CN114152601B - Method and kit for rapidly detecting mercury ions in water on site and application of kit - Google Patents
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Abstract
The application relates to a method for rapidly detecting mercury ions in water on site, a kit and application thereof, and belongs to the technical field of heavy metal detection. The application is based on cell-free synthesis technology, and a simple in vitro transcription system with mild reaction conditions is prepared, and the system uses an allosteric transcription factor MerR capable of specifically recognizing and combining mercury ions as a control element to control the expression of a downstream fluorescent gene so as to generate green fluorescence. The method disclosed by the application has the characteristics of high sensitivity and strong specificity by utilizing the characteristic of specifically recognizing mercury ions by using the allosteric transcription factor. The application has the advantages of simple system, definite components, no secondary pollution to the environment in field detection, simple preparation, quick field detection, low cost and quick response speed, and can realize the on-site instant detection without instruments.
Description
Technical Field
The application relates to the technical field of heavy metal detection, in particular to a method and a kit for rapidly detecting mercury ions in water on site and application thereof.
Background
With the rapid development of economy and the acceleration of the urban process, the problem of heavy metal pollution in water bodies in China is increasingly serious. Heavy metal pollution is undegradable and easy to be biologically enriched, so that the heavy metal pollution can harm ecological environment, animal and plant systems when reaching a certain concentration in water, and finally, human health can be directly or indirectly endangered through a food chain. Among toxic metal elements, mercury has a first toxicity arrangement, and even if the concentration of mercury is low, the mercury can have serious influence on the neuroendocrine system of the organism. Currently, mercury detection is commonly performed by atomic absorption spectrometry (afm), inductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence spectrometry (R-FS), cold vapor atomic fluorescence spectrometry (CVAF), high Performance Liquid Chromatography (HPLC), and the like, which all require expensive instruments and specialized operators, and can only be performed under laboratory conditions, which is inconvenient for field detection. Allosteric transcription factors (allosteric transcription factor, aTF) are a class of regulatory proteins that are widely distributed in bacteria, typically comprising two domains, an effector sensing domain (EBD) and a DNA Binding Domain (DBD). The small molecule effector alters the conformation of aTF by binding to EBD to enhance or attenuate the binding capacity of aTF to DNA, thereby regulating transcription and expression of the gene. At present, no relevant record is made on realizing the on-site rapid detection of mercury ions by using a cell-free synthesis technology.
Disclosure of Invention
The application aims to provide a method for rapidly detecting mercury ions in water on site, which is based on a cell-free synthesis technology, has the advantages of simple preparation, rapid on-site detection, low cost and rapid response speed, and can remarkably improve the sensitivity and the specificity of mercury ion detection in water.
In order to solve the technical problems, the application provides the following technical scheme:
the application provides a method for rapidly detecting mercury ions on site, which comprises the steps of dripping a solution to be detected into an in-vitro transcription system, and performing fluorescence detection after incubation; the in vitro transcription system takes an allosteric transcription factor MerR as a control element to control the expression of fluorescent genes in downstream double-stranded DNA.
Preferably, the amino acid sequence of the allosteric transcription factor MerR is shown as SEQ ID NO. 1.
Preferably, the volume ratio of the solution to be tested to the in vitro transcription system is 1:10.
preferably, the double-stranded DNA contains an allosteric transcription factor MerR specific recognition sequence.
More preferably, the nucleotide sequence of the double-stranded DNA is shown as SEQ ID NO. 2.
Preferably, the in vitro transcription system further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7RNA polymerase.
The application also provides a kit for rapidly detecting mercury ions on site, which comprises an allosteric transcription factor MerR and double-stranded DNA containing an allosteric transcription factor MerR specific recognition sequence, wherein the downstream of the MerR specific recognition site of the double-stranded DNA contains a fluorescent gene.
Preferably, the kit further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7RNA polymerase.
