CN114152601B - A method, kit and application for rapid on-site detection of mercury ions in water - Google Patents

Abstract

The invention relates to a method, a kit and an application for rapid on-site detection of mercury ions in water, and belongs to the technical field of heavy metal detection. Based on cell-free synthesis technology, the present invention is an in vitro transcription system that is simple to prepare and has mild reaction conditions. This system uses MerR, an allosteric transcription factor that can specifically recognize and bind mercury ions, as a control element to control the expression of downstream fluorescent genes and produce Green fluorescence. The method of the present invention uses the characteristics of allosteric transcription factors to specifically recognize mercury ions, and has the characteristics of high sensitivity and strong specificity. Moreover, the present invention has a simple system and clear ingredients. On-site detection will not cause secondary pollution to the environment. It has the advantages of simple preparation, rapid on-site detection, low cost, and fast response speed, and can realize on-site instant detection without instruments.

Description

A method, kit and application for rapid on-site detection of mercury ions in water

Technical field

The invention relates to the technical field of heavy metal detection, and in particular to a method, a kit and an application for rapid on-site detection of mercury ions in water.

Background technique

With the rapid development of economy and acceleration of urbanization, the problem of heavy metal pollution in water bodies in my country has become increasingly serious. Heavy metal pollution is non-degradable and easy to bioaccumulate. When it reaches a certain concentration in water, it will harm the ecological environment, animal and plant systems, and ultimately harm human health directly or indirectly through the food chain. Among toxic metal elements, mercury ranks first in toxicity. Even at very low concentrations, it can have serious effects on the body's neuroendocrine and other systems. At present, mercury is commonly detected by atomic absorption spectrometry, inductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence spectrometry (R-FS), cold vapor atomic fluorescence spectrometry (CVAF) and high performance liquid chromatography ( HPLC), etc. These detection methods require expensive instruments and professional operators, and can only be carried out under laboratory conditions, making it inconvenient for on-site detection. Allosteric transcription factor (aTF) is a type of regulatory protein widely distributed in bacteria, which usually contains two domains: effector sensing domain (EBD) and DNA binding domain (DBD). Small molecule effectors change the conformation of aTF by binding to EBD, thereby enhancing or weakening the binding ability of aTF to DNA, thereby regulating gene transcription and expression. At present, there are no relevant records of using cell-free synthesis technology to achieve rapid on-site detection of mercury ions.

Contents of the invention

The purpose of the present invention is to provide a method for rapid on-site detection of mercury ions in water. Based on cell-free synthesis technology, it has the advantages of simple preparation, rapid on-site detection, low cost, and fast response speed, and can significantly improve the sensitivity of mercury ion detection in water. and specificity.

In order to solve the above technical problems, the present invention provides the following technical solutions:

The invention provides a method for rapid on-site detection of mercury ions. The solution to be tested is dropped into an in vitro transcription system, and fluorescence detection is performed after incubation. The in vitro transcription system uses the allosteric transcription factor MerR as a control element to control the downstream dual Expression of fluorescent genes in 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 test solution to the in vitro transcription system is 1:10.

Preferably, the double-stranded DNA contains a specific recognition sequence for the allosteric transcription factor MerR.

More preferably, the nucleotide sequence of the double-stranded DNA is shown in SEQ ID NO. 2.

Preferably, the in vitro transcription system also includes: double-distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase.

The invention also provides a kit for rapid on-site detection of mercury ions. The kit includes an allosteric transcription factor MerR and a double-stranded DNA containing a specific recognition sequence of the allosteric transcription factor MerR. The MerR of the double-stranded DNA A fluorescent gene is contained downstream of the specific recognition site.

Preferably, the kit further includes: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase.

More preferably, the buffer includes: Spermidine 20mM, Tris-HCl 400mM, MgCl ₂ 80mM, NaCl 200mM and DTT 100mM.

The present invention also provides the application of the above-mentioned method or the above-mentioned kit in the detection of mercury pollutants.

The present invention provides a method for rapid on-site detection of mercury ions in water. Based on cell-free synthesis technology, the in vitro transcription system is simple to prepare and has mild reaction conditions. The system uses an allosteric transcription factor that can specifically recognize and bind mercury ions. MerR is a control element that controls the expression of downstream fluorescent genes to produce green fluorescence. It has been verified that the method of the present invention uses the characteristics of allosteric transcription factors to specifically recognize mercury ions, and has the characteristics of high sensitivity and strong specificity. Moreover, the present invention has a simple system and clear ingredients. On-site detection will not cause secondary pollution to the environment. It has the advantages of simple preparation, rapid on-site detection, low cost, and fast response speed, and can realize on-site instant detection without instruments.

Description of the drawings

Figure 1 shows the map of template plasmid pUC57.

Figure 2 shows the sequencing results after purification of PCR amplification products.

Figure 3 shows the fluorescence intensity results of reactions with different mercury ion concentrations for 60 minutes.

Figure 4 is the regression curve of mercury ion concentration and corresponding fluorescence intensity.

Figure 5 shows the detection results of other heavy metals.

Figure 6 is a fluorescence comparison chart generated by other heavy metal detection; among them, PC is a positive control and NC is a negative control.

Detailed ways

The invention provides a method for rapid on-site detection of mercury ions. The solution to be tested is dropped into an in vitro transcription system, and fluorescence detection is performed after incubation. The in vitro transcription system uses the allosteric transcription factor MerR as a control element to control the downstream dual Expression of fluorescent genes in stranded DNA.

In the present invention, the transformation factor Merr is a protein composed of 144 amino acids. DGTHCEEASSLKLKLKLKDVREKMADLMETVLSELVCHARKNVSCPLIASLQGEAGLARSAMP (SEQ ID No.1). In the present invention, the allosteric transcription factor MerR can specifically recognize and bind mercury ions, and realizes the detection of mercury ions by controlling the expression of fluorescent groups downstream of double-stranded DNA in the in vitro recording system. The present invention has no special limitation on the synthesis method of the allosteric transcription factor MerR. In specific embodiments of the present invention, the synthesis of the allosteric transcription factor MerR is completed by Genscript Biotechnology Co., Ltd.

In the present invention, the length of the double-stranded DNA is 343 bp, which contains the specific recognition sequence of the allosteric transcription factor MerR. The nucleotide sequence of the double-stranded DNA is: GCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGATAATACGACTCACTATAGGAGGATATTTTACCCTGTACTAAGGTACGTGGTTTATGCTGTAAGTGAGGCCCACATACTCTGATGATCCGAGACGGTCGGGTCCAG ATATTCGTATCTGTCGAGTAGAGTGTGGGCTCGGATCATTCATGGCAAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCTTGCCATGTGTATGTGGGTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG (SEQ ID NO. 2). The present invention has no special limitations on the synthesis method of the double-stranded DNA. In specific embodiments of the present invention, the construction of the vector for obtaining the double-stranded DNA is completed by Genscript Biotechnology Co., Ltd.

In the present invention, the in vitro transcription system also includes: double-distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase. The buffer includes Spermidine with a final concentration of 20mM, Tris-HCl with a final concentration of 400mM, _MgCl2 with a final concentration of 80mM, NaCl with a final concentration of 200mM and DTT with a final concentration of 100mM.

In the present invention, the solution to be tested is dropped into the in vitro transcription system, and fluorescence detection is performed after incubation. In the present invention, the volume ratio of the test solution to the in vitro transcription system is preferably 1:10. In the present invention, the incubation temperature is preferably 35-40°C, more preferably 37°C; the incubation time is preferably 10-70 min, more preferably 60 min. In the present invention, the fluorescence detection is preferably ultraviolet detection. Under ultraviolet conditions, if green fluorescence visible to the naked eye is produced, it indicates that the solution to be detected contains mercury ions.

The invention also provides a kit for rapid on-site detection of mercury ions. The kit includes an allosteric transcription factor MerR and a double-stranded DNA containing a specific recognition sequence of the allosteric transcription factor MerR. The MerR of the double-stranded DNA A fluorescent gene is contained downstream of the specific recognition site. In the present invention, the kit also includes: 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 20mM. In the present invention, the concentration of the dNTPs is preferably 100mM; the concentration of the TIPP is preferably 0.3U; and the concentration of the T7 RNA polymerase is preferably 200U/μL.

The present invention also provides the application of the above-mentioned method or the above-mentioned kit in the detection of mercury pollutants. The kit system of the invention has a simple system, clear components, high detection sensitivity and strong specificity, can be used for on-site detection, and has the advantages of rapidity, low cost and fast response speed.

In the present invention, the raw materials, reagents and equipment used are all known products, and conventional commercially available products can be used.

In the present invention, biological methods such as vector construction, extraction, and purification are all conventional technical means in the field, unless otherwise specified.

