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A rapid detection kit for diarrheal shellfish toxin and its detection method

A technology for detecting kits and shellfish toxins, applied in biochemical equipment and methods, enzymes, measuring devices, etc., can solve the problems of difficult on-site, rapid detection, cumbersome detection methods, expensive instruments, etc., and avoid the application limitations of the method , Improve storage stability, easy operation

Active Publication Date: 2022-03-01
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem solved by the present invention is to provide a rapid detection kit for diarrheal shellfish toxin and its detection method, so as to overcome the defects of existing detection methods such as cumbersome detection methods, time-consuming detection, expensive instruments, poor stability, and difficulty in realizing on-site and rapid detection.

Method used

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  • A rapid detection kit for diarrheal shellfish toxin and its detection method
  • A rapid detection kit for diarrheal shellfish toxin and its detection method
  • A rapid detection kit for diarrheal shellfish toxin and its detection method

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Embodiment 1

[0034] A kind of diarrhea shellfish toxin rapid detection kit, it comprises:

[0035](1) 96-well microwell plate embedded with PP2A enzyme gel; it can be disassembled into 12 microwell strips, which can test multiple samples at one time, or can be disassembled for separate testing, and the remaining microwell strips can be stored in - 18°C, use next time. The embedding steps of the PP2A enzyme gel are as follows:

[0036] (a) Accurately pipette tetramethoxysilane, 20% D-glucose solution, 1mM HCl solution and polyethylene glycol 600 at a volume ratio of 15:41.3:40:3.7, vortex and mix for 1min, and ultrasonicate at room temperature for 15min to obtain Uniform and transparent sol-gel solution, ready for use;

[0037] (b) After adding 30 μL protein phosphatase 2A enzyme solution, 15 μL 1% hydroxyethyl cellulose solution and 15 μL sol-gel solution prepared in step (1) into a 96-well microwell plate, mix immediately to make it evenly Lay it on the bottom of the microwell plate an...

Embodiment 2

[0044] In the present invention, D-glucose is introduced into the sol-gel preparation process to adjust the micropore volume of the gel, mainly considering that the sol-gel embedding can provide a suitable microenvironment for the enzyme and avoid the occurrence of enzymes due to the spatial structure. However, the barrier effect brought by the embedding material will cause the enzyme, substrate and inhibitor to not be fully contacted, thereby affecting the sensitivity of the method. Therefore the present invention intends to optimize the factors affecting the gel pore size to enhance the contact of PP2A enzymes, substrates and inhibitors and improve the sensitivity of the method. Specifically, 0, 10%, 20% and 30% D-glucose solutions were investigated. effect on enzyme activity.

[0045] figure 1 It is the diagram of the influence of different concentrations of D-glucose solutions on the enzyme activity. When the concentration of D-glucose solution was 0 to 30%, it was found...

Embodiment 3

[0047] Apply the method for the total toxic equivalent of test kit of the present invention to measure diarrhea shellfish toxin, may further comprise the steps:

[0048] (1) Preparation of sample solution: Weigh 2.00g of homogenized shellfish sample, extract the toxin twice with 18mL methanol, vortex for 2min, centrifuge at 8000r / min for 5min, combine the two supernatants and dilute to volume with methanol to 20mL. Accurately pipette 1 mL of the extract, add 125 μL of 2.5 mol / L NaOH solution, seal and mix well, and incubate at 76°C for 40 min. After the solution was cooled to room temperature, 125 μL of 2.5 mol / L HCl solution was added to neutralize it, and blown to nearly dry with nitrogen at 40 °C. Add buffer solution A to the residue to make up to 5mL, vortex and mix well, then test.

[0049] (2) Adding buffer solution B: adding buffer solution B to a 96-well microplate at an amount of 100 μL / well.

[0050] (3) Add okadaic acid concentration gradient series standard solu...

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Abstract

The invention relates to a rapid detection kit for diarrhea shellfish toxin and a detection method thereof. Belonging to the field of aquatic product safety testing, the kit includes: (1) 96-well microwell plates embedded with protein phosphatase 2A (PP2A) enzyme gel; (2) okadaic acid concentration gradient series standard solutions; (3) Buffer solution A; (4) Buffer solution B; (5) Substrate chromogenic solution; (6) Quality control sample solution. The invention adopts improved sol-gel technology to immobilize PP2A enzyme on the bottom of microporous plate, and utilizes diarrhea shellfish toxin to inhibit PP2A enzyme from catalyzing the dephosphorylation of colorless substrate disodium p-nitrophenyl phosphate under alkaline conditions. The yellow product reacts to nitrophenol (pNP), and the toxin is quantitatively detected according to the dose-response relationship between the absorbance of pNP at 405nm and the concentration of the toxin. The invention has simple sample pretreatment and detection steps, low operational technical requirements, short time consumption, low cost and high accuracy, and is suitable for rapid screening of diarrheal shellfish toxins in large batches of shellfish samples.

Description

technical field [0001] The invention belongs to the field of aquatic product safety detection, relates to a rapid detection kit for diarrheal shellfish toxin and a detection method thereof, and is a method for detecting diarrheal shellfish toxin (including okadaic acid and various derivatives thereof) in shellfish. ) total toxic equivalent protein phosphatase inhibition detection kit and detection method thereof. Background technique [0002] Diarrhetic shellfish poisoning (DSP) is the first type of fat-soluble shellfish toxin discovered, which can cause nausea, vomiting, diarrhea and other poisoning symptoms in those who eat it by mistake. The main components include Okadaic acid, OA) and its derivatives Dinophysistoxins (DTXs). The toxic mechanism of diarrheal shellfish toxin lies in its ability to strongly inhibit the activity of protein phosphatases in the cytoplasm, especially protein phosphatase 2A (Proteinphosphatase 2A, PP2A), resulting in the failure of dephosphory...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/42C12N11/12C12N11/089C12N11/04C12N9/16
CPCC12N9/16C12N11/04C12N11/08C12N11/12C12Q1/42C12Y301/03G01N2333/916
Inventor 郭萌萌谭志军陈佳琦吴海燕郑关超彭吉星姚琳翟毓秀
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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