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Mycoplasma hyopneumoniae multi-recombination antigen ELISA (enzyme-linked immunosorbent assay) detection kit

A technology of mycoplasma hyopneumoniae and a detection kit, which is applied in the direction of biological testing, material inspection products, etc., can solve problems such as failure to achieve the desired effect, and achieve the effects of easy large-scale promotion and application, high sensitivity, and broad market prospects

Active Publication Date: 2014-12-31
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used ELISA method, but due to cross-reaction and other effects, the desired effect cannot be achieved

Method used

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  • Mycoplasma hyopneumoniae multi-recombination antigen ELISA (enzyme-linked immunosorbent assay) detection kit
  • Mycoplasma hyopneumoniae multi-recombination antigen ELISA (enzyme-linked immunosorbent assay) detection kit
  • Mycoplasma hyopneumoniae multi-recombination antigen ELISA (enzyme-linked immunosorbent assay) detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] An ELISA detection kit for Mycoplasma hyopneumoniae multiple recombinant antigens, which is equipped with:

[0036] 1. ELISA plate coated with multiple recombinant antigens:

[0037] Mycoplasma hyopneumoniae protein P46 and DnaK were based on literature [Cloning and expression of Mycoplasma hyopneumoniae P46 gene, Jiangsu Journal of Agricultural Sciences, 2008,24(3):288-292] and [Expression of Mycoplasma hyopneumoniae DnaK gene and establishment of ELISA method, Jinling Journal of the Academy of Science and Technology, 2011,27(4):79-84] method to construct the expression bacteria of Mycoplasma hyopneumoniae P46 and DnaK, and connect the bacteria to LB (Amp + / Kan + ) Liquid culture medium, shake culture to OD value 0.6-1.0, add IPTG (final concentration 1mM) to induce expression for 5h. At the same time, uninduced bacteria and induced empty vector bacteria were used as controls for SDS-PAGE electrophoresis. The supernatant was collected by centrifugation, washed once with l...

Embodiment 2

[0058] 1 Test kit operation procedure

[0059] Preparation before experiment: add 10× washing solution to ddH 2 O is diluted into washing working solution (1×). Dilute the sample to be tested 40 times with the diluent.

[0060] (1) Set two positive control and negative control wells on the recombinant antigen-coated plate, and add the corresponding control serum sample (diluted), 100μL per well;

[0061] (2) Add the sample to be tested to the test sample well, 100μL per well, and leave it at room temperature for 60 minutes;

[0062] (3) Shake off the liquid in each sample well, use 1× washing solution, 300μL per well, wash 3-5 times, and spin dry;

[0063] (4) Add enzyme-labeled secondary antibody working solution, 100 μL per well, repeat step 3 after 30 minutes at room temperature;

[0064] (5) Add 100μL of color developing working solution, and avoid light at room temperature for 10min;

[0065] (6) Add 50μL of stop solution to each sample well to stop the color development, and measur...

Embodiment 3

[0070] 1 Serum sample:

[0071] Mycoplasma hyopneumoniae positive serum, Mycoplasma hyopneumoniae positive serum, Porcine circovirus positive serum, Haemophilus parasuis type 4 positive serum, Haemophilus parasuis type 5 positive serum, Mycoplasma hyopneumoniae positive serum, Mycoplasma hyopneumoniae negative serum .

[0072] 2 Detection method:

[0073] According to Examples 1 and 2, the P36, P46, P97, DnaK mixed protein coating kit was prepared and tested.

[0074] 3 results:

[0075] Use positive serum for Mycoplasma hyopneumoniae, Mycoplasma hyopneumoniae positive serum, Porcine circovirus positive serum, Haemophilus parasuis type 4 positive serum, Haemophilus parasuis type 5 positive serum, Mycoplasma hyopneumoniae positive serum, Mycoplasma hyopneumoniae negative serum Table 1 shows the test results of serum specificity of the kit of the present invention.

[0076] Table 1 Mycoplasma hyopneumoniae multiple recombinant antigen ELISA test kit

[0077]

[0078] It can be seen from T...

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Abstract

The invention belongs to the health inspection field, and discloses a mycoplasma hyopneumoniae multi-recombination antigen ELISA (enzyme-linked immunosorbent assay) detection kit. The kit is internally provided with a multi-recombination antigen-coated ELISA plate, enzyme conjugated working solution, positive control blood serum, negative control blood serum, sample diluent, secondary antibody diluent, 10*washing concentrate, color development solution A, color development solution B and stop solution, wherein the multi-recombination antigen is mycoplasma hyopneumoniae protein P36, mycoplasma hyopneumoniae protein P46, mycoplasma hyopneumoniae protein P97R1 and mycoplasma hyopneumoniae protein DnaK. Verified by tests, the kit is high in specificity and sensitivity, and good in repeatability and operability, can be applied for mycoplasma hyopneumoniae antibody laboratory study and clinical detection, is easy to popularize and apply in large range, and has wide market prospects and more economic and social benefits.

Description

Technical field [0001] The invention relates to a kit for detecting animal infectious diseases, belonging to the field of veterinary biological products. Background technique [0002] Mycoplasma hyopneumoniae (Mhp) is the main pathogen that causes Mycoplasmal pneumoniae of swine (Mps, also known as swine asthma), and it is also an important primary pathogen of swine respiratory disease syndrome. Mhp mainly infects the respiratory tract of pigs and is not very pathogenic. The clinical symptoms are mainly cough and asthma. The lesions are mainly characterized by the apical, cardiac, intermediate, and septal lobes of the lungs that are "meat-like" or "shrimp". Flesh-like" consolidation. After Mhp infects the body, it mainly adheres to the cilia on the inner wall of the respiratory tract, causing ciliary cell pathology and apoptosis, leading to cilia rupture and shedding, causing disease in the body, and seriously affecting the growth and development of pigs and the feed conversion ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
Inventor 刘茂军杜改梅邵国青韦艳娜冯志新白方方熊祺琰
Owner JIANGSU ACAD OF AGRI SCI