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Rapid detection kit for diarrhetic shellfish toxin and detection method thereof

A technology for detection kits and shellfish toxins, applied in biochemical equipment and methods, enzymes, measuring devices, etc., can solve the problems of difficult to achieve on-site, rapid detection, cumbersome detection methods, poor stability, etc., and avoid method application limitations. , Improve storage stability and maintain good activity

Active Publication Date: 2018-07-31
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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  • Rapid detection kit for diarrhetic shellfish toxin and detection method thereof
  • Rapid detection kit for diarrhetic shellfish toxin and detection method thereof
  • Rapid detection kit for diarrhetic shellfish toxin and detection method thereof

Examples

Experimental program
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Effect test

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 diarrhetic shellfish toxin and a detection method thereof, and belongs to the field of aquatic product safety testing. The kit includes (1) 96-well microplate with protein phosphatase 2A (PP2A) enzyme gel embedded; (2) an okadaic acid concentration gradient series standard solution; (3) a buffer solution A; (4) a buffer solution B; (5) a substratecolor development solution; and (6) a quality control sample solution. PP2A is immobilized at the bottom of the microplate by use of an improved sol-gel technique, on the basis that the diarrhetic shellfish toxin can inhibit PP2A catalysis effect under alkaline conditions on dephosphorylation reaction of colourless substrate p-nitrophenyl phosphate for production of yellow product p-nitrophenol (pNP), the diarrhetic shellfish toxin can be quantitatively detected according to the dose-response relationship of the absorbance of the pNP at 405 nm and the concentration of the toxin. Sample pretreatment and detection steps are simple, the operation technology is not high in demand, the time is short, the cost is low, the accuracy is high, and the rapid detection kit is suitable for rapid screening of the diarrhetic shellfish toxin in large-scale 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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IPC IPC(8): C12Q1/42C12N11/12C12N11/08C12N11/04
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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