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Brucella antibody immune colloidal gold detection test paper strip and preparing method thereof

A technology for brucellosis and detection test paper, applied in the field of serological detection, can solve the problems of low specificity and low sensitivity of brucellosis detection, and achieve the effects of easy judgment and preservation, high sensitivity and good stability

Inactive Publication Date: 2013-06-26
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to disclose a kind of immunocolloidal gold test strip for detecting Brucella antibody, which solves the shortcomings of low specificity and low sensitivity of current Brucellosis detection

Method used

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  • Brucella antibody immune colloidal gold detection test paper strip and preparing method thereof
  • Brucella antibody immune colloidal gold detection test paper strip and preparing method thereof
  • Brucella antibody immune colloidal gold detection test paper strip and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Preparation of colloidal gold probes

[0027] 1. Materials and methods

[0028] (1) Rabbit anti-bovine IgG was purchased from Beijing Dingguo Biotechnology Co., Ltd.; rabbit anti-bovine IgG and rabbit anti-sheep IgG were purchased from Beijing Yihongan Biotechnology Co., Ltd.; protein quantification kits were purchased from Lianxing Biotechnology Company; The consumables for the colloidal gold immunochromatography method were provided by Shanghai Jiening Biotechnology Company;

[0029] (2) Preparation of colloidal gold

[0030] Using the sodium citrate reduction method, take the silicified Erlenmeyer flask, add 100mL deionized and 1mL 1% chloroauric acid, and heat to boil in a microwave oven; quickly add different doses of 1% trisodium citrate, and a light yellow The aqueous solution of chloroauric acid quickly turns gray, then turns black, and then gradually stabilizes red. The whole process is about 3 minutes, continue to boil for 15 minutes, and make up the volume...

Embodiment 2

[0043] Cloning and expression of Brucella L7 / L12 protein gene and purification of L7 / L12 protein

[0044] 1. Materials and methods

[0045] (1) Strains and plasmids

[0046] Brucella S19, Escherichia coli DH5a, Escherichia coli BL21(DE3) and pET28a+ vectors were preserved by Room 5, No. 11 Academy of Military Medical Sciences, and pMD18-T simple vector was purchased from TakaRa Company.

[0047] (2) Related molecular biology operations

[0048] Bacterial total DNA extraction, PCR amplification, plasmid recombination, Escherichia coli competent preparation, transformation, plasmid extraction and other operations were carried out according to literature [6]; T4 ligase and DNA recovery and purification were carried out according to the kit instructions (purchased from TakaRa Company) ; DNA sequencing was entrusted to Shanghai Sangon Bioengineering Company.

[0049] (3) Amplification and cloning of L7 / L12 gene

[0050] Design an upstream primer with a restriction endonuclease ...

Embodiment 3

[0059] Assembly and testing of highly specific immunocolloidal gold antibody detection test strips for rucelliosis

[0060] 1. Material method

[0061] (1) Material 2992, 903, 8964 type sample pad (sample pad), Ahlstrom 8964 type glass fiber membrane (conjugate release membrane), Sartorius CN140, AE99 and PRIMA60 type nitrocellulose membrane (nitrocellulose membrane), 470 and 2727 type water absorption Pad (absorbent pad), 6cm×30cm, 8cm×30cm type PVC floor are all products of Shanghai Jinbiao Biotechnology Co., Ltd.

[0062] (2) Handling of the sample pad

[0063] Take a high-quality sample pad and immerse it in a solution prepared by adding 0.01mol / L PBS (pH 7.4) and 0.05% Tween-20, soak at 37°C for 30 minutes, take it out and ventilate and dry it at room temperature, and store it in a dry condition for later use.

[0064] (3) Treatment of nitrocellulose membrane

[0065] Soak the nitrocellulose membrane in a methanol solution for 10 minutes, take 0.01mol / LPBS (pH7.4), BSA...

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Abstract

The invention discloses a Brucella antibody immune colloidal-gold detection test strip, wherein, staphylococcus aureus A proteion (SPA) labeled colloidal-gold is made into the colloidal-gold pad of the colloidal-gold antibody detection test strip. L7 / L12 protein is a Brucella protein with high specificity and immunogenicity. L7 / L12 gene is cloned from bovine Brucella genome and is connected to pET-28a to construct the prokaryotic expression recombinant plasmid, then the plasmid is transferred into the Escherichia coli to express L7 / L12 protein, after the protein is purified, the purified protein is used as antigen to coat the nitrate cellulose film to be used as the check line (T line), and the check line and the colloidal-gold pad are assembled to the immune colloidal-gold detection teststrip of the invention. The test strip of the invention can be used for the detection of Brucella antibody in animal serum with strong specificity, high sensitivity, and good stability, and has considerable meaning and actual application value for the monitoring, diagnosis, purification, and control of the Brucella.

Description

technical field [0001] The invention provides a Brucella antibody immune colloidal gold detection test strip, which is used for the detection of Brucella antibody in animals. The invention also discloses a preparation method of the test strip, which belongs to the technical field of serological detection. Background technique [0002] Brucellosis (Brucellosis) is a zoonotic infectious disease caused by small club-shaped Brucella, which is widely prevalent all over the world. The economic loss caused by this disease is as high as billions of dollars every year, and this disease will also pose a serious threat to human health. Since the 1990s, the incidence of human and animal diseases has been on the rise again. [0003] The most effective way to prevent and control the disease is to deploy control, purify the pathogen and early diagnosis, which inevitably requires rapid and effective detection of the disease, but there are two difficulties in the detection of the disease. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/558
Inventor 闫广谋张楠张西臣李建华宫鹏涛
Owner JILIN UNIV
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