Detection method and detection kit for thyroid hormone disrupter

A thyroid hormone and detection method technology, applied in the field of detection methods and detection kits for thyroid hormone interferers, can solve the problems of restricting on-site application, weakening the time and efficiency advantages of the method, and achieving the requirements of maintaining the activity of yeast cells and reducing the detection conditions , the effect of system stability

Pending Publication Date: 2022-03-04
BEIJING NORMAL UNIVERSITY
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0004] However, the application of the traditional recombinant TR gene yeast method in the field of water environment usually needs to be combined with complex sample pretreatment processes, such as enrichment, purification, and concentration of water samples, which greatly weakens the time and efficiency of the method. Advantage
In addition, the traditional method requires the cultivation of yeast cells, and its aseptic operation requirements severely restrict the field application of this method, so it is urgent to improve the method to meet the needs of ready-made rapid and efficient detection

Method used

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  • Detection method and detection kit for thyroid hormone disrupter
  • Detection method and detection kit for thyroid hormone disrupter
  • Detection method and detection kit for thyroid hormone disrupter

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Experimental program
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Effect test

Embodiment 1

[0052] The selection of embodiment 1 yeast immobilization material

[0053] Gelatin (Sigma, USA), polyvinyl alcohol (Shanghai Sinopharm, China), and sodium alginate (Sigma, USA) were selected as alternative materials for yeast immobilization. In order to characterize the toxicity of the material to yeast cells, the acute toxicity test of yeast cells was carried out. The mass concentration of sodium alginate is selected as follows: 1%, 2%, 3%; the mass concentration of polyvinyl alcohol is selected as follows: 0.5%, 1%, 1.5%; the mass concentration of gelatin is selected as follows: 18%, 19%, 20% %; Agar medium with a mass concentration of 2% was selected as a blank control.

[0054] The acute toxicity test procedure is briefly described as follows: sterilize the dissolved culture medium at 121°C in a high-temperature and humid environment for 20 minutes, and then pour (12-15) mL / dish into a plate while it is still hot. Adjust the OD600 value (absorbance value at 600nm) to ab...

Embodiment 2

[0058] The selection of embodiment 2 yeast immobilization mode

[0059] Embedded immobilization method and surface immobilization method were selected as alternative yeast immobilization methods. Prepare OD 600 Values ​​of 0.65, 1 and 2 yeast liquid, take 500 μL of yeast liquid of different densities into sterile eppendorf (EP) tubes. The embedding and fixation experiment group was centrifuged at 4000 rpm for 1 min (Sigma Laborzentrifugen 2K15, Germany), removed the supernatant, added 500 μL of unfixed 2% (w / v) sodium alginate medium after high temperature sterilization, vortexed and oscillated to cool and solidify. In the surface immobilization experiment group, take 500 μL of high-temperature sterilized sodium alginate medium, centrifuge the yeast liquid as described above to remove the supernatant, add 30 μL of SD culture medium, and inoculate the yeast liquid into medium surface. After completing the yeast immobilization by the above two methods, add 500 μL of deionized...

Embodiment 3

[0061] The selection of embodiment 3 yeast breaking method

[0062] The following yeast crushing methods were selected for experiments: papain combined with dextranase crushing method, lysate crushing method, chemical crushing method and physical crushing method, and the crushing method was selected based on the experimental results. The test process can be briefly described as follows: Prepare OD 600 The yeast liquid of 2 was fixed by the surface immobilization method described above, and the reaction system of papain combined with dextranase fragmentation method was: 48 μL 0.8% (w / v, dissolved in DMSO) papain (Beijing Suolaibao, China ), then add 192 μL 0.8% (w / v, dissolved in DMSO) dextranase (Beijing Suolaibao, China), vortex shaker for 1 min, add 40 μL 0.05% NaCl solution (w / v), vortex Shake for more than 1min. The reaction system of the lysate crushing method is as follows: add 40 μL pre-cooled lysate (Beijing Huayueyang Biotechnology Co., Ltd., China), mix well, centr...

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Abstract

The invention relates to a thyroid hormone disrupter detection method and a detection kit, and the detection method comprises the following steps: fixing a yeast on the surface of a culture medium, the yeast containing a thyroid hormone receptor gene, a co-activator gene and a reporter gene; adding a sample to be detected to the surface of the culture medium to react with the yeast; breaking cell walls of the yeast acted on the to-be-detected sample; and detecting the expression level of the reporter gene in the yeast. According to the method for detecting the interference effect of the environmental thyroid hormone of the immobilized yeast, the yeast cells with relatively high density can be directly fixed and covered on the surface of the culture medium, sample testing can be carried out only by directly adding an environmental sample within the storage period of 90 days, water sample pretreatment and temporary yeast cell pre-culture are not needed, and the detection cost is reduced. The detection condition requirements are greatly reduced, and the method can be suitable for field on-site detection.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a detection method and a detection kit for thyroid hormone interferents. Background technique [0002] At this stage, the detection of environmental thyroid hormone disruptors (TDCs) in environmental water samples is mainly based on ectopic detection, and qualitative and quantitative instrumental detection is carried out according to the physical and chemical characteristics of TDCs. It is usually necessary to collect environmental water samples, transport them back to the laboratory, and carry out analysis and testing after pretreatment. It can be seen that the detection of TDCs in environmental water samples at this stage is usually complicated and time-consuming. The most important thing is that the above methods cannot directly give environmental water samples. The information of thyroid hormone interference effect cannot directly judge the thyroid hormone interference toxi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10C12N11/084C12N1/19C12N15/12C12Q1/34C12R1/645
CPCC12N11/10C12N11/084C07K14/721C12Q1/34G01N2333/938
Inventor 刘芸易皓李剑
Owner BEIJING NORMAL UNIVERSITY
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