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. Low matrix effect, high sensitivity effect

Inactive Publication Date: 2012-05-09
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
  • Description
  • Claims
  • Application Information

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0048] In this example, HL-7702 hepatocytes were used as the research object, and OA standard was used to study the DSP cell detection method.

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

[0050] experimental method:

[0051] After the cells grew to 80% confluence, 0.25% trypsin-EDTA was digested to collect the cells, centrifuged and resuspended (at a density of 5 × 10). 5 cells / mL), inoculate 2 mL of cell suspension in a 6-well plate with coverslips in advance, 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 control was set. 24h after OA exposure, washed twice with PBS, and stained according to the method of Phallotoxins (Cat. No. O7465).

[005...

Embodiment 2

[0071] Example 2 Diarrheal shellfish toxin detection

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

Embodiment 3

[0075] Example 3 Repeated experiment of cell F-actin detection method

[0076] 6 shellfish samples were repeatedly tested for 3 times, the depolymerization rate was calculated, and the OA concentration was calculated according to the standard curve of the cellular F-actin detection method. The results are shown in Table 3. The coefficient is 7.97%.

[0077] Table 3 Repeatability of F-actin assay in cells

[0078]

[0079]

[0080] Table 3 shows that the cellular 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 method for detecting diarrheal shellfish toxins with high sensitivity. Background technique [0002] At present, the detection methods for diarrheal shellfish poisoning (DSP) in my country mainly include mouse biological method and enzyme linked immunosorbent assay (ELISA). [0003] Detection principle of mouse biological method: Shellfish samples were extracted with acetone and ether, evaporated to dryness under reduced pressure, dissolved in 1% Tween-60 normal saline and injected into the abdominal cavity of mice to observe the survival of mice and calculate the toxicity. force. [0004] The detection principle of ELISA: The basis of the determination is the antigen-antibody reaction. The microplate is coated with the capture antibody against the okadaic acid (OA) (the main component of DSP) antibody, and the standard or sample solution and OA enzyme label are add...

Claims

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Application Information

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Patent Type & Authority Patents(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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