More preferably, the buffer comprises: sperminine 20mM, tris-HCl400mM, mgCl 2 80mM, naCl200mM and DTT100 mM.
The application also provides an application of the method or the kit in mercury pollutant detection.
The application provides a method for rapidly detecting mercury ions in water on site, which is based on a cell-free synthesis technology and is simple in preparation and mild in reaction condition, and an allosteric transcription factor MerR capable of specifically recognizing and combining mercury ions is used as a control element for controlling the expression of a downstream fluorescent gene to generate green fluorescence. Proved by verification, the method disclosed by the application has the characteristics of high sensitivity and strong specificity by utilizing the characteristic of specifically recognizing mercury ions by using the allosteric transcription factor. The application has the advantages of simple system, definite components, no secondary pollution to the environment in field detection, simple preparation, quick field detection, low cost and quick response speed, and can realize the on-site instant detection without instruments.
Drawings
FIG. 1 is a map of the template plasmid pUC 57.
FIG. 2 is a graph showing the sequencing results after purification of PCR amplification products.
FIG. 3 is a graph showing the results of the reaction for 60min at different mercury ion concentrations.
FIG. 4 is a regression curve of mercury ion concentration versus corresponding fluorescence intensity.
FIG. 5 is a graph showing the results of other heavy metal detection.
FIG. 6 is a graph showing fluorescence contrast generated by other heavy metal detection; wherein PC is positive control and NC is negative control.
Detailed Description
The application provides a method for rapidly detecting mercury ions on site, which comprises the steps of dripping a solution to be detected into an in-vitro transcription system, and performing fluorescence detection after incubation; the in vitro transcription system takes an allosteric transcription factor MerR as a control element to control the expression of fluorescent genes in downstream double-stranded DNA.
In the present application, the allosteric transcription factor MerR is a protein consisting of 144 amino acids, and the amino acid sequence of the MerR is: MENNLENLTIGVFAKAAGVNVETIRFYQRKGLLREPDKPYGSIRRYGEADVVRVKFVKSAQRLGFSLDEIAELLRLDDGTHCEEASSLAEHKLKDVREKMADLARMETVLSELVCACHARKGNVSCPLIASLQGEAGLARSAMP (SEQ ID NO. 1). In the application, the allosteric transcription factor MerR can specifically identify and combine mercury ions, and the detection of the mercury ions is realized by controlling the expression of a fluorescent group at the downstream of double-stranded DNA in an external transcription system. The method for synthesizing the allosteric transcription factor MerR is not particularly limited in the present application, and in the specific embodiment of the present application, the synthesis of the allosteric transcription factor MerR is completed by Kirsrui Biotechnology Co.
In the application, the length of the double-stranded DNA is 343bp, the double-stranded DNA contains an allosteric transcription factor MerR specific recognition sequence, and the nucleotide sequence of the double-stranded DNA is as follows: GCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGATAATACGACTCACTATAGGAGGATATTTACCCTGTACTAAGGTACGTGGTTTATGCTGTAAGTGAGGCCCACATACTCTGATGATCCGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCGGATCATTCATGGCAAGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCTTGCCATGTGTATGTGGGTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG (SEQ ID NO. 2). The method for synthesizing the double-stranded DNA is not particularly limited in the present application, and in the specific embodiment of the present application, the construction work of the vector for obtaining double-stranded DNA is performed by Kirschner Biotech Co., ltd.
In the present application, the in vitro transcription system further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7RNA polymerase. The buffer comprises Sperminine with a final concentration of 20mM, tris-HCl with a final concentration of 400mM, mgCl with a final concentration of 80mM 2 NaCl at a final concentration of 200mM and DTT at a final concentration of 100mM.
