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A mercury ion detection method and detection device

A detection method and detection device technology, applied in the field of environmental biology, can solve the problems of bulky equipment, high cost, and high technical requirements for operators, and achieve the effects of reducing manpower, high accuracy, and reducing costs

Inactive Publication Date: 2016-08-31
HAIKOU EXPERIMENTAL STATION CHINESE ACAD OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These detection methods are expensive, the equipment is bulky, the operator has high technical requirements, and the samples need to be transported and pre-treated over long distances.

Method used

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  • A mercury ion detection method and detection device
  • A mercury ion detection method and detection device
  • A mercury ion detection method and detection device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The construction method of merR1-GFP fusion gene in this embodiment is as follows:

[0046] (1) Construction of plasmid p1

[0047] LB liquid medium was prepared, and Bacillus megaterium (purchased from Shanghai Beinuo Biotechnology Co., Ltd.) was cultivated overnight at 37°C. For the extraction of Bacillus megaterium genomic DNA, refer to the instructions of the Genomic DNA Extraction Kit of Tienensis Bacteria (Cat. No.: DP302-02) for details. Primers were designed to amplify the merR1 gene, and the primer sequences were as follows:

[0048] P1F: 5'-ATGAAATTTCGTATCGGAGAACTGGCTGAC-3' (shown in SEQ ID NO:3),

[0049] P1R: 5'-TTATTTCTTCATCAGTGTTTCAATAATGGG-3' (as shown in SEQ ID NO: 4);

[0050] PCR reaction system: 2×Pfu MasterMix (Beijing Kangwei Century Biotechnology Co., Ltd., product number: CW0686A) 25μl, primer P1F1pmol, primer P1R1pmol, H 2 O22μl, DNA1μl;

[0051] PCR reaction program: 94°C, 5min, 40 cycles (94°C for 30s, 55°C for 30s, 72°C for 1min), 72°C fo...

Embodiment 2

[0070] The extraction and purification method of merR1-GFP fusion protein in this embodiment is as follows:

[0071] Pick a single clone of BL21-E from the LB plate, and culture it overnight at 37°C in LB medium containing 50 μg / ml kanamycin. Then expand culture in 4 Erlenmeyer flasks each containing 1 liter of LB culture medium at a ratio of 1:100. OD 600 When the growth rate reached 0.6, IPTG with a final concentration of 0.4 mmol / l was added to the culture medium, and the culture was continued for 4 hours. The culture solution was centrifuged at 6000 rpm at 4°C for 10 minutes. Discard the supernatant and wash the cell pellet twice with Buffer A. The formulation of buffer A is 50mmol / l Tris-HCl, pH 7.5, 2mmo / l 2-mercaptoethanol, 5% (v / v) glycerol, 0.2mol / l (NH 4 ) 2 SO4 . Resuspend the cell pellet with 50 mL of Buffer A. The cell suspension was injected into an M-110EH microfluidic homogenizer (Microfluidics, Newton, MA, USA), and the bacterial cells were lysed under ...

Embodiment 3

[0073] The binding properties of merR1-GFP fusion protein to O / Pmerry, the binding properties of fusion protein to mercury ions, and the binding properties of mercury ions to merR1-GFP / Pmerr1-Omerr1 complex:

[0074] (1) Binding test of merR1-GFP fusion protein and Pmerr1-Omerr1

[0075] The genomic DNA of Bacillus megaterium was used as a template for PCR amplification, and the synthesized primers were as follows:

[0076] O / Pmerr1-F: 5'-AGGGTAAGTAAAATCTCATGAATGAAGTAA-3' (shown in SEQ ID NO:9),

[0077] O / Pmerr1-R: 5'-TTCATCGCGATCGACAACCCCTAGCAATTT-3' (as shown in SEQ ID NO: 10);

[0078] PCR reaction system: 2×Pfu MasterMix (Beijing Kangwei Century Biotechnology Co., Ltd., product number: CW0686A) 25μl, primer O / Pmerr1-F1pmol, primer O / Pmerr1-R1pmol, H 2 O22μl, DNA1μl;

[0079] PCR reaction program: 94°C, 5min, 40 cycles (94°C for 30s, 55°C for 30s, 72°C for 1min), 72°C for 10min.

[0080] A 316bp fragment was amplified by PCR, which was Pmerr1-Omerr1. This fragment was...

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Abstract

The invention discloses a detection method and a detection device for mercury ions, and belongs to the technical field of environmental biology. The detection method for the mercury ions comprises the following steps of adding a mercury ion-containing solution in a Pmerr1-Omerr1-biotin double-stranded DNA system in combination with merR1-GFP fusion protein; standing; taking the solution out; measuring fluorescence intensity; and calculating mercury ion content in the solution by using a standard curve. The detection method for the mercury ions has the following advantages that (1) sample detection can be carried out at a sampling point without transporting the sample for a long distance to a specific detection mechanism or using large expensive equipment; time needed for detection is short, a process from sampling the sample to obtaining data at the end can be obtained in 30 minutes, the detection method is particularly suitable for detecting the mercury content in large-scale wild field oil samples, and manpower and material resources required by the detection can be greatly reduced; and (2) a limit value of the mercury ions detected by the method is 0.3 [mu]g / L, which is very close to that detected by a cold atomic absorption spectrometry, and accuracy is high.

Description

technical field [0001] The invention specifically relates to a mercury ion detection method and a detection device for implementing the method, belonging to the field of environmental biotechnology. Background technique [0002] Mercury is a highly toxic heavy metal that can cause neurotoxicity, visual organ defects, etc., and can even cause death in severe cases. Mercury that enters the human body mostly passes through food channels. Mercury in food comes primarily from the soil in which crops grow. The soil mercury content in many places in China has reached the warning level. According to the soil environmental quality standard formulated by my country in 1995 (standard number: GB15618-1995), 1.0mg / kg is the soil limit value for ensuring agricultural production and maintaining human health, and 1.5mg / kg is the soil for ensuring agricultural and forestry production and normal plant growth critical value. [0003] Real-time detection of mercury content in soil is very im...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C12N15/70C07K19/00
Inventor 舒海燕常胜合
Owner HAIKOU EXPERIMENTAL STATION CHINESE ACAD OF TROPICAL AGRI SCI
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