In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the protection scope of the present invention.

Example 1

Preparation of double-stranded DNA templates

(1) Selection of vector plasmid: Select pUC57 plasmid as the vector plasmid of the target gene. The map of pUC57 plasmid is shown in Figure 1;

(2) Synthesis and amplification of target gene: Use chemical synthesis method to obtain the full-length target gene through the cycle steps of deprotection-activation-coupling-blocking-oxidation. The target gene is then PCR amplified through upstream and downstream primers.

Among them, the primer sequence is:

Upstream primer: 5’-GCGGATAACAATTTCACACAGGAAACAGC-3’ (SEQ ID NO.3);

Downstream primer: 5’-CAAAAAACCCCTCAAGACCCG-3’ (SEQ ID NO. 4).

PCR amplification program: Set to pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 60°C for 50 s, extension at 72°C for 1 min, 30 cycles; terminate at 72°C for 10 min.

(3) Gene ligation: Plasmid pUC57 and the target gene were digested by Xbal and BamHI at the same time, and then ligated overnight at 4°C under the action of T4 DNA ligase.

(4) Transfer the plasmid into competent cells DH5α for storage. After obtaining the glycerol bacteria, the plasmid was extracted using a plasmid miniprep kit (TIANGEN, DP103).

(5) Use a high-fidelity PCR kit (BioLabs, 10104358) to amplify DNA by PCR, and use a purification kit (QIAGENDE, 28104) to purify the PCR product. After purification, use Nanodrop to measure the concentration, dilute with double-distilled water to a concentration of 0.5 μM and store at -20°C.

(6) Send the purified template for sequencing, and the sequencing work is completed by Sangon Bioengineering (Shanghai) Co., Ltd. The sequencing results are shown in Figure 2.

Example 2

Preparation of allosteric transcription factor MerR

MerR, an allosteric transcription factor that specifically recognizes mercury ions, is a protein composed of 144 amino acids. The amino acid sequence is: MENNLENLTIGVFAKAAGVNVETIRFYQRKGLLREPDKPYGSIRRYGEADVVRVKFVKSAQRLGFSLDEIAELLRLDDGTHCEEASSLAEHKLKDVREKMADLARMETVLSELVCACHARKGNVSCPLIASLQGEAGLARSAMP, completed by Genscript Biotechnology Co., Ltd.

Example 3

Establishment of in vitro transcription system

(1) Preparation of buffer: The buffer components and final concentration are: Spermidine 20mM, Tris-HCl 400mM with pH 8.0, MgCl ₂ 80mM, NaCl 200mM, and DTT 100mM; after preparation, store in the refrigerator at 4°C for later use;

(2) Establishment of in vitro transcription system: Add 6.57 μL of double-distilled water, 2 μL of buffer, 0.57 μL each of 100mM dNTPs (ATP, UTP, CTP, GTP), 0.3U TIPP 0.15 μL, and 20mM fluorescent dye in a 200 μL centrifuge tube. DFHBI-1T 3μL, 0.5μM double-stranded DNA template 1μL, 10μM allosteric transcription factor MerR 0.5μL, 200U/μL T7 RNA polymerase 2μL, mix evenly and incubate at 37°C for 15 minutes to fully combine the protein MerR with the DNA template. .

Example 4

Set up 10 groups of experiments, groups 1-10 are respectively 0nM (negative control group), 0.1nM, 0.5nM, 1nM, 5nM, 10nM, 50nM, 100nM, 500nM and 1000nM mercury ion concentration groups. Use laboratory ultrapure water to prepare a mercury ion standard solution with a final concentration of 100 μM, and obtain mercury ion water samples with corresponding concentrations by dilution. 2 μL of each was added dropwise to the in vitro transcription system described in Example 3 to construct 2-10 Group 1 reaction: Add 2 μL of mercury ion-free laboratory ultrapure water into the system to form the first group reaction as a negative control.

Transfer 20 μL of each reaction system to a 384-well plate (black bottom), and use a microplate reader to continuously measure the fluorescence intensity generated at 37°C. Select the FL mode of the microplate reader, set the microplate reader program: the excitation wavelength is 472nm, the emission wavelength is 507nm, select the area where the sampling hole is located to measure the fluorescence intensity. The fluorescence intensity results produced by each group at 60 minutes are shown in Figure 3.

