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Method for detecting diarrhea shellfish toxin

A shellfish toxin and detection method technology, applied in the preparation of test samples, fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of low sensitivity, poor accuracy, poor comparability and repeatability, etc., and achieve simple sample extraction, Low matrix effect and good specificity

Inactive Publication Date: 2011-02-16
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0005] The mouse biological method is currently the standard detection method for diarrheal shellfish toxins in my country, but its shortcomings are obvious, such as: (1) The ethical issues brought about by the use of animals are strongly opposed by animal protection organizations; (2) The sensitivity is low , poor comparability and repeatability, the test results are related to the strain, body weight and state of the mice, etc., it is difficult to form a unified test standard; (3) the accuracy is poor, the method can only detect the size of the virulence, and cannot determine the composition of the toxin and content, it is easy to cause false positives due to the presence of high-concentration zinc or unsaturated fatty acids; (4) the sample extraction process is complicated
[0006] The ELISA method mainly has the following disadvantages: (1) antibodies are often only aimed at the main components, and their analogs may produce cross-reactions, resulting in false positives or inaccurate estimates of toxicity; (2) the preparation of monoclonal antibodies is difficult, and kits Expensive; (3) There is no commercial ELISA kit available in my country

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  • Method for detecting diarrhea shellfish toxin
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  • Method for detecting diarrhea shellfish toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Establishment of a standard curve for OA-induced F-actin depolymerization in HL-7702 hepatocytes

[0048] In this example, HL-7702 hepatocytes were used as the research object, and the research on the DSP cell detection method was carried out using OA standard products.

[0049] Use Oregon After 514phalloidin fluorescently labeled F-actin, it was observed using a confocal laser microscope.

[0050] experimental method:

[0051] After the cells grew to 80% confluence, they were digested with 0.25% trypsin-EDTA to collect the cells, centrifuged and resuspended (at a density of 5×10 5 cells / mL), inoculate 2 mL of cell suspension into a 6-well plate covered with glass, and culture in an incubator. After culturing for 24 hours, the medium was discarded, and HL-7702 hepatocytes were infected with 5, 10, and 20 nmol / L OA respectively, and methanol was used as a control. After 24 hours of OA exposure, wash with PBS twice, and stain according to the method of Phal...

Embodiment 2

[0071] Example 2 Detection of diarrheal shellfish toxin

[0072] STX, the main component of paralytic shellfish toxin, and DA and YTX, the main components of amnestic shellfish toxin, were selected to act on HL-7702 hepatocytes at different concentrations (5, 10, 20, 40, 80, 160nmol / L) (experimental The method is the same as in Example 1), and the specificity detection of the fluorescence detection method is carried out. Take the different concentrations of STX, DA and YTX as the abscissa, and the fluorescence intensity of each treatment group as the ordinate, such as image 3 as shown, image 3 It shows that other shellfish toxins that may be mixed with diarrheal shellfish toxins will not cause the depolymerization of F-actin in cells, and will not interfere with diarrheal shellfish toxins. SPSS 13.0 statistical software was used for one-way analysis of variance. In the range of 5-160nM, the fluorescence intensity values ​​of STX, DA and YTX were not significantly different...

Embodiment 3

[0075] Repeatability experiment of embodiment 3 cell F-actin detection method

[0076] The 6 shellfish samples were repeatedly detected 3 times, the depolymerization rate was calculated, and the OA concentration was calculated corresponding to the standard curve of the cell F-actin detection method. The results are shown in Table 3. The coefficient of variation is 3.18% to 14.12%. The coefficient is 7.97%.

[0077] Table 3 Repeatability of cell F-actin detection method

[0078]

[0079]

[0080] Table 3 shows that the cell F-actin detection method has good stability and good repeatability in the detection of shellfish samples.

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Abstract

The invention discloses a method for detecting diarrhea shellfish toxin, which comprises the following steps: A) establishing a standard curve of depolymerization of okadaic acid (OA) induced HL-7702 hepatocyte F-actin; B) carrying out the calculation on the concentration of the OA according to the standard curve of the F-actin depolymerization of the HL-7702 hepatocyte, establishing a detection method of the hepatocyte F-actin of the diarrhea shellfish toxin to determine the possible detection limit range; C) selecting one or more of components (STX, DA and YTX) which are possibly coexist with the diarrhea shellfish to act on the HL-7702 hepatocyte, determining the specificity of the method for detecting the OA content by detecting the degree of damaging the polymerizing power of the HL-7702 hepatocyte F-actin by the above components. The detection method of the invention has the advantages: (1) taking cells as experimental subjects to avoid the using of experimental animals and conforming to the rule of 3R; (2) having high sensitivity; (3) having good specificity; (4) having good repeatability; and (5) having simple and convenient sample extraction and low matrix effects.

Description

technical field [0001] The invention belongs to the field of biological detection methods, in particular to a detection method for diarrhea shellfish toxin with high sensitivity. Background technique [0002] Currently, the detection methods for diarrheal shellfish poisoning (DSP) in my country mainly include mouse biological method and enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA). [0003] The detection principle of the mouse biological method: shellfish samples are extracted with acetone and ether, evaporated to dryness under reduced pressure, dissolved in 1% Tween-60 saline and injected into the abdominal cavity of mice, the survival of the mice is observed, and the toxicity is calculated. force. [0004] The detection principle of ELISA: the basis of the determination is the antigen-antibody reaction, the microwell plate is coated with the capture antibody against the okadaic acid (OA) (the main component of DSP) antibody, and the standar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N1/30
Inventor 刘建军黄海燕黄爱君黄薇庄志雄彭朝琼袁建辉
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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