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Method of utilizing diagnostic marker to evaluate early stage toxicity of trace exogenous chemicals and applications thereof

A technology of diagnostic markers and chemicals, applied in the field of detection, can solve the problems of environmental dose polluted water samples, abnormal zebrafish phenotype, and insufficient sensitivity, and achieve the effects of easy promotion and use, predictive significance, and high sensitivity

Inactive Publication Date: 2018-01-16
BIOLOGY INST OF SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for pollutants in water bodies, it is often necessary to reach a certain dose to cause phenotypic abnormalities in zebrafish
In the existing research, in order to highlight the abnormal biological response, the experimenter usually increases the dose, so that although the toxic response can be observed, the so-called environmental dose is often a sample of concentrated water
However, for the prediction of the toxicity of exogenous chemicals, especially the prediction of trace chemicals in the water environment, the existing methods have the defect of insufficient sensitivity

Method used

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  • Method of utilizing diagnostic marker to evaluate early stage toxicity of trace exogenous chemicals and applications thereof
  • Method of utilizing diagnostic marker to evaluate early stage toxicity of trace exogenous chemicals and applications thereof
  • Method of utilizing diagnostic marker to evaluate early stage toxicity of trace exogenous chemicals and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The inventive method compares with conventional general toxicity method:

[0046] (1) Processing method of zebrafish general toxicity test samples

[0047] Collect normally developed zebrafish 2hpf fertilized eggs, put them in a 6-well plate, add 30 eggs to each well, and set 0, 0.01, 0.05, 0.1, 0.25, 1, 4, 10, 40, 100 μM concentration of cadmium chloride exposure group , and each concentration was parallelized 3 times. The microwell plate was placed in a constant temperature and light incubator at 28.5°C to 96hpf, observed every 24 hours, and the number of deaths was recorded.

[0048] (2) Metabolome experiment plus drug treatment

[0049] Take healthy 2hpf (hours after fertilization) zebrafish embryos, set up a blank group (normal sample) and 0.05, 0.1, 0.25μM cadmium chloride exposure groups, and continue to incubate in a 28.5°C incubator after administration.

[0050] (3) Metabolome sample processing

[0051] Collect larvae from each group at 96hpf, 50 zebrafish...

Embodiment 2

[0068] The method established by the present invention compares with the zebrafish behavior evaluation method:

[0069] (1) Using the behavioral prediction method on the zebrafish model:

[0070] Take the juvenile zebrafish with 2hpf development in a 6-well plate, about 15 per well, set up the control group and the 0.05, 0.10, 0.25, 0.50 and 1.00μM cadmium chloride exposure groups, and put them in an incubator at 28.5°C after administration Continue to incubate and perform 3 experimental operations in parallel. Replace the new exposure solution every day, and remove dead fish eggs or juveniles in time. At 96dpf, the juvenile fish were moved into the behavioral movement instrument, and after 5 minutes of adaptation, the spontaneous movement trajectory of the juvenile fish was recorded, and the uninterrupted video was recorded for 10 minutes, and the detection was performed 3 times in parallel.

[0071] (2) Adopt the prediction method of the present invention on the zebrafish ...

Embodiment 3

[0080] A method for predicting early toxicity of exogenous chemicals using a zebrafish model, evaluating the developmental toxicity of lead nitrate:

[0081] Lead nitrate solution to be tested, set 0.001, 0.006, 0.012μM lead nitrate dosage groups, add to the petri dish, put zebrafish embryos 6h after fertilization (hpf), set up the blank group (normal sample) and the sample exposure group in parallel , 50 embryos in each group, put into 28.5 ℃ incubator after administration, when reaching 120hpf, collect the larvae of each group, put in the EP tube with cover, wash three times with pure water, dry the water, add 800 μ L of methanol water mixed solution ( 4:1, -20℃), add steel balls, place in a tissue grinder and crush at 70Hz for 1min; place on ice for 30min; centrifuge at 14000rpm 4℃ for 10min, transfer the supernatant to a new centrifuge tube, and blow with nitrogen Blow dry on the instrument, add 200 μL methanol aqueous solution (1:1, 4°C) to redissolve the sample, and obta...

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Abstract

The invention relates to the detection field, and specifically relates to a method of utilizing a diagnostic marker to evaluate the early stage toxicity of trace exogenous chemicals and applications thereof. The diagnostic marker is one or more of L-valine, N-actyl-L-aspartic acid, lysolecithin, creatinine, and 2-phosphoglyceric acid. People find that exogenous chemicals can break the balance of amino acids in zebra fish and influence the decomposition and metabolism of proteins; the disturbance in the growth period is associated with the mechanism of toxicity; and the inventor finds that thesensitivity of a toxicity predicting method can be largely improved by combining a zebra fish model with metabonomics. Thus, the change of the zebra fish metabolite index can be used to judge the early stage toxicity of exogenous chemicals.

Description

technical field [0001] The invention relates to the field of detection, in particular to a method for evaluating the early toxicity of trace exogenous chemicals by using diagnostic markers and its application. Background technique [0002] The continuous discharge of pollutants has brought huge environmental problems to the world. Pesticides and heavy metals are the main sources of environmental pollution. They reach the water ecosystem through industrial or urban effluents, accumulate in animals and plants, and cause serious harm to human health through the food chain. [0003] The early manifestations of the harm of pollutant exposure to the body are not obvious, and the traditional symptom warning method may not be able to express the occurrence of toxicity, especially in the initial or mild toxicity stage. Once clinically obvious symptoms of toxicity, such as certain cancers, tissue function damage, and neurological disorders, may become irreversible damage and eventual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/88G01N30/86
Inventor 韩利文刘可春何秋霞韩建刘发生李晓彬张轩铭张姗姗张云夏青王雪
Owner BIOLOGY INST OF SHANDONG ACAD OF SCI
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