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Sodium fluorescence probe detection method of ciguatoxin

A fluorescent probe and sodium ion technology, applied in fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve problems such as low sensitivity, low specificity, and easy interference of detection results, and achieve Sensitive detection, strong specific effect

Active Publication Date: 2013-04-17
SHENZHEN CENTER FOR DISEASE CONTROL AND PREVENTION (SHENZHEN HEALTH INSPECTION CENTER SHENZHEN INSTITUTE OF PREVENTIVE MEDICINE)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of low sensitivity, low specificity, and easily disturbed detection results in the prior art, the present invention provides a ciguatoxin sodium ion fluorescent probe detection method, which can directly detect the concentration of sodium ion flow in cells. Variation to detect ciguatoxin

Method used

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  • Sodium fluorescence probe detection method of ciguatoxin
  • Sodium fluorescence probe detection method of ciguatoxin
  • Sodium fluorescence probe detection method of ciguatoxin

Examples

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Embodiment 1

[0021] Example 1: Detection of ciguatoxin by sodium ion fluorescent probe detection method.

[0022] Cultivate mouse brain neuroma cells (N-2a), wait until the cells grow to 80-90% confluence, digest and collect the cells with 0.25% trypsin-EDTA, centrifuge and remove the supernatant, add RPMI-1640 medium to resuspend cells to a density of 3 x 10 5 cells / mL, inoculate 100 μL of cell suspension in a 96-well black plate, place at 37°C and 5% CO 2 Cultured in a saturated humidity cell incubator. After culturing for 24 h, discard the medium, wash with PBS 2-3 times, add 90 μL ouabain solution and 10 μL ciguatoxin standard (P-CTX-1) to each well of the experimental group. The concentration gradient is 1~10 7 pg / L, a solvent control group and a blank control group were set, and three parallels were set for each test group. After 12 h of exposure, the medium was discarded, and 100 μL of sodium ion fluorescent probe CoroNa at a concentration of 5 μmol / L was added. TM Green stai...

Embodiment 2

[0024] Example 2: Detection of ciguatoxin by sodium ion fluorescent probe detection method.

[0025] Cultivate mouse brain neuroma cells (N-2a), wait until the cells grow to 80-90% confluence, digest and collect the cells with 0.25% trypsin-EDTA, centrifuge and remove the supernatant, add RPMI-1640 medium to resuspend cells to a density of 3 x 10 5 cells / mL, inoculate 100 μL of cell suspension in a 96-well black plate, place at 37°C and 5% CO 2 Cultured in a saturated humidity cell incubator. After culturing for 24 h, discard the medium, wash with PBS 2-3 times, add 90 μL ouabain solution and 10 μL ciguatoxin standard (P-CTX-1) to each well of the experimental group. The concentration gradient is 1~10 7 pg / L, a solvent control group and a blank control group were set, and three parallels were set for each test group. After 18 h of exposure, the medium was discarded, and 100 μL of sodium ion fluorescent probe CoroNa at a concentration of 5 μmol / L was added. TM Green stai...

Embodiment 3

[0027] Example 3: Detection of ciguatoxin by sodium ion fluorescent probe detection method.

[0028] Cultivate mouse brain neuroma cells (N-2a), wait until the cells grow to 80-90% confluence, digest and collect the cells with 0.25% trypsin-EDTA, centrifuge and remove the supernatant, add RPMI-1640 medium to resuspend cells to a density of 3 x 10 5 cells / mL, inoculate 100 μL of cell suspension in a 96-well black plate, place at 37°C and 5% CO 2 Cultured in a saturated humidity cell incubator. After culturing for 24 h, discard the medium, wash with PBS 2-3 times, add 90 μL ouabain solution and 10 μL ciguatoxin standard (P-CTX-1) to each well of the experimental group. The concentration gradient is 1~10 7 pg / L, a solvent control group and a blank control group were set, and three parallels were set for each test group. After 24 h of exposure, the medium was discarded, and 100 μL of sodium ion fluorescent probe CoroNa at a concentration of 5 μmol / L was added. TM Green stai...

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Abstract

The invention relates to a sodium fluorescence probe detection method of ciguatoxin, belonging to the field of fluorescence detection methods. The method comprises the following steps: A) culturing cells, inoculating the cells into a 96-hole black plate and putting the black plate in a cell incubator to culture the cells; B) after culturing the cells for 24 hours, discarding the culture medium, washing the cells 2-3 times with phosphate buffered saline (PBS) and selecting ciguatoxin contaminated cells at different concentrations; C) after contaminating the cells for 12-24 hours, discarding the culture medium, adding a sodium fluorescence probe for dyeing and putting the cells in the cell incubator to be incubated in dark place; D) after washing the cells 2-3 times with balanced salt solution, using a multimode microplate reader to detect the fluorescence intensity of each hole; and E) establishing a standard curve for the sodium fluorescence probe detection method. The method is mainly used for detecting ciguatoxin.

Description

technical field [0001] The invention belongs to the field of fluorescence detection methods, in particular to a detection method of ciguatoxin sodium ion fluorescent probe. Background technique [0002] At present, countries all over the world are actively carrying out research on the detection and prevention of ciguatoxin. There have been many research results of detection and analysis methods. The detection methods of ciguatoxin mainly include: mouse biological method, immunological detection method, high performance liquid chromatography-mass spectrometry method, and traditional cell detection method. [0003] In the late 1990s, foreign researchers reported for the first time that ciguatera toxin can bind to sodium ion channel protein to open the sodium ion channel, and has the properties of sodium ion channel activator. The traditional cell detection method is a technology that utilizes the toxic effect of ciguatera toxin on cells to detect the toxin. The basic princip...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C12Q1/02
Inventor 袁建辉杨慧刘建军徐新云柯跃斌程锦泉庄志雄
Owner SHENZHEN CENTER FOR DISEASE CONTROL AND PREVENTION (SHENZHEN HEALTH INSPECTION CENTER SHENZHEN INSTITUTE OF PREVENTIVE MEDICINE)
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