CN114152601A - Method and kit for rapidly detecting mercury ions in water on site and application of kit - Google Patents
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Abstract
The invention relates to a method and a kit for rapidly detecting mercury ions in water on site and application thereof, and belongs to the technical field of heavy metal detection. The invention is based on the cell-free synthesis technology, prepares a simple in-vitro transcription system with mild reaction conditions, and controls the expression of downstream fluorescent genes to generate green fluorescence by taking an allosteric transcription factor MerR which can specifically recognize and combine mercury ions as a control element. The method provided by the invention utilizes the characteristic that the allosteric transcription factor specifically recognizes the mercury ions, and has the characteristics of high sensitivity and strong specificity. The invention has the advantages of simple system, clear components, no secondary pollution to the environment in field detection, simple preparation, quick field detection, low cost and high response speed, and can realize instrument-free field instant detection.
Description
Technical Field
The invention 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 urbanization process, the problem of heavy metal pollution in water in China is increasingly serious. Heavy metal pollution is not degradable and is easy to be biologically enriched, so that the heavy metal pollution can cause harm to the ecological environment and animal and plant systems when reaching a certain concentration in a water body, and finally, the heavy metal pollution can directly or indirectly harm human health through a food chain. Among toxic metal elements, mercury is the first toxic element, and even if the concentration of mercury is very low, the toxic element can have serious influence on systems of the body, such as neuroendocrine and the like. At present, atomic absorption spectrometry, 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 are commonly used for mercury detection, and expensive instruments and professional operators are required for the detection methods, and the detection methods can only be performed under the condition of a laboratory and are inconvenient for field detection. Allosteric transcription factors (aTF) are a class of regulatory proteins that are widely distributed in bacteria and generally comprise two domains, an effector-sensing domain (EBD) and a DNA-binding domain (DBD). The small molecule effector can enhance or reduce binding capacity of aTF and DNA by changing conformation of aTF through binding with EBD, and further regulates transcription and expression of genes. At present, no record related to the on-site rapid detection of mercury ions by using a cell-free synthesis technology exists.
Disclosure of Invention
The invention 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 high response speed, and can significantly improve the sensitivity and specificity of mercury ion detection in water.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a method for rapidly detecting mercury ions on site, which comprises the steps of dropwise adding a solution to be detected into an in vitro transcription system, and carrying out fluorescence detection after incubation; the in vitro transcription system takes an allosteric transcription factor MerR as a control element to control the expression of a fluorescent gene in downstream double-stranded DNA.
Preferably, the amino acid sequence of the allosteric transcription factor MerR is shown in 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 in SEQ ID NO. 2.
Preferably, the in vitro transcription system further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase.
The invention also provides a kit for rapidly detecting mercury ions on site, wherein the kit comprises the allosteric transcription factor MerR and the double-stranded DNA containing the allosteric transcription factor MerR specific recognition sequence, and 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 T7 RNA polymerase.
More preferably, the buffer comprises: spermidine 20mM, Tris-HCl 400mM, MgCl280mM, NaCl 200mM and DTT 100 mM.
The invention also provides the application of the method or the kit in mercury pollutant detection.
The invention provides a method for rapidly detecting mercury ions in water on site, which is based on a cell-free synthesis technology, is used for preparing an in vitro transcription system with simple preparation and mild reaction conditions, and the system takes an allosteric transcription factor MerR capable of specifically recognizing and combining the mercury ions as a control element to control the expression of downstream fluorescent genes and generate green fluorescence. Proved by verification, the method provided by the invention utilizes the characteristic that the allosteric transcription factor is used for specifically recognizing the mercury ions, and has the characteristics of high sensitivity and strong specificity. The invention has the advantages of simple system, clear components, no secondary pollution to the environment in field detection, simple preparation, quick field detection, low cost and high response speed, and can realize instrument-free field instant detection.
Drawings
FIG. 1 is a map of a template plasmid pUC 57.
FIG. 2 is a diagram showing the sequencing results after purification of PCR amplification products.
FIG. 3 is a graph showing the results of fluorescence intensity of 60min reactions with different concentrations of mercury ions.
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 tests.
FIG. 6 is a graph comparing fluorescence generated by detection of other heavy metals; wherein PC is a positive control, and NC is a negative control.
Detailed Description
The invention provides a method for rapidly detecting mercury ions on site, which comprises the steps of dropwise adding a solution to be detected into an in vitro transcription system, and carrying out fluorescence detection after incubation; the in vitro transcription system takes an allosteric transcription factor MerR as a control element to control the expression of a fluorescent gene in downstream double-stranded DNA.
In the invention, the allosteric transcription factor MerR is a protein consisting of 144 amino acids, and the amino acid sequence of the MerR is as follows: MENNLENLTIGVFAKAAGVNVETIRFYQRKGLLREPDKPYGSIRRYGEADVVRVKFVKSAQRLGFSLDEIAELLRLDDGTHCEEASSLAEHKLKDVREKMADLARMETVLSELVCACHARKGNVSCPLIASLQGEAGLARSAMP (SEQ ID NO. 1). In the invention, the allosteric transcription factor MerR can specifically recognize 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 in vitro transcription system. The synthesis method of the allosteric transcription factor MerR is not particularly limited in the present invention, and in the specific embodiment of the present invention, the synthesis of the allosteric transcription factor MerR is performed by kasei biotechnology limited.
In the invention, the length of the double-stranded DNA is 343bp, and 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, and in the specific embodiment of the present invention, the construction of the vector for obtaining the double-stranded DNA is performed by the Kinsley Biotechnology Ltd.
In the present invention, the in vitro transcription system further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase. The buffer solution comprises Spermidine with a final concentration of 20mM, Tris-HCl with a final concentration of 400mM, and MgCl with a final concentration of 80mM2NaCl at a final concentration of 200mM and DTT at a final concentration of 100 mM.
The invention adds the solution to be tested into the in vitro transcription system, and carries out fluorescence detection after incubation. In the present invention, the volume ratio of the solution to be tested to the in vitro transcription system is preferably 1: 10. in the present invention, the incubation temperature is preferably 35 to 40 ℃, more preferably 37 ℃; the incubation time is preferably 10-70min, more preferably 60 min. In the invention, the fluorescence detection is preferably ultraviolet detection, and under the ultraviolet condition, if green fluorescence which can be seen by naked eyes is generated, the mercury ions are contained in the solution to be detected.
The invention also provides a kit for rapidly detecting mercury ions on site, wherein the kit comprises the allosteric transcription factor MerR and the double-stranded DNA containing the allosteric transcription factor MerR specific recognition sequence, and the downstream of the MerR specific recognition site of the double-stranded DNA contains a fluorescent gene. In the present invention, the kit further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase. In the present invention, the fluorescent dye is preferably DFHBI-1T, and the concentration of the fluorescent dye is preferably 20 mM. In the present invention, the concentration of the dNTPs is preferably 100 mM; the concentration of the TIPP is preferably 0.3U; the concentration of the T7 RNA polymerase is preferably 200U/. mu.L.
The invention also provides the application of the method or the kit in mercury pollutant detection. The kit disclosed by the invention is simple in system, clear in components, high in detection sensitivity, strong in specificity, capable of being used for field detection, and has the advantages of high speed, low cost and high response speed.
In the invention, the raw materials, reagents and equipment are known products, and conventional commercial products can be adopted.
In the invention, biological methods such as vector construction, extraction, purification and the like are all performed by adopting conventional technical means in the field if no special description is provided.
In order to further illustrate the present invention, the following embodiments are described in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of double-stranded DNA template
(1) Selection of vector plasmids: selecting a pUC57 plasmid as a vector plasmid of a target gene, wherein the map of the pUC57 plasmid is shown in figure 1;
(2) synthesis and amplification of target gene: the full-length target gene is obtained by a chemical synthesis method through the cyclic steps of deprotection-activation-coupling-blocking-oxidation. And then carrying out PCR amplification on the target gene through upstream and downstream primers.
Wherein, the primer sequence is as follows:
an upstream primer: 5'-GCGGATAACAATTTCACACAGGAAACAGC-3' (SEQ ID NO. 3);
a downstream primer: 5'-CAAAAAACCCCTCAAGACCCG-3' (SEQ ID NO. 4).
PCR amplification procedure: setting the pre-denaturation at 95 ℃ for 5min, the denaturation at 95 ℃ for 30s, the annealing at 60 ℃ for 50s, the extension at 72 ℃ for 1min, and 30 cycles; the reaction was terminated at 72 ℃ for 10 min.
(3) Gene connection: 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 DH5 α for storage. After the glycerol strain is obtained, a plasmid is extracted by using a plasmid miniextraction kit (TIANGEN, DP 103).
(5) The DNA was amplified by PCR using a high fidelity PCR kit (BioLabs,10104358) and the PCR product was purified using a purification kit (QIAGENDE, 28104). After purification, the concentration was measured using Nanodrop and diluted to a concentration of 0.5. mu.M with double distilled water and stored at-20 ℃.
(6) The purified template was sent to sequencing, and the sequencing work was performed by Biotechnology engineering (Shanghai) Co., Ltd. and the sequencing results are 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, manufactured by Kinsley Biotechnology, Inc.
Example 3
Establishment of in vitro transcription System
(1) Preparation of a buffer: the buffer components and final concentrations were: spermidine 20mM, Tris-HCl 400mM at pH 8.0, MgCl280mM, NaCl 200mM, DTT 100 mM; after the preparation is finished, storing the mixture in a refrigerator at 4 ℃ for later use;
(2) establishment of in vitro transcription System: 6.57. mu.L of double distilled water, 2. mu.L of a buffer, 0.57. mu.L of 100mM dNTPs (ATP, UTP, CTP, GTP), 0.3U of TIPP, 0.15. mu.L of 20mM fluorescent dye DFHBI-1T, 3. mu.L of 0.5. mu.M double-stranded DNA template, 0.5. mu.L of 10. mu.M allosteric transcription factor MerR, and 2. mu.L of 200U/. mu. L T7 RNA polymerase were sequentially added to a 200. mu.L centrifuge tube, and after mixing uniformly, the mixture was incubated at 37 ℃ for 15min to allow the protein MerR to bind to the DNA template sufficiently.
Example 4
10 sets of experiments were set up, and 1-10 sets were 0nM (negative control), 0.1nM, 0.5nM, 1nM, 5nM, 10nM, 50nM, 100nM, 500nM and 1000nM mercury ion concentration sets, respectively. Preparing a mercury ion standard solution with the final concentration of 100 mu M by using ultrapure water for a laboratory, diluting to obtain mercury ion water samples with corresponding concentrations, dropwise adding 2 mu L of each mercury ion standard solution into the in-vitro transcription system described in the embodiment 3, and constructing 2-10 groups of reactions; as a negative control, 2. mu.L of laboratory ultrapure water containing no mercury ion was added dropwise to the system to constitute a group 1 reaction.
20. mu.L of each reaction was transferred to a 384-well plate (black matrix), and the intensity of fluorescence generated was continuously measured at 37 ℃ using a microplate reader. Selecting a FL mode of the microplate reader, and setting a microplate reader program: the excitation wavelength is 472nm, the emission wavelength is 507nm, and the area of the sample adding hole is selected to measure the fluorescence intensity. 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 by 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 an obvious linear relation with the mercury ion concentration within the range of 0.5-500nM, and the regression equation is obtained after taking logarithm of the mercury ion concentration: 1.701X +2.121, R20.9916, wherein Y represents fluorescence intensity, X represents the logarithm of mercury ion concentration, and the regression equation curve is shown in FIG. 4.
It can be seen that the detection method can realize quantitative detection of mercury ions in water within the range of 0.5-500nM, and detection sensitivity can be improved by prolonging detection time.
Example 5
Detection of other heavy metals
In order to verify the detection specificity of the detection technology, heavy metals of zinc, copper, lead, cadmium and nickel are added into a detection system as interferents to observe a reaction result.
Set up 8 sets 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; dripping 2 mul of zinc and copper ion standard solution with the concentration of 200 mul into the reaction system to be used as an experiment 3 group and an experiment 4 group; 2 mul of lead, cadmium, nickel and mercury ion standard solution with the concentration of 10 mul is dripped into the reaction system to be used as 5-8 groups. After incubation at 37 ℃ for 15min, the results were examined under UV light and are shown in FIG. 6.
Finally, 20. mu.L of each group of reaction system was transferred to a 384-well plate (black matrix), and the intensity of the generated fluorescence was measured using a microplate reader. Selecting a FL mode of the microplate reader, and setting a microplate reader program: the excitation wavelength is 472nm, the emission wavelength is 507nm, the area of the sample adding hole is selected to measure the fluorescence intensity, and the result is shown in figure 5.
The embodiment shows that the method can realize the detection of 1nM mercury ions by using the characteristic of identifying the mercury ions by the allosteric transcription factor specificity, has the characteristics of high sensitivity and strong specificity, has the advantages of simple system, clear components, no secondary pollution to the environment in field detection, simple preparation, quick field detection, low cost and high response speed, and can realize the instrument-free field instant detection.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
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Claims (10)
1. A method for rapidly detecting mercury ions on site is characterized in that a solution to be detected is dropwise added into an in vitro transcription system, and fluorescence detection is performed after incubation; the in vitro transcription system takes an allosteric transcription factor MerR as a control element to control the expression of a fluorescent gene in downstream double-stranded DNA.
2. The method of claim 1, wherein the amino acid sequence of the allosteric transcription factor MerR is set forth in SEQ ID No. 1.
3. The method of claim 1, wherein the volume ratio of the test solution to the in vitro transcription system is 1: 10.
4. the method of claim 1, wherein the double-stranded DNA comprises an allosteric transcription factor MerR-specific recognition sequence.
5. The method according to claim 4, wherein the nucleotide sequence of the double-stranded DNA is represented by SEQ ID No. 2.
6. The method of claim 1 or 4, wherein the in vitro transcription system further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase.
7. The kit for rapidly detecting the mercury ions on site is characterized by comprising an allosteric transcription factor MerR and double-stranded DNA (deoxyribonucleic acid) containing an allosteric transcription factor MerR specific recognition sequence, wherein the downstream of a MerR specific recognition site of the double-stranded DNA contains a fluorescent gene.
8. The kit of claim 7, further comprising: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase.
9. The kit of claim 8, wherein the buffer comprises: spermidine 20mM, Tris-HCl 400mM, MgCl280mM, NaCl 200mM and DTT 100 mM.
10. Use of the method of any one of claims 1 to 6 or the kit of any one of claims 7 to 9 for 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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