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A Mycoplasma hyopneumoniae recombinant antigen ELISA detection kit

A technology of Mycoplasma hyopneumoniae and detection kit, which is applied in the directions of measuring devices, instruments, scientific instruments, etc., can solve the problems of unusable practical application, high manufacturing cost, low expression level of target protein, etc.

Active Publication Date: 2014-10-15
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For the detection of swine panting disease, there is currently only an indirect hemagglutination detection reagent for Mycoplasma hyopneumoniae in China. However, due to the high manufacturing cost, the detection results are easily affected by factors such as operators, environment and materials, and it is difficult to popularize it on a large scale.
[0005] Domestic Qin Qingsong et al. (2005) have mutated 3 TGAs encoding Trp in the P46 gene ORF of the international standard strain of Mycoplasma hyopneumoniae 232 strains to a gene TGG encoding Trp codon TGG that can be read through in Escherichia coli, and the mutation The post-Mycoplasma hyopneumoniae membrane protein P46 gene was expressed in Escherichia coli, but because the expression level of the target protein was low and the purification was difficult, it could not be used for practical application (Qin Qingsong, Ning Yibao, Shen Qingchun. Mycoplasma hyopneumoniae surface protein Expression of P46 gene in Escherichia coli. Chinese Journal of Preventive Veterinary Medicine , 2005, 27(2): 94-97)

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  • A Mycoplasma hyopneumoniae recombinant antigen ELISA detection kit
  • A Mycoplasma hyopneumoniae recombinant antigen ELISA detection kit
  • A Mycoplasma hyopneumoniae recombinant antigen ELISA detection kit

Examples

Experimental program
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Embodiment 1

[0044] 1. Solution preparation

[0045] (1) Preparation of sample diluent

[0046] Prepared according to the following formula: sodium chloride 8.0g, KH 2 PO 4 0.3g, Na 2 HPO 4 .12H 2 O 5.33g, KCl 0.2g, add water for injection to 900mL, add 5%-10% skimmed milk powder and fully dissolve, adjust pH to 7.0-7.2, add 0.01%-0.05% sodium azide, add water for injection to volume 1000mL.

[0047] (2) Preparation of enzyme-binding working solution

[0048] The enzyme-binding working solution is a commercially available horseradish peroxidase-labeled rabbit anti-pig antibody (referred to as the enzyme-labeled secondary antibody). The verification test is carried out according to the recommended dilution factor in the instructions for use, and the correction is made according to the test results. If the recommended dilution factor is 20000×, use working concentrations of 2500×, 5000×, 10000×, 20000× and 40000× to add to the secondary antibody diluent, and perform ELISA assays resp...

Embodiment 2

[0071] 1. Kit detection operating procedures

[0072] Preparation before the experiment: Dilute the 10× washing solution with distilled water to make 1× washing working solution. Dilute the sample to be tested 50 times with diluent.

[0073] (1) Set two positive control and negative control wells on the recombinant antigen-coated plate, and add corresponding control serum samples (diluted), 100 μL per well;

[0074] (2) Add each sample to be tested into the test sample well, 100 μL per well, put it in a self-sealing bag and place it at room temperature for 30 minutes;

[0075] (3) Pour off the liquid in each well and wash with 1× washing solution, 350 μL per well, and wash 3 to 5 times;

[0076] (4) Add enzyme-labeled secondary antibody working solution, 100 μL per well, pack it in a self-sealing bag, place it at room temperature for 30 minutes, and repeat step 3);

[0077] (5) Add 10 μL of color-developing working solution, and develop color at room temperature for 15 minu...

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Abstract

The invention discloses a pig mycoplasma pneumonia recombination antigen ELISA detection Kit. The Kit is provided with an antibody detection plate, enzyme conjugate treatment fluid, a positive control, a negative control, sample diluent, 10x condensed cleaning solution, developing solution A, developing solution B and termination solution. The detection plate of the Kit is a detachable 96-pore enzyme label plate enveloped by the mutational pig mycoplasma pneumonia membrane protein P46 gene protein antigen, the enzyme conjugate treatment fluid is a rabbit anti-pig antibody labeled by horse radish peroxidase, the positive control serum is taken from a pig which is detected positive through indirect hemagglutination and the ELISA Kit of IDEXX and has obvious pig mycoplasma pneumonia lesions in the lungs after anatomy, and the negative control serum is taken from a pig which is detected negative through indirect hemagglutination and the ELISA Kit of IDEXX and has no pig mycoplasma pneumonia lesions in the lungs after anatomy. The pig mycoplasma pneumonia recombination antigen ELISA detection Kit has the advantages of strong specificity, high sensitivity, simple operation, easy large-scale generation and application, and broad market prospect.

Description

technical field [0001] The invention relates to a kit for detecting animal infectious diseases, which belongs to the field of veterinary biological products. Background technique [0002] Mycoplasma hypneumoniae (Mycoplasma hypneumoniae, Mhp) is a pathogenic microorganism that causes swine mycoplasma pneumonia (also known as panting disease or porcine enzootic pneumonia). The disease is characterized by contact, highly contagious, chronic, high morbidity and low mortality. It is mainly manifested as cough and dyspnea. Anatomical findings are mainly lung lesions, especially the heart lobe, middle lobe and apex of the two lungs. Pancreatoid and sarcoid features appear in the leaves. Its main harm is that it causes the growth of pigs to be hindered and the feed conversion rate to drop sharply. In addition, its high morbidity rate seriously affects the economic benefits of raising pigs. Studies in recent years have found that mixed infection of Mycoplasma hyopneumoniae with po...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/531
Inventor 沈青春宁宜宝高和义覃青松
Owner CHINA INST OF VETERINARY DRUG CONTROL
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