The application adds the solution to be tested into the in vitro transcription system, and carries out fluorescence detection after incubation. In the application, the volume ratio of the solution to be tested to the in vitro transcription system is preferably 1:10. in the present application, the temperature of the incubation is preferably 35 to 40 ℃, more preferably 37 ℃; the incubation time is preferably 10 to 70min, more preferably 60min. In the application, the fluorescence detection is preferably ultraviolet detection, and under the ultraviolet condition, if green fluorescence visible by naked eyes is generated, mercury ions are contained in the solution to be detected.
The application also provides a kit for rapidly detecting mercury ions on site, which comprises an allosteric transcription factor MerR and double-stranded DNA containing an allosteric transcription factor MerR specific recognition sequence, wherein the downstream of the MerR specific recognition site of the double-stranded DNA contains a fluorescent gene. In the present application, the kit further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7RNA polymerase. In the present application, the fluorescent dye is preferably DFHBI-1T, and the concentration of the fluorescent dye is preferably 20mM. In the present application, the concentration of dNTPs is preferably 100mM; the concentration of TIPP is preferably 0.3U; the concentration of the T7RNA polymerase is preferably 200U/. Mu.L.
The application also provides an application of the method or the kit in mercury pollutant detection. The kit disclosed by the application is simple in system, clear in components, high in detection sensitivity and strong in specificity, can be used for on-site detection, and has the advantages of rapidness, low cost and high response speed.
In the application, all the raw materials, reagents and equipment are known products, and conventional commercial products are adopted.
In the application, the biological methods such as vector construction, extraction and purification are all adopted by the conventional technical means in the field unless specified.
The technical solutions provided by the present application are described in detail below in conjunction with examples for further illustrating the present application, but they should not be construed as limiting the scope of the present application.
Example 1
Preparation of double-stranded DNA templates
(1) Selection of vector plasmids: selecting pUC57 plasmid as the target gene vector plasmid, wherein the map of pUC57 plasmid is shown in FIG. 1;
(2) Synthesis and amplification of the target gene: the full-length target gene is obtained by the cycle steps of deprotection, activation, coupling, closure and oxidation by using a chemical synthesis method. And then carrying out PCR amplification on the target gene through the upstream primer and the downstream primer.
Wherein, the primer sequence is:
an upstream primer: 5'-GCGGATAACAATTTCACACAGGAAACAGC-3' (SEQ ID NO. 3);
a downstream primer: 5'-CAAAAAACCCCTCAAGACCCG-3' (SEQ ID NO. 4).
PCR amplification procedure: set to 95℃pre-denaturation for 5min,95℃denaturation for 30s,60℃annealing for 50s,72℃extension for 1min,30 cycles; stop at 72℃for 10min.
(3) Gene ligation: plasmid pUC57 and the target gene were digested simultaneously with Xbal and BamHI, and then ligated overnight at 4℃with T4 DNA ligase.
(4) The plasmid was transduced into competent cells DH 5. Alpha. For preservation. After glycerol bacteria were obtained, plasmids were extracted using a plasmid miniprep kit (TIANGEN, DP 103).
(5) The DNA was PCR amplified using a high fidelity PCR kit (biolab, 10104358) and the PCR product was purified using a purification kit (QIAGENDE, 28104). After purification, the concentration was measured using Nanodrop and diluted with double distilled water to a concentration of 0.5 μm and stored at-20 ℃.
(6) The purified template was subjected to sample sequencing, the sequencing work was completed by the biological engineering (Shanghai) Co., ltd, and the sequencing result was shown in FIG. 2.
Example 2
Preparation of allosteric transcription factor MerR
The allosteric transcription factor MerR for specifically recognizing mercury ions is a protein consisting of 144 amino acids, and the amino acid sequence is as follows: MENNLENLTIGVFAKAAGVNVETIRFYQRKGLLREPDKPYGSIRRYGEADVVRVKFVKSAQRLGFSLDEIAELLRLDDGTHCEEASSLAEHKLKDVREKMADLARMETVLSELVCACHARKGNVSCPLIASLQGEAGLARSAMP by Kirschner Biotech Co., ltd.
