Immune colloidal gold test paper for detecting mycobacterium bovis antibody and preparation method thereof

A technology for detecting Mycobacterium tuberculosis and test paper, which is applied in the field of serological detection, can solve the problems of inability to distinguish between wild virus infection and artificial infection, low specificity and low sensitivity of Mycobacterium tuberculosis detection, and achieve easy judgment and storage, rapid detection, The effect of high sensitivity

Inactive Publication Date: 2010-09-29
JILIN UNIV
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  • Description
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AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to disclose an immune colloidal gold test strip for detecting Mycobacterium tuberculosis antibody, which solves the shortcomings of low specificity, inacc

Method used

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  • Immune colloidal gold test paper for detecting mycobacterium bovis antibody and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Preparation of colloidal gold probes

[0017] 1. Materials and methods

[0018] (1) Rabbit anti-bovine IgG was purchased from Beijing Dingguo Biotechnology Co., Ltd.; rabbit anti-bovine IgG was purchased from Beijing Yihongan Biotechnology Co., Ltd.; protein quantification kit was purchased from Lianxing Biotechnology Company.

[0019] (2) Preparation of colloidal gold

[0020] 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 to 100mL with deionized water after cooling. The average diameter, degree of dispersion and uniformity of colloidal gold particles were observed under e...

Embodiment 2

[0033] Cloning, expression and purification of CFP10 and MPT64 protein genes of Mycobacterium tuberculosis

[0034] 1. Materials and methods

[0035] (1) Strains and plasmids

[0036] Mycobacterium tuberculosis S19, Escherichia coli DH5a, Escherichia coli BL21(DE3) and pET28a+ vectors were preserved by Room 5, No. 11 Institute of Military Medical Sciences, and pMD18-T simple vector was purchased from TakaRa Company.

[0037] (2) Related molecular biology operations

[0038] The extraction of total bacterial DNA, PCR amplification, plasmid recombination, Escherichia coli competent preparation, transformation, and plasmid extraction were carried out according to the methods described in "Molecular Cloning"; the recovery and purification of T4 ligase and DNA were carried out according to the kit instructions (purchased From TakaRa Company); DNA sequencing was entrusted to Shanghai Sangon Bioengineering Company.

[0039] (3) Amplification and cloning of CFP10 and MPT64 genes

...

Embodiment 3

[0053] Assembly and testing of tuberculosis highly specific immune colloidal gold antibody test strips

[0054] 1. Material method

[0055] (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 Absorbent pad, 6cm×30cm, 8em×30cm PVC floor are all products of Shanghai Jinbiao Biotechnology Co., Ltd.

[0056] (2) Handling of the sample pad

[0057] 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.

[0058] (3) Treatment of nitrocellulose membrane

[0059] Soak the nitrocellulose membrane in methanol solution for 10 minutes, take 0.01mol / 2222 LPBS (pH7.4), BSA, Tween-20, NaCl, su...

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Abstract

The invention discloses immune colloidal gold test paper for detecting mycobacterium bovis antibody and a preparation method thereof. Staphylococal Protein A (SPA) marks colloidal gold to prepare a gold colloidal pad of the immune colloidal gold test paper. The proteins of CFP10 and MPT64 are immunogenic proteins with high specificity to virulent mycobacterium tuberculosis. The genes of CFP10 andMPT64 are cloned from the genome of the mycobacterium tuberculosis, and are connected to pET-28a to construct two prokaryotic expression recombinant plasmids; the two plasmids are converted into Escherichia coli to express the proteins of CFP10 and MPT64; after the proteins are purified, the mixed proteins are taken as antigens to coat a nitrocellulose membrane to form a detection line; and the detection line is equipped with the gold colloidal pad to form the immune colloidal gold test paper. The test paper can distinguish that human bodies and animals are inoculated with BCG or subjected towild virus infection, can be used for detecting the mycobacterium tuberculosis antibody in animal serum, has the characteristics of strong specificity, high sensitivity, good stability, convenience and quickness, and has significance and actual application value for monitoring, diagnosing, purifying and controlling the mycobacterium tuberculosis.

Description

technical field [0001] The invention provides an immune colloidal gold detection test strip for the Mycobacterium bovis antibody, which is used for detecting the Mycobacterium bovis 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] The detection of bovine tuberculosis in my country mainly relies on serological allergy (GB / T 18645-2002). This detection method has a large number of human factors, mainly depends on visual inspection, has many subjective factors, and has a high error. In addition, the traditional method cannot distinguish the cattle inoculated with BCG from the cattle infected with wild virus, which has brought serious interference to the epidemiological investigation and the control of the epidemic source. Therefore, it is of great practical significance to invent a diagnostic method and a diagnostic tool that is convenient, fast,...

Claims

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Application Information

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IPC IPC(8): G01N33/544G01N33/531G01N33/558
Inventor 闫广谋张楠宫鹏涛李建华张西臣杨举
Owner JILIN UNIV
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