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Novel Brucella antibody detection method

A technology for detection of Brucella and antibodies, which is applied in measuring devices, color/spectral characteristic measurement, instruments, etc., can solve the problems of expensive reagent equipment, low specificity, and long detection cycle, so as to improve detection sensitivity and facilitate Test the use and interpret the objective effect

Pending Publication Date: 2021-11-02
WUXI ZODOLABS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the deficiencies in the prior art, the invention provides a novel Brucella antibody detection method, which solves the problem that the ELISA method and the PCR detection method have higher technical requirements for experimental operators, long detection cycle, and expensive reagent equipment; tiger red The plate agglutination test has low detection sensitivity, low specificity, naked eye interpretation, and large differences in results, which are not conducive to mass promotion and use at the grassroots level

Method used

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  • Novel Brucella antibody detection method

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Embodiment 1

[0020] A kind of novel brucella antibody detection method that an embodiment of the present invention proposes, the preparation method of brucella outer membrane protein OMP22, OMP25, OMP28 comprises the following steps:

[0021] (1) Use gene editing methods to implant OMP22, OMP25, OMP28 gene fragments into plasmids;

[0022] (2) screening single clones by test tube LB medium;

[0023] (3) Take it into a culture tank for expanded culture, and cultivate it at 37 degrees Celsius for 3 to 5 hours;

[0024] (4) IPTG induction, add 1 ml of IPTG (pre-prepared 1Mol / L IPTG, ratio 1:1000), and incubate at 37°C at 200 rpm for 4 hours;

[0025] (5) Centrifuge to collect the thallus precipitate, and ultrasonically break;

[0026] (6) Identify the expression form: Gel electrophoresis, take 40ul of supernatant; resuspend 40ul with 100ul1×PBS for precipitation, add 10ul protein sample buffer (5×) to boiling water for 10min, and run gel electrophoresis with 12% BT concentration ; Identify...

Embodiment 2

[0029] A kind of novel brucellosis antibody detection technique that an embodiment of the present invention proposes, brucellosis outer membrane protein label colloidal gold and latex microsphere comprise the following steps:

[0030] (1) Preparation of 40nm colloidal gold: it is prepared by reducing chloroauric acid with trisodium citrate; 0.5 mL of 1% chloroauric acid is added to 500 mL of deionized water, and after heating to boiling, 0.6 mL of 1% trisodium citrate is added rapidly, and stirred until After the solution turns red completely, continue to heat and stir for 2 minutes, turn off the heat and stop heating, after cooling, add deionized water to make the volume to 500mL, and measure the maximum absorption peak at 525-527.5nm;

[0031] (2) The latex microspheres are purchased commercial red latex microspheres with carboxyl groups, with a particle size of 3 μm, a solid content of 4%, uniformity CV<5%, and a carboxyl content of 180 μmol / g;

[0032] (3) Colloidal gold l...

Embodiment 3

[0035] A new type of brucellosis antibody detection technology proposed by an embodiment of the present invention, serum / plasma buffer solution preparation: take by weighing 2.9g disodium hydrogen phosphate dodecahydrate, 8.0g sodium chloride, 0.24g potassium dihydrogen phosphate, 0.2g Put potassium chloride in a 1L glass beaker, add 800mL ultrapure water and magnetically stir until it is completely dissolved, then set the volume to 1L (adjust the pH to pH7.4).

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Abstract

The invention belongs to the technical field of Brucella antibody detection, and particularly relates to a novel Brucella antibody detection method. According to the method, colloidal gold and latex microspheres are utilized to bridge a brucellosis antibody, the particle size is changed, the maximum absorption and scattering light wavelength change are detected through a spectrophotometer to identify the brucellosis infection condition. Colloidal gold and latex microspheres are respectively used for marking brucellosis outer membrane proteins, and form a 'cross-linked bridge' by capturing brucellosis antibodies, on the basis of an agglutination experiment, the agglutination principle of capturing brucellosis antibodies by adopting the colloidal gold and the latex microspheres is adopted, and a spectrophotometer method is combined for testing results, so that the detection sensitivity is greatly improved, and a result can be obtained within 3-5 minutes. The method has the advantages of high sensitivity, objective interpretation and simple operation, and is convenient for detection and use by basic level personnel.

Description

technical field [0001] The invention relates to the technical field of brucella antibody detection, in particular to a novel brucella antibody detection method. Background technique [0002] Brucellosis (abbreviated as brucellosis) is a zoonotic disease caused by Brucella. It mostly occurs in sheep, cattle, and pigs in livestock, causing high fever, abortion and other symptoms. People exposed to positive sick animals are highly infected risk. The World Health Organization OIE once listed it as a B-type animal disease, and my country listed it as a second-class animal disease. [0003] Commonly used detection methods for brucellosis include tiger bengal plate agglutination test, ELISA detection method, bacterial PCR detection method, etc. However, these methods such as ELISA method and PCR detection method have high technical requirements for experimental operators, long detection cycle, and expensive reagent equipment; tiger red plate agglutination test has low detection s...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N21/31
Inventor 游冬张卓菁梁如中王逸周丽邹晓兰李林
Owner WUXI ZODOLABS BIOTECH
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