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Immune colloidal gold test paper strip for detecting bovine tuberculosis antibody and its preparation method

A technology of colloidal gold test paper and bovine tuberculosis, which is applied in the field of animal bacteriology and zoonosis, can solve the problems of missed detection and low sensitivity, and achieve the effect of low temperature requirement, low detection cost and high specificity

Active Publication Date: 2009-06-03
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the application of colloidal gold immunochromatography technology to the in vitro detection of bovine tuberculosis antibody is still blank in China. The only commercialized kit abroad is the "Anigen Rapid Bovine TB Ab TestKit" produced by Animal Genetics, Inc. in Korea. The kit is Using MPB70 protein as the detection antigen has good specificity, but the sensitivity is not high, and it may be missed in clinical practice

Method used

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  • Immune colloidal gold test paper strip for detecting bovine tuberculosis antibody and its preparation method
  • Immune colloidal gold test paper strip for detecting bovine tuberculosis antibody and its preparation method
  • Immune colloidal gold test paper strip for detecting bovine tuberculosis antibody and its preparation method

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Embodiment 1

[0047] Embodiment 1 (preparation embodiment)

[0048] (1) Cloning of the Mycobacterium bovis-specific secretory protein MPB83 gene and its expression and purification in recombinant Escherichia coli 1 Cloning of the cDNA sequence of the Mycobacterium bovis-specific antigen gene mpb83

[0049] (1) PCR primer design and synthesis

[0050] The upstream and downstream primers were designed according to the mpb83 gene sequence in Genbank (accession number: GI: 31794050). The upstream primer introduced an EcoRI restriction site (as indicated by the underline in the primer), and designed 6 protective bases, and the downstream primer introduced a HindIII restriction site (as indicated by the underline in the primer), and designed 6 protective bases. The primers of the present invention were synthesized by Beijing Aoke Biological Company.

[0051] mpb83 upstream primer: 5'-AGTTCA GAATTC ATGATCAACGTTCAGGCCAAAC-3'

[0052] mpb83 downstream primer: 5'-ACGTCG AAGCTT TTACTGTGCCGGGGGC...

Embodiment 2

[0071] Embodiment 2 (preparation embodiment)

[0072] Mycobacterium bovis-specific secreted protein MPB83 and MPB70 antibody diagnostic kit assembly and preparation method 1. Kit assembly:

[0073] The kit of the present invention consists of the following: sample pad (1), binding pad (2), nitrocellulose membrane (3), water-absorbing pad (4) and PVC backing (7), and the specific structure and assembly sequence are: A sample pad (1), a binding pad (2), a nitrocellulose membrane (3), and an absorbent pad (4) are adhered to the backing (7) in sequence; the binding pad (2) is coated with MPB83 protein-colloidal gold marker; said nitrocellulose membrane (3) is respectively coated with a detection line (5) made of purified MPB70 protein and a quality control line (6) made of purified rabbit anti-MPB83 protein IgG .

[0074] 2 Preparation of colloidal gold:

[0075] Dilute 1% chloroauric acid to 0.01% with ultrapure water, stir and boil on a magnetic heating stirrer, add 2.0ml 1% ...

Embodiment 3

[0086] Embodiment 3 (application embodiment)

[0087] How to use the immune colloidal gold test strip for bovine tuberculosis antibody

[0088] 1. Pretreatment of serum samples

[0089] The bovine serum samples were centrifuged at 4000rpm / min for 10min to remove blood cells, stored at 20°C for a long time and at 4°C for a short term.

[0090] 2. Detection steps

[0091] Use a pipette to draw 150 μl of the serum to be tested and slowly drop it on the sample hole of the colloidal gold test strip, and observe the result after 20 minutes.

[0092] 3. Result judgment

[0093] like image 3 As shown, if the color of the detection line of the test strip of the sample to be tested is obviously darker than that of the test strip of the positive standard product, and a red band appears on the quality control line, it is judged as a positive sample, that is, there is bovine tuberculosis in the sample to be tested. Antibodies to Mycobacterium-specific secretory proteins MPB83 and MPB...

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Abstract

The invention discloses an immune colloidal gold indicator paper and making method to test cow tuberculosis antibody, which comprises the following parts: sample pad, connecting pad, cellulose nitrate film, absorbent pad and PVC backing liner, wherein the sample pad, connecting pad, cellulose nitrate film and absorbent pad are attached on the PVC backing liner; the connecting pad clads MPB83 protein-colloidal gold marker; the purified MPB70 protein is cladded on the cellulose nitrate film, which constitutes detecting line and quality control line formed by pure rabbit-anti-MPB83 protein lgG. The invention also provides the making method of cow mycobacterium astopic antigen MPB83, which possesses the recombinant Escherichia coli BL21 / pET28a-MPB83 in the CCTCC with reserving number at CCTCC NO:M206142.

Description

technical field [0001] The invention relates to the technical fields of animal bacteriology and animal infectious disease. Specifically relates to an immune colloidal gold test strip for detecting bovine tuberculosis antibody and a preparation method thereof. Background technique [0002] Bovine tuberculosis is a zoonotic infectious disease that seriously endangers the dairy industry and is caused by Mycobacterium bovis infection. It is classified as Class B zoonotic disease by the International Organization for Animal Health (OIE). Bovine tuberculosis costs world agriculture more than all other diseases of cattle combined, at more than $3 billion a year. Mycobacterium bovis can infect not only cattle and other mammals, but also humans. The etiological surveys of human tuberculosis at home and abroad have found that Mycobacterium bovis is one of the important causes of human tuberculosis. Therefore, the report of the Seventh Meeting of the World Health Organization Exper...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31G01N33/544G01N33/532C12R1/19
Inventor 郭爱珍张书环陈焕春谭亚娣晁彦杰陈颖钰钦博吕文匡有吉
Owner HUAZHONG AGRI UNIV
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