor of-T Idiotype structure novel mercury ion sensor is selective good, the advantage that specificity is good, becomes the focus of nearest people research.
Exonuclease III can act on the closed double-stranded DNA of 3' end, and progressively cuts mononucleotide along the direction of 3' → 5'.This character of exonuclease III has been applied to the detection of nucleic acid molecule and organic molecule.In the present invention, we devise a kind of novel method of the fluorometry super sensitivity detection mercury ion based on exonucleaseⅲ.The closed double-stranded DNA of 3' end is formed based on T-T base mispairing two DNA fragmentations, utilize exonuclease III can act on the closed double-stranded DNA of 3' end, and the character of mononucleotide is progressively cut along the direction of 3' → 5', in conjunction with the Fluorescence amplification effect of real-time fluorescence quantitative PCR, establish a kind of novel method detecting mercury ion in the aqueous solution.
Summary of the invention
The object of this invention is to provide a kind of mercury ion detecting novel method based on exonucleaseⅲ, realize simple, quick, the highly sensitive detection of mercury ion in water.
The present invention is achieved through the following technical solutions: a kind of mercury ion detecting novel method based on exonucleaseⅲ, comprising:
(1) by two sections can to mercury ion specific recognition and the DNA fragmentation being rich in T base mix with testing sample, two Hgs of segment DNA fragment in testing sample 2+the double-stranded DNA that 3' end is closed is combined under effect;
(2) exonuclease III digestion double-stranded DNA;
(3) digested DNA fragmentation is not had to carry out fluorescent quantitative PCR to remaining;
(4) Hg is determined according to fluoroscopic examination result 2+concentration.
Further, described two sections can to mercury ion specific recognition and the sequence being rich in the DNA fragmentation of T base has nucleotide sequence as shown in SEQ ID NO:1 and SEQ ID NO:2.Thus, the efficiency and the sensitivity that utilize the inventive method to carry out ion concentration of mercury detection can be improved further.
Further, the primer sequence of described fluorescent quantitative PCR has the nucleotide sequence as shown in SEQ ID NO:3 and SEQ ID NO:4.Thus, the efficiency and the sensitivity that utilize the inventive method to carry out ion concentration of mercury detection can be improved further.
Further, the concentration of described mercury ion is 2-200nM.Thus, the efficiency and the sensitivity that utilize the inventive method to carry out ion concentration of mercury detection can be improved further.
Further, the concentration of described mercury ion is 5-100nM.Thus, the efficiency and the sensitivity that utilize the inventive method to carry out ion concentration of mercury detection can be improved further.
Further, based on following linear equation, determine the concentration of mercury ion in described sample:
y=1.03x+4.66，
Y is the cycle number of amplification curve under different ion concentration of mercury, and x is the concentration of corresponding mercury ion.Thus, the efficiency and the sensitivity that utilize the inventive method to carry out ion concentration of mercury detection can be improved further.
According to embodiments of the invention, determine that ion concentration of mercury in described sample is by having compared the fluoroscopic examination result of described system and typical curve, wherein, described typical curve based on known ion concentration of mercury be respectively 2 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM standard model carry out parallel laboratory test and set up.Thus, the efficiency and the sensitivity that utilize the inventive method to carry out ion concentration of mercury detection can be improved further.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Fig. 1 adds the mercury ion of different concns, and does real-time fluorescence quantitative PCR, obtains its amplification curve (ion concentration of mercury gets 2 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM respectively).
