Immune colloidal gold test paper strip for detecting bovine tuberculosis antibody and its preparation method
A technology of colloidal gold and test strips, which is applied in the fields of animal bacteriology and zoonotic diseases, can solve the problems of missed detection and low sensitivity, and achieve the effects of low temperature requirements, low detection cost, and short detection time
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Embodiment 1
[0050] Embodiment 1 (preparation embodiment)
[0051] (1) Cloning of Mycobacterium bovis-specific secretory protein MPB83 gene and its expression and purification in recombinant Escherichia coli
[0052] 1 Cloning of cDNA sequence of Mycobacterium bovis specific antigen gene mpb83
[0053] (1) PCR primer design and synthesis
[0054] 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.
[0055] mpb83 upstream primer: 5'-AGTTCA GAATTC ATGATCAACGTTCAGGCCAAAC-3'
[0056] mpb83 downstream primer: 5'-ACGTCG AAGCTT TTACTGTGCCGGGGGCATC...
Embodiment 2
[0075] Embodiment 2 (preparation embodiment)
[0076] Method for assembly and preparation of Mycobacterium bovis-specific secretion protein MPB83 and MPB70 antibody diagnostic kit
[0077] 1. Kit assembly:
[0078] 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; described nitrocellulose membrane (3) is respectively coated with the detection line (5) that the purified MPB70 protein constitutes and the quality control line (6) that the purified rabbit anti-MPB83 protein IgG constitutes .
[0079] 2 Preparation of colloidal gold:
[0080] Dilute 1% chloroauric acid to 0.01% with ultrapure water, stir ...
Embodiment 3
[0091] Embodiment 3 (application embodiment)
[0092] How to use the immune colloidal gold test strip for bovine tuberculosis antibody
[0093] 1. Pretreatment of serum samples
[0094] 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.
[0095] 2. Detection steps
[0096] 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.
[0097] 3. Result judgment
[0098] As shown in Figure 3, if the color of the detection line of the test strip of the sample to be tested is obviously darker than the color of the detection line 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 Antibodies of Mycobacterium tuberculosis-specific secretory proteins MPB83 and MPB70 indicate...
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