CN114152601A - Method and kit for rapidly detecting mercury ions in water on site and application of kit - Google Patents

Method and kit for rapidly detecting mercury ions in water on site and application of kit Download PDF

Info

Publication number
CN114152601A
CN114152601A CN202111628198.4A CN202111628198A CN114152601A CN 114152601 A CN114152601 A CN 114152601A CN 202111628198 A CN202111628198 A CN 202111628198A CN 114152601 A CN114152601 A CN 114152601A
Authority
CN
China
Prior art keywords
detection
kit
double
merr
site
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202111628198.4A
Other languages
Chinese (zh)
Other versions
CN114152601B (en
Inventor
王景峰
赵辰
张永康
谌志强
邱志刚
薛斌
李辰宇
王尚
张曦
杨晓波
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Environmental Medicine and Operational Medicine Institute of Military Medicine Institute of Academy of Military Sciences
Original Assignee
Environmental Medicine and Operational Medicine Institute of Military Medicine Institute of Academy of Military Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Environmental Medicine and Operational Medicine Institute of Military Medicine Institute of Academy of Military Sciences filed Critical Environmental Medicine and Operational Medicine Institute of Military Medicine Institute of Academy of Military Sciences
Priority to CN202111628198.4A priority Critical patent/CN114152601B/en
Publication of CN114152601A publication Critical patent/CN114152601A/en
Priority to ZA2022/04603A priority patent/ZA202204603B/en
Application granted granted Critical
Publication of CN114152601B publication Critical patent/CN114152601B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明涉及一种现场快速检测水中汞离子的方法、试剂盒及其应用，属于重金属检测技术领域。本发明以无细胞合成技术为基础，制备简单、反应条件温和的体外转录体系，该体系以能够特异性识别和结合汞离子的变构转录因子MerR为控制元件，控制下游荧光基因的表达，产生绿色荧光。本发明所述方法运用变构转录因子特异性识别汞离子的特性，具有灵敏度高和特异性强的特点。并且本发明体系简单，成分明确，在现场检测不会对环境造成二次污染，具有制备简单、可快速现场检测、成本低、响应速度快的优势，能够实现免仪器现场即时检测。

Figure 202111628198

The invention relates to a method, a kit and an application for on-site rapid detection of mercury ions in water, and belongs to the technical field of heavy metal detection. The invention is based on cell-free synthesis technology, and has simple preparation and mild reaction conditions in vitro transcription system. Green fluorescence. The method of the invention uses the characteristics of allosteric transcription factors to specifically identify mercury ions, and has the characteristics of high sensitivity and strong specificity. In addition, the system of the invention is simple, the components are clear, the on-site detection will not cause secondary pollution to the environment, and the invention has the advantages of simple preparation, fast on-site detection, low cost and fast response speed, and can realize on-site instant detection without instruments.

Figure 202111628198

Description

Method and kit for rapidly detecting mercury ions in water on site and application of kit
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
<110> institute of environmental and operational medicine of military medical research institute of military science institute
<120> method and kit for rapidly detecting mercury ions in water on site and application of kit
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 144
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Glu Asn Asn Leu Glu Asn Leu Thr Ile Gly Val Phe Ala Lys Ala
1 5 10 15
Ala Gly Val Asn Val Glu Thr Ile Arg Phe Tyr Gln Arg Lys Gly Leu
20 25 30
Leu Arg Glu Pro Asp Lys Pro Tyr Gly Ser Ile Arg Arg Tyr Gly Glu
35 40 45
Ala Asp Val Val Arg Val Lys Phe Val Lys Ser Ala Gln Arg Leu Gly
50 55 60
Phe Ser Leu Asp Glu Ile Ala Glu Leu Leu Arg Leu Asp Asp Gly Thr
65 70 75 80
His Cys Glu Glu Ala Ser Ser Leu Ala Glu His Lys Leu Lys Asp Val
85 90 95
Arg Glu Lys Met Ala Asp Leu Ala Arg Met Glu Thr Val Leu Ser Glu
100 105 110
Leu Val Cys Ala Cys His Ala Arg Lys Gly Asn Val Ser Cys Pro Leu
115 120 125
Ile Ala Ser Leu Gln Gly Glu Ala Gly Leu Ala Arg Ser Ala Met Pro
130 135 140
<210> 2
<211> 343
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc 60
tgcaggtcga ctctagataa tacgactcac tataggagga tatttaccct gtactaaggt 120
acgtggttta tgctgtaagt gaggcccaca tactctgatg atccgagacg gtcgggtcca 180
gatattcgta tctgtcgagt agagtgtggg ctcggatcat tcatggcaag agacggtcgg 240
gtccagatat tcgtatctgt cgagtagagt gtgggctctt gccatgtgta tgtgggtagc 300
ataacccctt ggggcctcta aacgggtctt gaggggtttt ttg 343
<210> 3
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcggataaca atttcacaca ggaaacagc 29
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
caaaaaaccc ctcaagaccc g 21