The typical curve of Fig. 2 mercury ion detecting.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described further, but should not be construed as limitation of the present invention:
The corresponding oligonucleotide fragment of embodiment 1 Design and synthesis
By consulting pertinent literature, designing two sections and can be rich in the DNA fragmentation of T base to mercury ion specific recognition, designing the upstream and downstream primer of real-time fluorescence quantitative PCR according to this sequence.Sequence is prepared by DNA synthesizer.
Masterplate DNA:5 '-AATCTGGTTTAGCTACGCCTTCCCCGTGGCGATGTTTCTT
AGCGCCTTACTTGTTTGTTG-3’（SEQ ID NO：1）
Complementary DNA: 5 '-CTTCTTTCTTGTAAGGCGCTAAGAAACATCGCCACGGG
GAAGGCGTACTCGCG -3’ （SEQ ID NO：2）
Upstream primer: 5 '-AATCTGGTTTAGCTACGCCTTC-3 ' (SEQ ID NO:3)
Downstream primer: 5 '-GTAAGGCGCTAAGAAACATCG-3 ' (SEQ ID NO:4)
Embodiment 2 T-Hg 2+-T hybridization
First PCR pipe adds the mixed solution of template DNA and complementary DNA, adds appropriate Hg successively subsequently 2+ion standardized solution makes its ultimate density be respectively 2,5,10,20,50,100,200 nM, after reacting 30 min.Template DNA and complementary DNA pass through T-Hg 2+-T specificity structure forms the closed double-stranded DNA of 3' end.
The enzyme catalysis of embodiment 3 exonucleaseⅲ and the foundation of typical curve
In above-mentioned PCR pipe, add exonucleaseⅲ 0.1 μ L successively, react 90 s, whole system 98 DEG C of reaction 10 min in constant-temperature metal bath, allow exonucleaseⅲ inactivation, terminate exonucleaseⅲ to the shearing of template DNA thereupon.Add appropriate PCR mixed solution subsequently, at CFX96 after the primer of upstream and downstream tMexperiment is completed in real-time fluorescence quantitative PCR.Due to the Hg added 2+ionic concn is different, and the template DNA that result in different quantities is cut by exonucleaseⅲ, and the concentration for the template DNA of pcr amplification is also thereupon different, so obtain different amplification curves (Fig. 1).The foundation of mercury ion typical curve: utilize real-time fluorescence quantitative PCR instrument to measure the cycle number of amplification curve under different ion concentration of mercury, according to the cycle number of amplification curve under the different ion concentration of mercury measured, draw the typical curve (Fig. 2) of ion concentration of mercury, the linearity range 5-100 nM of this sensor, detects and is limited to 3 nM.The cycle number of the linear equation of typical curve to be y=1.03x+4.66, y be amplification curve under different ion concentration of mercury, x is the concentration of corresponding mercury ion, linear dependence >0.99.
The specific assay of embodiment 4 mercury ion
Step with (2), adds Hg successively in the mixed solution of template DNA and complementary DNA 2+, Cu 2+, Cd 2+, Pb 2+, Ni 2+, Fe 2+reference liquid, makes its ultimate density be 100 nM, reacts 30 minutes at 37 ° of C.Experiment is completed subsequently according to step (3).Learn that the mercury ion detecting new technology of this PCR-based technology has very high specificity by experimental result.
Embodiment 5 reality adds sample tests
Appropriate mercury ion reference liquid is added respectively in tap water sample, its ultimate density is made to be respectively 5,10,20,50 nM, the mercury ion detecting novel method based on exonucleaseⅲ is adopted to measure the rate of recovery of the mercury ion in actual sample, result shows that its rate of recovery is between 96.50%-104.00%, standard deviation is less than 5.06%, can meet the detection demand to mercury ion in actual life completely.Result is as table 1:
Table 1. tap water sample adds the mensuration of mercury ion
Sequence table
 