Example 3
Establishment of in vitro transcription System
(1) Preparation of the buffer: the buffer solution comprises the following components in percentage by weight: sperminine 20mM, tris-HCl400mM pH 8.0, mgCl 2 80mM, 200mM NaCl, 100mM DTT; after the preparation is completed, the refrigerator is preserved at 4 ℃ for standby;
(2) Establishment of an in vitro transcription system: 6.57 mu L of double distilled water, 2 mu L of buffer solution, 0.57 mu L of 100mM dNTPs (ATP, UTP, CTP, GTP) respectively, 0.3U of TIPP (TIPP) 0.15 mu L, 20mM of fluorescent dye DFHBI-1T 3 mu L, 0.5 mu M of double-stranded DNA template 1 mu L, 10 mu M of allosteric transcription factor MerR 0.5 mu L and 200U/mu L T RNA polymerase 2 mu L are sequentially added into a 200 mu L centrifuge tube, and the mixture is uniformly mixed and incubated for 15min at 37 ℃ to ensure that the protein MerR and the DNA template are fully combined.
Example 4
10 experiments were set up, with groups 1-10 being 0nM (negative control), 0.1nM, 0.5nM, 1nM, 5nM, 10nM, 50nM, 100nM, 500nM and 1000nM mercuric ion concentration groups, respectively. Preparing a standard mercury ion solution with a final concentration of 100 mu M by using ultrapure water in a laboratory, obtaining a mercury ion water sample with a corresponding concentration by dilution, and dripping 2 mu L of each sample into the in vitro transcription system described in the example 3 to construct 2-10 groups of reactions; to the system, 2. Mu.L of laboratory ultrapure water containing no mercury ions was added dropwise to constitute a group 1 reaction as a negative control.
20. Mu.L of each reaction system was transferred to 384-well plates (black matrixes), and the generated fluorescence intensities were continuously measured at 37℃using an enzyme-labeled instrument. Selecting a microplate reader FL mode, and setting a microplate reader program: the excitation wavelength is 472nm, the emission wavelength is 507nm, and the fluorescence intensity is measured by selecting the area where the sample adding hole is located. The results of fluorescence intensity generated at 60min for each group are shown in FIG. 3.
When the standard fluorescence intensity value reaches 1, the appearance of fluorescence can be observed with naked eyes, and as can be seen from fig. 3, the detection time is prolonged to 60min, the detection sensitivity can be further improved, and the detection of 0.5nM mercury ions in water can be realized. And the fluorescence intensity has obvious linear relation with the mercury ion concentration within the range of 0.5-500nM, and the regression equation is obtained after taking the logarithm of the mercury ion concentration: y=1.701 x+2.121, r 2 = 0.9916, where Y represents fluorescence intensity, X represents mercury ion concentration logarithm, and regression equation curve is shown in fig. 4.
The detection method can realize quantitative detection of mercury ions in water within the range of 0.5-500nM, and the detection sensitivity can be improved by prolonging the detection time.
Example 5
Detection of other heavy metals
In order to verify the detection specificity of the detection technology, heavy metals such as zinc, copper, lead, cadmium and nickel are used as interferents to be added into a detection system for observing the reaction result.
Setting 8 groups of reactions, respectively: positive control group, negative control group, zinc, copper, lead, cadmium, nickel and mercury ion group. The positive control and negative control groups were the same as in example 4; 2 mu L of zinc and copper ion standard solution with the concentration of 200 mu M is dropwise added into the reaction system to serve as experiment 3 groups and 4 groups; 2. Mu.L of a 10. Mu.M standard solution of lead, cadmium, nickel and mercury ions was added dropwise to the reaction system as 5-8 groups. After incubation at 37℃for 15min, the results were then viewed under UV light and are shown in FIG. 6.
