Mycoplasma Gallisepticum immune body immune colloidal gold fast detecting reagent kit and its application

A technology of mycoplasma and kit, which is applied in the fields of animal molecular biology, immunology, and rapid detection kit of mycoplasma gallisepticum antibody, achieving the effect of high biological safety and low production cost

Inactive Publication Date: 2008-06-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In terms of chicken epidemic diagnosis and detection, the Chinese invention patent (Patent No. ZL99101537.1) discloses the preparation and application of a quick diagnostic test strip for livestock ...

Method used

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  • Mycoplasma Gallisepticum immune body immune colloidal gold fast detecting reagent kit and its application
  • Mycoplasma Gallisepticum immune body immune colloidal gold fast detecting reagent kit and its application
  • Mycoplasma Gallisepticum immune body immune colloidal gold fast detecting reagent kit and its application

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Experimental program
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Effect test

Embodiment 1

[0027] Preparation of Mycoplasma gallisepticum pMGA1.2 protein:

[0028] For the molecular biology methods used, please refer to the literature: Sambrook J, Fritsch EF, Mani Artis T, editors (translated by Jin Dongyan, etc.), Molecular Cloning Experiment Guide. The second edition, provided by Beijing Science Press, 1992 method is carried out.

[0029] 1. PCR and cloning of pMGA gene fragment

[0030] The pMGA1.2 gene involved in the present invention is obtained from the applicant's pMGA recombinant plasmid MgW17 (Genbank accession number is AF275312, and the construction of the plasmid is shown in the literature: Weng Changjiang et al. Research on the pMGA multigene family of Mycoplasma gallisepticum HS strain, Chinese Journal of Veterinary Medicine, 2003, 23(2): 149-152) obtained by subcloning itself. The whole MgW17 recombinant plasmid is about 10.6kb in total, and the Mycoplasma gallisepticum DNA fragment contained is 7434bp, which contains two complete pMGA genes (pMGA1...

Embodiment 2

[0052] Preparation of rabbit anti-mouse IgG antibody:

[0053] 1. Extraction of Balb / C mouse IgG

[0054] The extraction and purification of IgG are carried out according to the methods described in Shen Guan et al. (Shen Guan et al., Modern Immunology Experimental Technology (Second Edition) [M], Hubei Science and Technology Press, 2002): Take 10.0mL healthy Balb / C small After the mouse serum was mixed with an equal volume of normal saline, saturated (NH 4 ) 2 SO 4 Solution 20.0mL, made to 50% saturation (NH 4 ) 2 SO 4 solution. Take it out after standing at 4°C for 30min; centrifuge at 3,000r / min for 30min at 4°C, discard the supernatant, add 20.0mL normal saline to the precipitate; after the precipitate dissolves, slowly add 10.0mL saturated (NH 4 ) 2 SO 4 solution, made into 33% saturation (NH 4 ) 2 SO 4 solution. Take it out after standing at 4°C for 30min; centrifuge at 3,000r / min for 30min at 4°C. Discard the supernatant and precipitate. Repeat the above o...

Embodiment 3

[0069] Preparation of nitrocellulose membrane

[0070] 1 Preparation of coating buffer: 0.01M pH7.2 PBS buffer containing 3% methanol is used as coating buffer, filtered through 0.22μ membrane, set at 4°C for later use, valid for one week. 1000ml 3% methanol 0.01M pH7.2 PBS buffer formulation: NaCl 8g, KCl 0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 30ml, dilute to 1000ml with double distilled deionized water.

[0071] 2 Preparation of nitrocellulose membrane: use the coating buffer in step 1 of this example to dilute the pMGA1.2 protein to 50-100 μg / ml, adjust the machine, draw the T line, which is the detection line, and the T line is close to The end of the gold standard pad is about 5mm away from the end of the gold standard pad; dilute the rabbit anti-mouse IgG antibody to 50-100 μg / ml with coating buffer, adjust the machine, and mark the line C, which is the quality control line, and the C line is close to Absorbent pad, about 3mm from the absorbent ...

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Abstract

The invention belongs to the immunity application field, which relates to the related fields of animal molecular biochemistry and the immunology, etc. The invention discloses a reagent kit respectively used for fast testing antibody of Mycoplasma Gallisepticum Strain, which comprises a case body, and the test paper card and sample diluent equipped inside the case body, wherein, the test paper card is formed by the absorption pad, nitrocellulose membrane, gold-label pad and sample pad affixed in turn on the non-absorbent supporting flake by taking the sample pad, absorption pad and the pMGA1.2 recombinant proteins coated with the Mycoplasma Gallisepticum Strain as the detection line and the pMGA1.2 recombinant proteins coated with anti-mouse IgG as nitrocellulose membrane of the quality control line. The reagent kit used for testing the corresponding antibody has the obvious advantages of strong specificity, high sensitivity, easy operation and fast diagnosis.

Description

technical field [0001] The invention belongs to the field of immune application and relates to related fields such as animal molecular biology and immunology. Specifically, the present invention belongs to a rapid detection kit for Mycoplasma gallisepticum antibodies, which are respectively used for the detection of Mycoplasma gallisepticum antibodies in chicken serum to determine whether the tested chickens are infected with Mycoplasma gallisepticum or whether the immunized chickens produce the corresponding antibodies. Background technique [0002] As we all know, avian influenza, Newcastle disease, bursa, infectious bronchitis, infectious laryngotracheitis, mycoplasma gallisepticum, Marek's disease, egg drop syndrome and other diseases are important diseases that endanger the global poultry industry. [0003] Mycoplasma gallisepticum (Mycoplasma gallisepticum) is a class of the smallest single-celled prokaryotic microorganisms lacking cell walls and able to reproduce ind...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/558
Inventor 毕丁仁张进良胡思顺张文通张展英王政李自力肖运才王喜亮石德时刘梅许青荣
Owner HUAZHONG AGRI UNIV
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