<110> Changshu Institute of Technology
 
<120> mono-kind is based on the mercury ion detecting novel method of exonucleaseⅲ
 
<130> xb15062902
 
<160> 4
 
<170> PatentIn version 3.3
 
<210> 1
<211> 60
<212> DNA
<213> Artificial
 
<220>
<223> can to the template DNA of mercury ion specific recognition
 
<400> 1
aatctggttt agctacgcct tccccgtggc gatgtttctt agcgccttac ttgtttgttg 60
 
 
<210> 2
<211> 53
<212> DNA
<213> Artificial
 
<220>
<223> can to the complementary DNA of mercury ion specific recognition
 
<400> 2
cttctttctt gtaaggcgct aagaaacatc gccacgggga aggcgtactc gcg 53
 
 
<210> 3
<211> 22
<212> DNA
<213> Artificial
 
<220>
<223> is used for the upstream primer of quantitative fluorescent PCR
 
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aatctggttt agctacgcct tc 22
 
 
<210> 4
<211> 21
<212> DNA
<213> Artificial
 
<220>
<223> is used for the downstream primer of quantitative fluorescent PCR
 
<400> 4
gtaaggcgct aagaaacatc g

Claims (6)

1., based on a mercury ion detecting novel method for exonucleaseⅲ, it is characterized in that, comprising:
(1) by two sections can to mercury ion specific recognition and the DNA fragmentation being rich in T base mix with testing sample, two Hgs of segment DNA fragment in testing sample 2+the double-stranded DNA that 3' end is closed is combined under effect;
(2) exonuclease III digestion double-stranded DNA;
(3) digested DNA fragmentation is not had to carry out fluorescent quantitative PCR to remaining;
(4) Hg is determined according to fluoroscopic examination result 2+concentration.
2. the mercury ion detecting novel method based on exonucleaseⅲ according to claim 1, is characterized in that: described two sections can to mercury ion specific recognition and the sequence being rich in the DNA fragmentation of T base has nucleotide sequence as shown in SEQ ID NO:1 and SEQ ID NO:2.
3. the mercury ion detecting novel method based on exonucleaseⅲ according to claim 1, is characterized in that: the primer sequence of described fluorescent quantitative PCR has the nucleotide sequence as shown in SEQ ID NO:3 and SEQ ID NO:4.
4. the mercury ion detecting novel method based on exonucleaseⅲ according to claim 1, is characterized in that: the concentration of described mercury ion is 2-200nM.
5. the mercury ion detecting novel method based on exonucleaseⅲ according to claim 4, is characterized in that: the concentration of described mercury ion is 5-100nM.
6. the mercury ion detecting novel method based on exonucleaseⅲ according to claim 1, is characterized in that: based on following linear equation, determines the concentration of mercury ion in described sample:
y=1.03x+4.66，
Y is the cycle number of amplification curve under different ion concentration of mercury, and x is the concentration of corresponding mercury ion.
CN201510382389.5A 2015-07-02 2015-07-02 Novel exonuclease-III-based mercury ion detection method Pending CN104946770A (en)

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CN105352922A (en) * 2016-01-04 2016-02-24 广东省生态环境与土壤研究所 Mercury ion detection method and detection kit using Y-shaped DNA assembling and bonding signal amplification
CN106872682A (en) * 2017-02-17 2017-06-20 济南大学 A kind of colorimetric bio sensor for detecting mercury ion and preparation method thereof
CN107941797A (en) * 2017-12-11 2018-04-20 福州大学 A kind of visual colorimetric determination sensor for detecting mercury ion
CN113088564A (en) * 2021-04-29 2021-07-09 长江大学 Method for detecting mercury ions based on PCR
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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CN103695540A (en) * 2013-12-13 2014-04-02 常熟理工学院 Method for detecting lead ions based on combination of fluorescence quantitative PCR (polymerase chain reaction) and nuclease GR-5
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105352922A (en) * 2016-01-04 2016-02-24 广东省生态环境与土壤研究所 Mercury ion detection method and detection kit using Y-shaped DNA assembling and bonding signal amplification
CN105352922B (en) * 2016-01-04 2017-12-19 广东省生态环境与土壤研究所 A kind of Y shape DNA is assembled the detection method and detection kit that amplification of signal is used for mercury ion
CN106872682A (en) * 2017-02-17 2017-06-20 济南大学 A kind of colorimetric bio sensor for detecting mercury ion and preparation method thereof
CN107941797A (en) * 2017-12-11 2018-04-20 福州大学 A kind of visual colorimetric determination sensor for detecting mercury ion
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
CN113088564A (en) * 2021-04-29 2021-07-09 长江大学 Method for detecting mercury ions based on PCR

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