Claims (10)

1.一种现场快速检测汞离子的方法，其特征在于，将待测溶液滴加到体外转录体系中，孵育后进行荧光检测；所述体外转录体系以变构转录因子MerR为控制元件，控制下游双链DNA中荧光基因的表达。1. a method for quick detection of mercury ions on site, is characterized in that, solution to be tested is added dropwise to in vitro transcription system, after incubation, fluorescence detection is carried out; Described in vitro transcription system takes allosteric transcription factor MerR as a control element, controls Expression of fluorescent genes in downstream double-stranded DNA. 2.根据权利要求1所述的方法，其特征在于，所述变构转录因子MerR的氨基酸序列如SEQ ID NO.1所示。2 . The method according to claim 1 , wherein the amino acid sequence of the allosteric transcription factor MerR is shown in SEQ ID NO.1. 3 . 3.根据权利要求1所述的方法，其特征在于，所述待测溶液与体外转录体系的体积比为1：10。3 . The method according to claim 1 , wherein the volume ratio of the solution to be tested to the in vitro transcription system is 1:10. 4 . 4.根据权利要求1所述的方法，其特征在于，所述双链DNA中含有变构转录因子MerR特异性识别序列。4 . The method according to claim 1 , wherein the double-stranded DNA contains a specific recognition sequence for the allosteric transcription factor MerR. 5 . 5.根据权利要求4所述的方法，其特征在于，所述双链DNA的核苷酸序列如SEQ ID NO.2所示。5. The method according to claim 4, wherein the nucleotide sequence of the double-stranded DNA is shown in SEQ ID NO.2. 6.根据权利要求1或4所述的方法，其特征在于，所述体外转录体系还包括：双蒸水、缓冲液、dNTPs、TIPP、荧光染料以及T7 RNA聚合酶。The method according to claim 1 or 4, wherein the in vitro transcription system further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase. 7.一种现场快速检测汞离子的试剂盒，其特征在于，所述试剂盒中包括变构转录因子MerR以及含有变构转录因子MerR特异性识别序列的双链DNA，所述双链DNA的MerR特异性识别位点下游含有荧光基因。7. A test kit for on-site rapid detection of mercury ions, characterized in that the test kit comprises allosteric transcription factor MerR and the double-stranded DNA containing the specific recognition sequence of the allosteric transcription factor MerR, and the double-stranded DNA of the double-stranded DNA is The MerR-specific recognition site contains fluorescent genes downstream. 8.根据权利要求7所述的试剂盒，其特征在于，所述试剂盒还包括：双蒸水、缓冲液、dNTPs、TIPP、荧光染料以及T7 RNA聚合酶。8. The kit according to claim 7, wherein the kit further comprises: double distilled water, buffer, dNTPs, TIPP, fluorescent dye and T7 RNA polymerase. 9.根据权利要求8所述的试剂盒，其特征在于，所述缓冲液包括：Spermidine 20mM、Tris-HCl 400mM、MgCl280mM、NaCl 200mM和DTT 100mM。9. The kit according to claim 8, wherein the buffer comprises: Spermidine 20mM, Tris-HCl 400mM, MgCl2 80mM, NaCl 200mM and DTT 100mM. 10.权利要求1-6任一项所述的方法或权利要求7-9任一项所述的试剂盒在汞污染物检测中的应用。10. Use of the method according to any one of claims 1-6 or the kit according to any one of claims 7-9 in the detection of mercury pollutants.
CN202111628198.4A 2021-12-28 2021-12-28 A method, kit and application for rapid on-site detection of mercury ions in water Active CN114152601B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202111628198.4A CN114152601B (en) 2021-12-28 2021-12-28 A method, kit and application for rapid on-site detection of mercury ions in water
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

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111628198.4A CN114152601B (en) 2021-12-28 2021-12-28 A method, kit and application for rapid on-site detection of mercury ions in water

Publications (2)

Publication Number Publication Date
CN114152601A true CN114152601A (en) 2022-03-08
CN114152601B CN114152601B (en) 2023-09-15

