Preparation method for detecting biotoxicity of nodularin

A biotoxic, nodular algae technology, applied in biochemical equipment and methods, microbial determination/inspection, testing of organic pollutants in water, etc., can solve the problems of low sensitivity, rough results, large consumption of toxins, etc. The effect of good reproducibility, few influencing factors, and simple mortality

Inactive Publication Date: 2017-02-22
TONGJI UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a method for testing nodular toxins in water, aiming at problems such as rough results, low sensitivity, large toxin consumption, and the need for a large number of animal test products in the existing detection methods for nodular toxin biotoxicity. Detection method of coccitoxin biotoxicity

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  • Preparation method for detecting biotoxicity of nodularin
  • Preparation method for detecting biotoxicity of nodularin

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Embodiment 1

[0021] The present invention is applied to the biotoxicity detection of 5 μg / mL nodular toxin (NOD):

[0022] (1) Human umbilical vein endothelial cells were placed at a temperature of 37°C and an air humidity of 95% (containing 5% CO 2 ) in a cell culture box, cultivated in RPMI1640 culture medium containing 20% ​​fetal bovine serum, 60 μg / mL endothelial cell growth supplement and 1% penicillin-streptomycin double antibody.

[0023] (2) Take the cells of passage 3, add phosphate buffer solution (PBS) respectively as blank sample A, add nodularin water sample as test sample B, place on a mixer and shake for 2 hours.

[0024] (3) Wash the cells of samples A and B twice with PBS, and prepare the concentration of about 1×10 5 cells / mL cell suspension;

[0025] (4) Add 5 μL Annexin V-FITC to A and B samples and mix well, then add 5 μL propidium iodide (PI) and mix well; room temperature, dark, reaction 5-15min; within 1 hour, perform flow cytometry Cytometer detection.

[0026...

Embodiment 2

[0031] The present invention is applied to the biotoxicity detection of 10 μg / mL nodular toxin (NOD):

[0032] (1) Human umbilical vein endothelial cells were placed at a temperature of 37°C and an air humidity of 95% (containing 5% CO 2 ) in a cell culture box, cultured in RPMI1640 culture medium containing 10% fetal bovine serum, 60 μg / mL endothelial cell growth supplement and 1% penicillin-streptomycin double antibody.

[0033] (2) Take the cells of passage 3, add phosphate buffer solution (PBS) respectively as blank sample A, add nodularin water sample as test sample B, place on a mixer and shake for 2 hours.

[0034] (3) Wash the cells of samples A and B twice with PBS, and prepare the concentration of about 5×10 5 cells / mL cell suspension,;

[0035] (4) Add 5 μL Annexin V-FITC to A and B samples and mix well, then add 5 μL propidium iodide (PI) and mix well; room temperature, dark, reaction 5-15min; within 1 hour, perform flow cytometry Cytometer detection.

[0036] ...

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Abstract

The invention discloses a preparation method for detecting biotoxicity of nodularin, and provides a detection method for testing the biotoxicity of the nodularin in water. The method comprises the following steps: (1) adding human umbilical vein endothelial cells to a cell incubator that a temperature is at 37 DEG C and an air humidity is 95% (containing 5% of CO2), and cultivating the human umbilical vein endothelial cells in an RPMI1640 culture solution which consists of 10-20% of fetal calf serum, 60 [mu]g/mL of an endothelial cell growth additive and 1% of penicillin-streptomycin double antibodies; and (2) adding a phosphate buffer solution (PBS) to cells which undergo passage for 2-3 generations, so as to obtain a blank sample A, adding a nodularin water sample as a test sample B, conducting oscillating on a mixer for 2h, and the like. According to the method, the biotoxicity of the nodularin is detected by virtue of the human umbilical vein endothelial cells, so that a final result is accurate, stable, good in repeatability and low in content of influencing factors; and the method, which is effective improvement of an existing method, is relatively high in practical value and application potential.

Description

technical field [0001] The invention belongs to the field of detection methods of environmental pollutants, and in particular relates to a detection method of nodular toxin biotoxicity. The method uses human umbilical vein endothelial cells (HUVECs) for detection, and establishes a method for accurately detecting the water sample biotoxicity of nodularin. The final result of the method is accurate, stable and reproducible, and is an effective improvement to the existing method. . Background technique [0002] Cyanotoxins produced during freshwater cyanobacterial blooms threaten the health of aquatic ecosystems, and cyanotoxins are the most serious hazards. Among them, nodularin is a new type of algal toxin discovered recently. Nodular toxin is a cyclic pentapeptide hepatotoxin produced by Nodular algae, which has severe hepatotoxicity, genotoxicity, embryotoxicity, and genotoxicity. It is also a tumor initiator and accelerator, and can be used in biological and Enrichmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N33/18
CPCG01N33/5014G01N33/1826G01N33/5064
Inventor 史俊邓慧萍张为丁超
Owner TONGJI UNIV
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