When the standard fluorescence intensity value reaches 1, the appearance of fluorescence can be observed with the naked eye. As can be seen from Figure 3, extending the detection time to 60 minutes can further improve the detection sensitivity and achieve the detection of 0.5nM mercury ions in water. And the fluorescence intensity has an obvious linear relationship with mercury ion concentration in the range of 0.5-500nM. After taking the logarithm of the mercury ion concentration, the regression equation is obtained: Y = 1.701*X + 2.121, R ² = 0.9916, where Y represents the fluorescence intensity. , X represents the logarithm of mercury ion concentration, and the regression equation curve is shown in Figure 4.

It can be seen that the detection method of the present application can achieve quantitative detection of mercury ions in water in the range of 0.5-500nM, and extending the detection time can improve the detection sensitivity.

Example 5

Detection of other heavy metals

In order to verify the detection specificity of this detection technology, heavy metals zinc, copper, lead, cadmium, and nickel were added into the detection system as interference substances to observe the reaction results.

Set up 8 groups of reactions, namely: positive control group, negative control group, zinc, copper, lead, cadmium, nickel and mercury ion groups. The positive control and negative control groups were the same as those in Example 4; 2 μL of zinc and copper ion standard solutions with a concentration of 200 μM were added dropwise to the reaction system as experimental groups 3 and 4; 2 μL of a standard solution of zinc and copper ions with a concentration of 10 μM was added dropwise to the reaction system. Standard solutions of lead, cadmium, nickel, and mercury ions are used as groups 5-8. Then, after incubating at 37°C for 15 minutes, the results were viewed under UV. The results are shown in Figure 6.

Finally, transfer 20 μL of the reaction system from each group to a 384-well plate (black bottom), and use a microplate reader to measure the fluorescence intensity generated. Select the FL mode of the microplate reader and set the microplate reader program: the excitation wavelength is 472nm and the emission wavelength is 507nm. Select the area where the sampling hole is located to measure the fluorescence intensity. The results are shown in Figure 5.

It can be seen from the above examples that the method of the present invention uses the characteristics of allosteric transcription factors to specifically recognize mercury ions, can realize the detection of 1 nM mercury ions, and has the characteristics of high sensitivity and strong specificity, and the system of the present invention It is simple and has clear ingredients. On-site detection will not cause secondary pollution to the environment. It has the advantages of simple preparation, rapid on-site detection, low cost, and fast response speed, and can achieve on-site instant detection without instruments.

The above are only examples of the present invention, and do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the content of the description of the present invention, or directly or indirectly applied in other related technical fields, shall be regarded as Likewise, it is included in the patent protection scope of the present invention.

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Claims (3)

1. A method for rapid on-site detection of mercury ions, characterized in that the solution to be tested is added dropwise to an in vitro transcription system, and fluorescence detection is performed after incubation; the in vitro transcription system uses the allosteric transcription factor MerR as a control element to control Expression of fluorescent genes in downstream double-stranded DNA; the in vitro transcription system also includes: double-distilled water, buffer, dNTPs, TIPP, fluorescent dyes and T7 RNA polymerase; the buffer includes: Spermidine20mM, 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 T7 RNA polymerase is 200U /μL; The amino acid sequence of the allosteric transcription factor MerR is shown in SEQ ID NO.1; the volume ratio of the test solution to the in vitro transcription system is 1:10; the double-stranded DNA contains the specificity of the allosteric transcription factor MerR Recognition sequence; the nucleotide sequence of the double-stranded DNA is shown in SEQ ID NO.2. 2. A kit for rapid on-site detection of mercury ions, characterized in that the kit includes the allosteric transcription factor MerR and a double-stranded DNA containing a specific recognition sequence of the allosteric transcription factor MerR, and the double-stranded DNA The MerR specific recognition site contains a fluorescent gene downstream; The amino acid sequence of the allosteric transcription factor MerR is shown in SEQ ID NO. 1; the double-stranded DNA contains a specific recognition sequence for the allosteric transcription factor MerR; the nucleotide sequence of the double-stranded DNA is shown in SEQ ID NO. .2 shown; The kit also includes: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase; the buffer includes: Spermidine 20mM, Tris-HCl 400mM, MgCl ₂ 80mM, NaCl 200mM and DTT 100mM; the fluorescent dye is DFHBI-1T, the concentration of the fluorescent dye is 20mM; the concentration of dNTPs is 100mM; the concentration of TIPP is 0.3U; the concentration of T7RNA polymerase is 200U/μL. 3. Application of the method of claim 1 or the kit of claim 2 in the detection of mercury contaminants.