Family

ID=80449360

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111628198.4A Active CN114152601B (en) 2021-12-28 2021-12-28 A method, kit and application for rapid on-site detection of mercury ions in water

Country Status (2)

Country Link
CN (1) CN114152601B (en)
ZA (1) ZA202204603B (en)

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4966090A (en) * 1989-02-01 1990-08-24 Genlux Forschungsgesellschaft Fur Biologische Verfahren Mbh Method for detecting the presence of mercury using microorganisms with mercury-enhanced bioluminescence
WO2009045632A2 (en) * 2007-08-10 2009-04-09 The Board Of Trustees Of The University Of Illinois Nucleic acid based fluorescent sensor for mercury detection
WO2011022590A2 (en) * 2009-08-20 2011-02-24 Inter American University Of Puerto Rico Heavy metal remediation system
US8048623B1 (en) * 2002-04-24 2011-11-01 The University Of North Carolina At Greensboro Compositions, products, methods and systems to monitor water and other ecosystems
CN102250819A (en) * 2010-05-18 2011-11-23 天津工业生物技术研究所 Highly-sensitive biosensor cell for detecting heavy metal mercury and manufacturing method thereof
CN102762726A (en) * 2009-11-27 2012-10-31 巴斯夫植物科学有限公司 Chimeric endonucleases and uses thereof
CN104297220A (en) * 2014-04-18 2015-01-21 中国热带农业科学院海口实验站 Detection method and detection device for mercury ions
CN107916243A (en) * 2016-10-10 2018-04-17 中国科学院微生物研究所 For handling microbial cell and albumen, the corresponding processing method and kit of heavy metal pollution
CN108949659A (en) * 2018-07-05 2018-12-07 广西师范学院 Bioengineered strain and its preparation method and application for detecting dimercurion
CN108998400A (en) * 2018-07-05 2018-12-14 广西师范学院 Bioengineered strain and its preparation method and application for restoring dimercurion
CN110241064A (en) * 2019-07-05 2019-09-17 中国农业大学 Construction and application of a nucleic acid-protein complex allosteric microbial whole-cell sensor for mercury ion detection
CN112301154A (en) * 2020-02-06 2021-02-02 广州普世君安生物科技有限公司 RDA method and kit for rapidly detecting respiratory syncytial virus
CN112986197A (en) * 2021-02-18 2021-06-18 军事科学院军事医学研究院环境医学与作业医学研究所 Ratiometric fluorescent probe for detecting mercury ions, fluorescent paper chip and detection method
CN113215327A (en) * 2021-06-25 2021-08-06 军事科学院军事医学研究院环境医学与作业医学研究所 Primer probe group, kit and method for detecting G II type norovirus
CN113234651A (en) * 2021-03-31 2021-08-10 深圳市职业病防治院 Construction and application of mercury ion microorganism whole-cell biosensor taking violacein as output signal
WO2021194567A2 (en) * 2019-12-03 2021-09-30 Northwestern University Ultrasensitive and multiplexed cell-free biosensors using cascaded amplification and positive feedback