Finally, 20. Mu.L of each reaction system was transferred to 384-well plates (black matrixes), and the generated fluorescence intensities were measured using an enzyme-labeled instrument. Selecting a microplate reader FL mode, and setting a microplate reader program: the excitation wavelength was 472nm, the emission wavelength was 507nm, and the fluorescence intensity was measured by selecting the region where the sample well was located, as shown in FIG. 5.
The embodiment shows that the method can realize detection of 1nM mercury ions by utilizing the characteristic of specifically recognizing the mercury ions by the allosteric transcription factors, has the characteristics of high sensitivity and strong specificity, has the advantages of simple system, definite components, no secondary pollution to the environment in field detection, simple preparation, quick field detection, low cost and quick response speed, and can realize the instrument-free field instant detection.
The foregoing description is only illustrative of the present application and is not intended to limit the scope of the application, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present application.
Sequence listing
<110> environmental medical and working medical institute of military medical institute of academy of military science
<120> method for rapidly detecting mercury ions in water on site, kit and application thereof
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gtccagatat tcgtatctgt cgagtagagt gtgggctctt gccatgtgta tgtgggtagc 300
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Claims (3)
1. A method for rapidly detecting mercury ions on site is characterized in that a solution to be detected is dripped into an in-vitro transcription system, and fluorescence detection is carried out after incubation; the in vitro transcription system takes an allosteric transcription factor MerR as a control element to control the expression of fluorescent genes in downstream double-stranded DNA; the in vitro transcription system further comprises: double distilled water, buffer solution, dNTPs, TIPP, fluorescent dye and T7RNA polymerase; the buffer solution comprises: sperminine 20mM, tris-HCl400mM, mgCl 2 80mM, naCl200mM and DTT100mM; the fluorescent dye is DFHBI-1T, and the concentration of the fluorescent dye is 20mM; the concentration of dNTPs is 100mM; the concentration of TIPP is 0.3U; the concentration of the T7RNA polymerase is200U/μL；
The amino acid sequence of the allosteric transcription factor MerR is shown as SEQ ID NO. 1; the volume ratio of the solution to be tested to the in vitro transcription system is 1:10; the double-stranded DNA contains an allosteric transcription factor MerR specific recognition sequence; the nucleotide sequence of the double-stranded DNA is shown as SEQ ID NO. 2.
2. A kit for rapidly detecting mercury ions on site, which is characterized by comprising an allosteric transcription factor MerR and double-stranded DNA containing an allosteric transcription factor MerR specific recognition sequence, wherein the downstream of the MerR specific recognition site of the double-stranded DNA contains a fluorescent gene;
the amino acid sequence of the allosteric transcription factor MerR is shown as SEQ ID NO. 1; the double-stranded DNA contains an allosteric transcription factor MerR specific recognition sequence; the nucleotide sequence of the double-stranded DNA is shown as SEQ ID NO. 2;
the kit further comprises: double distilled water, buffer solution, dNTPs, TIPP, fluorescent dye and T7RNA polymerase; the buffer solution comprises: sperminine 20mM, tris-HCl400mM, mgCl 2 80mM, naCl200mM and DTT100mM; the fluorescent dye is DFHBI-1T, and the concentration of the fluorescent dye is 20mM; the concentration of dNTPs is 100mM; the concentration of TIPP is 0.3U; the concentration of the T7RNA polymerase was 200U/. Mu.L.
3. Use of the method of claim 1 or the kit of claim 2 in the detection of mercury contaminants.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111628198.4A CN114152601B (en) | 2021-12-28 | 2021-12-28 | Method and kit for rapidly detecting mercury ions in water on site and application of kit |
| ZA2022/04603A ZA202204603B (en) | 2021-12-28 | 2022-04-25 | Method and kit for rapid in-situ detection of mercury ions in water, and use thereof |
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| CN202111628198.4A CN114152601B (en) | 2021-12-28 | 2021-12-28 | Method and kit for rapidly detecting mercury ions in water on site and application of kit |
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| CN114152601B true CN114152601B (en) | 2023-09-15 |
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