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Enzyme-linked immunoassay reagent kit for detecting aquatic animal spiroplasma

An enzyme-linked immunosorbent reagent and aquatic animal technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex detection experiment operation and difficulty in popularization and application, and achieve convenient use, easy popularization and application, and good repeatability Effect

Inactive Publication Date: 2011-02-09
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of spiroplasma pathogens in aquatic animals can use light microscopy, electron microscopy, microbiology, and molecular biology methods. These techniques have been applied to the routine detection of spiroplasma pathogens in shrimp and crabs in our laboratory. It is complex, requires specific professional skills and equipment, and is difficult to promote and apply in actual production

Method used

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  • Enzyme-linked immunoassay reagent kit for detecting aquatic animal spiroplasma
  • Enzyme-linked immunoassay reagent kit for detecting aquatic animal spiroplasma
  • Enzyme-linked immunoassay reagent kit for detecting aquatic animal spiroplasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: the enzyme-linked immunosorbent assay kit that detects spiroplasma in aquatic animals

[0016] (1) Antigen preparation

[0017] Crab Spiroplasma.CRAB, Lobster Spiroplasma.CRAYFISH and Penaeus vannamei Spiroplasma.SHRIMP were isolated and cultured from Chinese mitten crab with shivering disease, lobster with systemic disease and Penaeus vannamei , the strains after 2-3 generations in R2 medium were freeze-dried or stored at -70°C.

[0018] Antigen preparation: the isolated and cultured shrimp and crab Spiroplasma (Crab Spiroplasma.CRAB, Lobster Spiroplasma.CRAYFISH or Penaeus vannamei Spiroplasma.SHRIMP pathogens, using 0.4% formaldehyde for 12-15 hours Inactivation. Inactivate the pathogen by centrifuging at 12000r / min for 50min, washing, centrifuging, and repeating 3 times to prepare the antigen, and identify the purity of the antigen with an electron microscope, and measure the concentration with a UV spectrophotometer. Then store it in a refrigerator a...

Embodiment 2

[0051] Embodiment 2: the establishment of optimum reaction condition:

[0052] Determination of working concentration in indirect ELISA

[0053] The concentration of fixed antigen is 150μg / ml, and the concentration of serially diluted antiserum is 1:50, 1:250, 1:1250, 1:6250, 1:31250, 1:156250, and the antibody titer and optimal working concentration are determined by indirect ELISA technique ;Use the cross serial dilution method to determine the optimal working concentration of the immune serum, the antigen concentration is constant at 183 μg / ml, and the dilution of the immune serum is adjusted to 1:100, 1:200, 1:400, 1:800, 1:1600, 1: 3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400. After the indirect ELISA reaction, the absorption value at λ=450 was obtained; as shown in Table 1, the titer of the prepared immune serum measured by indirect ELISA was 1:31250; as shown in Table 2, the optimal working concentration was selected as positive / Negative>2.1, the positive result...

Embodiment 3

[0059] Example 3: Sensitivity, specificity, repeatability, shelf life verification of indirect ELISA method

[0060] (1) Sensitivity

[0061] According to the optimal working concentration of the primary antibody determined in Example 2 and the recommended working concentration of the secondary antibody, serially dilute the antigen concentration to determine the minimum detection antigen concentration; the concentration of the immune serum antibody is constant at 1:400; the work of the enzyme-labeled secondary antibody The concentration is constant at 1:3000, and the range of the antigen concentration is 0.573μg / ml-34.2μg / ml to measure the sensitivity of the immune serum to the antigen; the results can be seen in Table 3, the antigen that can be measured by this method is 0.573μg / ml protein quantity.

[0062] Table 3 ELISA detection of different antigen dilutions

[0063]

[0064] ①From the observation of color reaction, the lowest detection level of this method is at the...

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Abstract

The invention relates to an enzyme-linked immunity reagent kit which detects an aquatic animal spiroplasma; the reagent kit comprises peridium solution, cleaning solution, calf serum, one-antiserum, enzyme binder working solution, visualization solution, stop solution, positive control, negative control and an enzyme-labeled plate; the visualization solutions are tetramethy benzidine, citric acid-phosphate buffer solution and hydrogen peroxide; the positive control is a sample comprising spiroplasma; the negative control is the sample which doesn't comprise spiroplasma; the one-antiserum is polyclonal antibody of the aquatic animal spiroplasma; the enzyme binder working solution is a horseradish peroxide enzyme-labeled goat anti-rabbit IgG polyclonal antibody; indirect ELISA method is mainly adopted to detect the spiroplasmas in the blood sample and muscle tissue sample of an aquatic animal. The reagent kit has the advantages of convenience, swiftness, economy, high sensitivity, strong specificity and sound repeatability; furthermore, the reagent kit is in favor of popularization and application and can effectively detect the spiroplasmas in blood sample and muscle tissue sample.

Description

technical field [0001] The invention belongs to the field of biological technology of aquatic products, and in particular relates to an enzyme-linked immunosorbent kit for detecting spiroplasma in aquatic animals. Background technique [0002] Spiroplasma microbes can spread among freshwater crustaceans and other economic aquatic animals (Procambarus clarkii and Penaeus vannamei, etc.), posing a great threat to aquaculture. At present, the detection of spiroplasma pathogens in aquatic animals can use light microscopy, electron microscopy, microbiology, and molecular biology methods. These techniques have been applied to the routine detection of spiroplasma pathogens in shrimp and crabs in our laboratory. It is complex, requires specific professional skills and equipment, and is difficult to promote and apply in actual production. The enzyme-linked immunosorbent assay (ELISA) detection method based on the principle of serological reactions can directly identify the detection...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/543
Inventor 王文王俊海黄桦顾伟吴霆
Owner NANJING NORMAL UNIVERSITY