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4966090A (en) * 1989-02-01 1990-08-24 Genlux Forschungsgesellschaft Fur Biologische Verfahren Mbh Method for detecting the presence of mercury using microorganisms with mercury-enhanced bioluminescence
US8048623B1 (en) * 2002-04-24 2011-11-01 The University Of North Carolina At Greensboro Compositions, products, methods and systems to monitor water and other ecosystems
WO2009045632A2 (en) * 2007-08-10 2009-04-09 The Board Of Trustees Of The University Of Illinois Nucleic acid based fluorescent sensor for mercury detection
WO2011022590A2 (en) * 2009-08-20 2011-02-24 Inter American University Of Puerto Rico Heavy metal remediation system
CN102762726A (en) * 2009-11-27 2012-10-31 巴斯夫植物科学有限公司 Chimeric endonucleases and uses thereof
CN102250819A (en) * 2010-05-18 2011-11-23 天津工业生物技术研究所 Highly-sensitive biosensor cell for detecting heavy metal mercury and manufacturing method thereof
CN104297220A (en) * 2014-04-18 2015-01-21 中国热带农业科学院海口实验站 Detection method and detection device for mercury ions
CN107916243A (en) * 2016-10-10 2018-04-17 中国科学院微生物研究所 For handling microbial cell and albumen, the corresponding processing method and kit of heavy metal pollution
CN108949659A (en) * 2018-07-05 2018-12-07 广西师范学院 Bioengineered strain and its preparation method and application for detecting dimercurion
CN108998400A (en) * 2018-07-05 2018-12-14 广西师范学院 Bioengineered strain and its preparation method and application for restoring dimercurion
CN110241064A (en) * 2019-07-05 2019-09-17 中国农业大学 Construction and application of a nucleic acid-protein complex allosteric microbial whole-cell sensor for mercury ion detection
WO2021194567A2 (en) * 2019-12-03 2021-09-30 Northwestern University Ultrasensitive and multiplexed cell-free biosensors using cascaded amplification and positive feedback
CN112301154A (en) * 2020-02-06 2021-02-02 广州普世君安生物科技有限公司 RDA method and kit for rapidly detecting respiratory syncytial virus
CN112986197A (en) * 2021-02-18 2021-06-18 军事科学院军事医学研究院环境医学与作业医学研究所 Ratiometric fluorescent probe for detecting mercury ions, fluorescent paper chip and detection method
CN113234651A (en) * 2021-03-31 2021-08-10 深圳市职业病防治院 Construction and application of mercury ion microorganism whole-cell biosensor taking violacein as output signal
CN113215327A (en) * 2021-06-25 2021-08-06 军事科学院军事医学研究院环境医学与作业医学研究所 Primer probe group, kit and method for detecting G II type norovirus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JAEYOUNG K.JUNG 等: "Cell-free biosensors for rapid detection of water contaminants", 《NAT BIOTECHNOL》, vol. 38, no. 12, pages 1451 - 1459, XP037311063, DOI: 10.1038/s41587-020-0571-7 *
SERAPHINE V. WEGNER等: "Design of an Emission Ratiometric Biosensor from MerR Family Proteins:A Sensitive and Selective Sensor for Hg2+", 《J. AM. CHEM. SOC》, no. 129, pages 3474 - 3475 *
冯瑜祥;李相阳;邵向丽;许文涛;: "汞离子传感器检测技术研究进展", 生物技术通报, no. 09, pages 122 - 134 *

Also Published As

Publication number Publication date
CN114152601B (en) 2023-09-15
ZA202204603B (en) 2022-05-25

Similar Documents

Publication Publication Date Title
CN111812066B (en) Biosensors and kits based on CRISPR/Cas12a system and their use in small molecule detection
Li et al. Increasingly branched rolling circle amplification for the cancer gene detection
CN111175268A (en) A kind of fluorescent sensor with double signal amplification for detecting mercury ions and preparation method thereof
CN110468238A (en) A kind of primed probe group, kit and the application of constant-temperature amplification detection A type and influenza B virus
CN106093023A (en) A kind of colorimetric sensor detecting mercury ion and preparation method thereof
US20240141414A1 (en) Cell-free biosensors with dna strand displacement circuits
Zhao et al. Triple-helix molecular-switch-actuated exponential rolling circular amplification for ultrasensitive fluorescence detection of miRNAs
CN114152601A (en) Method and kit for rapidly detecting mercury ions in water on site and application of kit
CN112280877B (en) A CRISPR-Cas13a system for nucleic acid detection of Coxiella béziae
CN112011597B (en) Cadmium ion sensing method combining induced allosteric probe with rolling circle amplification
CN102827933B (en) Kit for qualitative detection of pinewood nematode and detection method thereof
WO2024067478A1 (en) Method for measuring single-molecule rna force spectrum and use thereof
TW201215681A (en) Micro electrochemical multiplex real-time PCR system
CN102925464A (en) Tetrahymena cell line containing luciferase gene, construction method and applications thereof
CN114292955B (en) Method and kit for rapidly detecting lead ions in water on site and application of kit
CN116179654B (en) Rolling circle amplification detection system for detecting cadmium ions in water and application thereof
Duval et al. Site-Directed Chemical Probing to map transient RNA/protein interactions
CN110863033A (en) Standard substance for quantitatively detecting microcystis and preparation and detection methods thereof
Wang et al. An rolling circle amplification-assisted CRISPR/Cas12a-based biosensor for protein detection
CN115725756A (en) RPA primer group, reagent and kit for detecting treponema pallidum and application of RPA primer group, reagent and kit
CN111471749B (en) A kind of ATP detection method and kit
Li et al. Ultrasensitive electrochemical aptasensor based on palindromic sequence mediated bidirectional SDA and a DNAzyme walker for kanamycin detection
CN101975776B (en) Genetic engineering kit for detecting environmental estrogen pollutants and application thereof
CN113832259A (en) Novel rapid detection kit for coronavirus and application thereof
TWI779550B (en) Method and biosensor for lead ions detection

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant