Paralytic shellfish toxin cell detection method based on alpha-enolase

A technology of α-enolase and shellfish toxin, which can be used in measurement devices, instruments, scientific instruments, etc., can solve the problems of complex sample processing, complicated operation, and high detection cost, and achieve widening application scope, avoiding cross-reaction, and sample processing. simple effect

Active Publication Date: 2016-07-20
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a semi-qualitative analysis method, the mouse biological method is currently the most widely used method in most countries, but it has poor sensitivity and accuracy, which may easily lead to animal ethics issues, etc.
[0005] High-performance liquid chromatography and associated techniques are currently the most widely used non-biological detection methods for paralytic shellfish toxins. The detection accuracy of this method is good, but professional instruments and staff are required. High cost, long detection cycle, analysis of target toxins requires corresponding toxin standards
Chinese patent application 201510772443.7 discloses a method for the determination of paralytic shellfish toxins in aquatic products, which can simultaneously detect 8 paralytic shellfish toxin components, but the operation is more complicated
[0006] The ELISA method is currently a popular and mature antigen-antibody binding experimental method, which has the advantage of convenient operation, but its detection results are easily affected by cross-reactions, resulting in false negative or false positive results. For the quantification of complex matrix or multi-component PSP Detection is difficult and expensive

Method used

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  • Paralytic shellfish toxin cell detection method based on alpha-enolase
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  • Paralytic shellfish toxin cell detection method based on alpha-enolase

Examples

Experimental program
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Effect test

Embodiment 2

[0035] Example two cell culture

[0036] Mouse neuroblastoma N2a cells were cultured in DMED complete medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, at 37°C and 5% CO 2 cultured in a saturated humidity incubator. After the cells grew to a confluence of 80%, the cells were subcultured or exposed to STX, and the subculture ratio was 1:3. Cells used for experiments were in logarithmic growth phase.

Embodiment 3

[0037] Example 3 Extraction of exposed cells and protein samples

[0038]After cultivating N2a cells for 24 hours in a 6-well plate, the STX standard (final concentrations were 0, 1nmol / L, 2nmol / L, 4nmol / L, 6nmol / L, 8nmol / L, 10nmol / L) or the sample was detected Dilute 60 μL of solution with DMEM serum-free medium to a total volume of 2 mL, and then expose N2a cells. After 24 hours of exposure, the exposure is terminated, and the cells in each well are collected by trypsinization. After washing with PBS, the cells are collected by centrifugation, and 200 μL of PhosphoSate is added. , after being lysed on ice for 30 minutes, centrifuged at 14000g for 40 minutes at 4°C, and the supernatant was taken to obtain a protein sample. Store in -20°C refrigerator until use.

Embodiment 4

[0039] Example 4 Investigation and Screening of Differentially Expressed Proteins

[0040] To ensure the repeatability and reliability of 2D-DIGE results, 3 batches of N2a cells were used in each group. The extracted protein samples were quantified using a 2-D protein quantification kit. According to the protein quantification and labeling test results, and in accordance with the CyDyeDIGEFluorLabelingKit instructions, samples were labeled with Cy3 and Cy5, and Cy2 was used as an internal standard to label all sample mixtures. Equal volume of protein is mixed with 2×LysisBuffer, and isoelectric focusing is performed automatically according to the following conditions: Stp30V12h, Stp100V1h, Stp500V1h, Stp1000V1h, Stp5000V4h, Grd8000V4h, Stp8000V4h, Stp500V10h, and the voltage can reach 100000vhs. After focusing, take out the gel strip and carry out the second vertical SDS-PAGE electrophoresis.

[0041] The gel after electrophoresis was scanned by TyphoonTrio fluorescence scann...

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Abstract

The invention provides a paralytic shellfish toxin cell detection method based on alpha-enolase.The detection method comprises the following steps: S1, sample treatment; S2, cell explosure and protein sample extraction; S3, detection, wherein an alpha-enolase ELISA test kit is used for detecting the content of alpha-enolase in the protein sample, standard curves are established, the content of alpha-enolase is calculated through the OD value, and then the content of paralytic shellfish toxin is obtained.The detection method belongs to the technical field of food safety testing.The cell detection method has the advantages that sample treatment is easy, limit of detection is low, repeatability is high, and cross reactions are avoided.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to a cell detection method for paralytic shellfish toxin based on α-enolase. Background technique [0002] Dozens of shellfish toxins have been discovered so far, and the common shellfish toxins mainly include diarrheal shellfish poisoning, paralytic shellfish poisoning (PSP), amnestic shellfish poisoning and neurogenic shellfish poisoning. The composition of PSP is complex, and more than 28 specific toxin components have been identified so far. Among them, saxitoxin (STX) has the most content and is highly toxic. Therefore, researchers generally use STX as the representative of PSP for research. [0003] According to the Mouse Biology Law (MBA), the United Nations Health Organization stipulates that the limit standard of PSP is 80μgSTXeq / 100g, that is, 400MU. After eating aquatic products containing PSP, even if the toxin content does not exceed the stand...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573
CPCG01N33/573G01N2333/988
Inventor 刘建军黄海燕孙烨刘威任晓虎彭朝琼黄新凤
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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