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Method for quantitatively detecting thyroid hormone in seashell larvae

A technology for quantitative detection of thyroid hormone, applied in the field of biology, can solve the problems of difficult homogenization and different shell content, and achieve the effect of simple method and simple pretreatment steps.

Active Publication Date: 2014-01-22
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems that shellfish larvae samples contain seawater, and the shell content is different in different development stages, and it is not easy to homogenize.

Method used

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  • Method for quantitatively detecting thyroid hormone in seashell larvae
  • Method for quantitatively detecting thyroid hormone in seashell larvae
  • Method for quantitatively detecting thyroid hormone in seashell larvae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] High performance liquid chromatography (HPLC) qualitatively determine thyroid hormones in oysters.

[0032] In order to ensure that the thyroid hormones in oysters are the same substance as human thyroid hormones, the inventors used high performance liquid chromatography to qualitatively analyze the thyroid hormones in oysters. Method conditions refer to [Samanidou, VF, HGGika, et al. (2000). "Rapid Hplc Analysis of ThyroidGland Hormones Tri-Iodothyronine(T3) and Thyroxine(T4) in HumanBiological Fluids after Spe." Journal of Liquid Chromatography&RelatedTechnologies 23(5 ):681-692.]. The present invention uses high-performance liquid chromatograph (Shimadzu, LC-20A), YMC-Pack ODS-AQ chromatographic column (YMC company), and the mobile phase (the substance that carries the component to be tested forward during the chromatographic process) contains 2 % Acetic acid in CH 3 OH-H 2 O (65:35 by volume). The loading volume is 20ul, the flow rate is 1mL / min, and the 240nm ultrav...

Embodiment 2

[0036] Detection of thyroid hormones T4 and T3 in oysters.

[0037] Sample collection: Collect oyster larvae in the oyster nursery, the volume is about 1ml. Directly freeze in liquid nitrogen and transfer to -80℃ refrigerator for storage, and the storage time should not exceed 3 months as much as possible. The present invention uses samples stored for 2 months.

[0038] The sample information is as follows:

[0039] Table 1. Sampling information table of oyster larvae

[0040]

[0041] Test after sample pretreatment:

[0042] After freezing the water-containing larva samples Bla and U7 into solids in liquid nitrogen, grind them into powder with a mortar;

[0043] 1) Put the ground powder sample in a 5ml EP tube, and use a freeze dryer (Beijing Bo Yikang, Model: FD-1-50) to dry for 10 hours to complete dryness at -50°C under dark conditions. Each sample is divided into The two parts of AB, BlaA, BlaB, U7A, U7B are used in the following steps respectively;

[0044] 2) Accurately weigh pa...

Embodiment 3

[0087] Evaluation of thyroid hormone recovery efficiency

[0088] Adopting the method of immersion and standard addition, adding T4 and T3 standard solutions to each sample after grinding with liquid nitrogen, and pretreating the content of T4 and T3 according to the method in Example 2.

[0089] Standard solution configuration: weigh about 0.0010g T4 (Sigma company, product number: T1775) and T3 (Sigma company, product number: T2877) with a one-tenths balance, and dissolve it with the extract (0.01mol / L NaOH solution), Adjust the concentration to 1000ng / ml and 50ng / ml as the spiked solution. After the above-mentioned spiked solution was diluted 10 times, its concentration was detected by the Roche 2010 automatic biochemical analyzer, and the concentration of T4 spiked solution was 1201.36ng / ml, and the concentration of T3 spiked solution was 36.42ng / ml.

[0090] Soaking and adding standard: Grind and freeze-dry the sample U7 at the top of the shell, and divide it into 3 parts (C, D...

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Abstract

The invention relates to the field of biology, and specifically discloses a method for simply, rapidly and accurately measuring thyroid hormone content in a seashell. The method comprises the steps of grinding a larvae sample into powder, drying the larvae sample at lower temperature, dividing the larvae sample into an A part and a B part, accurately weighing the dry weight of the sample of the A part, adding a NaOH solution, soaking for more than 1h, extracting the thyroid hormone, and centrifuging a supernatant to be used for accurately measuring the content of the thyroid hormone; accurately weighing the dry weight of the sample of the B part, then adding a cell cracking solution and PMSF (Phenylmethanesulfonyl fluoride) and standing at room temperature for more than 1h, cracking the cells to release proteins, and detecting the total protein content of a sample; integrating the data, calculating thyroid hormone x gram of protein of each gram in the larvae, namely, xg / g protein. The method provided by the invention has the advantages of being simple, being capable of accurately measuring the thyroid hormone content in a seashell, and being simple in preprocessing step, innoxious in operation and suitable for the ration detection of two thyroid hormones.

Description

Technical field [0001] The invention relates to the field of biology, in particular to a simple, rapid and accurate method for determining the content of thyroid hormone in shellfish. Background technique [0002] Thyroid hormones including thyroxine (T4) and 3,5,3'-triiodothyronine (T3) are widely present in vertebrates and play an important role in growth and development, energy metabolism and metamorphosis. There are many diseases related to thyroid hormones in humans, such as paralysis, goiter, hyperthyroidism and so on. In order to prevent, diagnose, and treat these diseases, there are already a set of very mature detection methods in higher animals such as humans, such as radioimmunoassay, enzyme-linked immunoassay, electrochemiluminescence, etc. [0003] In recent years, the evolution of thyroid hormones has been a hot spot. However, thyroid hormones are rarely studied in the branch of crown animals, especially in shellfish. Direct detection of its content in the research...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/78G01N30/02
CPCG01N5/04G01N30/02G01N2030/027G01N2333/46G01N2333/723
Inventor 黄雯阙华勇许飞李莉张国范
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI