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Kit for detecting alpaca mycobacterium paratuberculosis antibody and detection method thereof

A technology for mycobacteria and paratuberculosis, which is applied in measurement devices, analytical materials, instruments, etc., can solve the problems of inconvenient detection and low sensitivity, and achieve the effects of good specificity and sensitivity, simple method and strong specificity.

Inactive Publication Date: 2011-01-19
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection of paratuberculosis often uses intradermal allergy, complement fixation test, etc., but intradermal allergy needs to detect live animals, and the sensitivity is low; the sensitivity of complement fixation test is low, and the detection is very inconvenient

Method used

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  • Kit for detecting alpaca mycobacterium paratuberculosis antibody and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of Mycobacterium paratuberculosis recombinant hed protein

[0024] 1. Primer Design

[0025] The MAP gene sequence (X16293) was retrieved from GenBank, and the antigenicity of the hed (Host expression dependent) gene was analyzed using DNAStar software, and the 840bp fragment with better antigenicity was selected as the target gene for amplification, and the upstream primer P1: 5'-ATA GGATCC GGTAGTTACCGCGGCGAA-3', downstream primer P2: 5'-CGC AAGCTT TTAGGTGTGGCGTT TTC-3', restriction endonuclease sites are BamH I and Hind III.

[0026] 2. Amplification and cloning of hed gene

[0027] Genomic DNA of Mycobacterium paratuberculosis K-10 was extracted and amplified by PCR using a 50 μL system volume: 10×Ex Taq Buffer 5 μL, dNTPs (10 mM) 4 μL, Mg 2+ (25mM) 4μL, TaKaRaEx Taq enzyme (5U / μL) 0.3μL, upstream primer P1 (20pmol / L) 1μL, downstream primer P2 (20pmol / L) 1μL, ultrapure water 32.7μL, template DNA 2μL. The reaction program was: pre-denaturati...

Embodiment 2

[0030] Example 2 Identification of reactogenicity and immunogenicity of recombinant hed protein of Mycobacterium paratuberculosis

[0031] 1. Immunoblotting

[0032] After the recombinant hed protein purified in Example 1 was subjected to SDS-PAGE electrophoresis, the gel was electroblotted to a PVNF membrane (Osmonics company) using a semi-dry transfer apparatus (Bio-Rad). The transferred PVNF membrane was blocked with 1% (m / v) bovine serum albumin, and a 1:100 dilution of goat anti-paratuberculosis positive serum (China Veterinary Drug Control Institute) was added to wash the membrane after acting at 37°C for 1.5h; Horseradish peroxidase (HRP)-labeled rabbit anti-goat IgG diluted 1:10000, reacted at 37°C for 1 hour and then washed the membrane; after 5 minutes of color development with 3,3-diaminobenzidine (DAB), a single specific The band was similar in size to the recombinant protein, indicating that the recombinant protein had good reactogenicity.

[0033] 2. Mouse Immu...

Embodiment 3

[0036] Embodiment 3 Composition and preparation of the kit of the present invention

[0037] The kit for detecting antibodies to Mycobacterium paratuberculosis in alpaca includes the following reagents:

[0038] (1), Mycobacterium paratuberculosis recombinant hed protein (see Example 1 for the preparation method);

[0039] (2), alpaca paratuberculosis positive control serum, negative control serum;

[0040] (3), enzyme plate;

[0041] (4), coating solution: Tris-HCl buffer solution with a concentration of 1mol / L, pH 7.5;

[0042] (5), blocking solution: the gelatin solution that mass concentration is 2%;

[0043] (6), PBST: Na 2 HPO 4 2.9g / L, KH 2 PO 4 0.2g / L, NaCl 8.0g / L, KCl 0.2g / L, Tween-20 volume concentration is 0.05%, and its pH is 7.4;

[0044] (7), horseradish peroxidase-labeled Staphylococcus aureus protein A (HRP-SPA);

[0045] (8), chromogenic liquid preparation raw material: ① by Na 2 HPO 4 12H 2 Phosphate-citrate buffer composed of O 18.408g / L and 5....

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Abstract

The invention provides a kit for detecting an alpaca mycobacterium paratuberculosis antibody and a detection method thereof, belonging to the technical field of biology. The kit comprises mycobacterium paratuberculosis recombinant hed protein, positive control serum and negative control serum of alpaca paratuberculosis, an ELISA plate, coating liquid, confining liquid, PBST, staphylococcal protein A labeled by horseradish peroxidase (HRP-SPA), a raw material for preparing developing solution and stop solution. The kit of the invention introduces the high-expression mycobacterium paratuberculosis hed protein with strong specificity as a detection antigen for directly detecting the alpaca mycobacterium paratuberculosis antibody, thus having good specificity and sensitivity. The method for detecting the alpaca mycobacterium paratuberculosis antibody by the kit is simple, convenient, fast and accurate, thus being applicable to sample detection in batches.

Description

technical field [0001] The invention relates to an indirect ELISA detection kit for the hed (Host expression dependent) protein of alpaca mycobacterium paratuberculosis (Mycobacterium paratuberculosis, MAP) and a detection method thereof, belonging to the field of biotechnology. Background technique [0002] Alpacas are native to the Andes Mountains of South America. They belong to the genus Llama together with llamas, llamas, and guanacos. They are called South American miniature camelids. Camel hair has 22 natural colors and is a high-grade wool spinning raw material, known as "soft gold"; its skin can be made into carpets, quilts, etc.; its meat has high protein content and delicious taste. Alpacas are economic animals with great market prospects. Shanxi introduced alpacas from Australia for the first time in 2000, and then introduced alpacas in Xinjiang Uygur Autonomous Region and other regions, adding new highlights to the income increase of farmers and herdsmen in our ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
Inventor 唐泰山廉慧锋张常印王凯民姜焱祝长青张睿
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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