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Establishment method of mycoplasma ovipneumoniae indirect hemagglutination detection method

A Mycoplasma pneumoniae and method establishment technology, applied in the field of veterinary biological product detection, can solve the problems of long culture period, inapplicability of routine clinical diagnosis and epidemiological investigation, difficult detection methods, etc.

Pending Publication Date: 2020-10-27
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In summary, the diagnostic methods for Mycoplasma pneumoniae infection in ovis are divided into three categories: first, pathogen isolation and identification, which is a traditional diagnostic method and can also be considered as the "gold standard", but due to the influence of some antibiotics, and Mycoplasma grows slowly, has harsh nutritional requirements, and has a long culture period, which makes isolation and culture very difficult and difficult to use as a detection method; second, although the corresponding specific PCR technology, fluorescent quantitative PCR technology, has been established, the PCR method requires operation The personnel have a high technical level, and the cost is high, relatively time-consuming, and it is not suitable for routine clinical diagnosis and epidemiological investigation; third, although there are reports in China on the establishment of ELISA methods and HA techniques using whole bacterial antigens, However, foreign studies have shown that there are obvious cross-reactions between the whole bacterial antigens of Mycoplasma and Haemophilus parasuis and the corresponding pathogens of other infected animals

Method used

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  • Establishment method of mycoplasma ovipneumoniae indirect hemagglutination detection method
  • Establishment method of mycoplasma ovipneumoniae indirect hemagglutination detection method
  • Establishment method of mycoplasma ovipneumoniae indirect hemagglutination detection method

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Effect test

Embodiment 1

[0030] Example 1 Establishment of Indirect Serum Detection Method for Ovine Mycoplasma Pneumoniae Antibody

[0031] 1. Materials

[0032] (1) Serum and strains: positive serum of Mycoplasma ovis pneumoniae and negative serum of Mycoplasma ovis pneumoniae, positive serum of Pasteurella, Staphylococcus aureus, Mannella haemolyticus and serum of Mycoplasma mycoplasma goat subspecies, positive serum of Mycoplasma hyopneumoniae, Sera of Mycoplasma filamentous subspecies were all existing materials, which were preserved at -20°C by the Laboratory of Veterinary Medicine, Southwest University for Nationalities; Escherichia coli and Salmonella factor serum were purchased from Sichuan Institute of Biological Products; C-terminal recombinant engineering bacteria (Yang Falong, Zhang Xianyu, Tang Cheng, et al. Expression and immunogenicity of C-terminal repeat region of Mycoplasma ovis pneumoniae P113 protein[J]. Chinese Veterinary Science, 2013(7):733-737.) by Stored at -20°C in the Labo...

Embodiment 2

[0060] The best reaction system and condition of embodiment 2 screening this method

[0061] ① Screen the best reaction system

[0062] Choose two rows on a 96-well V-type plate, add 10 μL of diluent to each well of the first row, and add 10 μL of Mycoplasma ovis positive serum to the first well, then perform multiple dilutions on the positive serum, and finally add the following to each well The optimal sensitization concentration is 10 μL of antigen after sensitization. Add 25 μL of diluent to each well in the second row, add 25 μL of Mycoplasma pneumoniae positive serum to the first well, then perform multiple dilutions on the positive serum, and add 25 μL of antigen sensitized at the optimal sensitizing concentration to each well after dilution. Place the 96-well V-shaped reaction plate on a shaker for 1 to 2 minutes, react at room temperature for 30 minutes to 1 hour, and record the positive serum reaction results of the two groups of experiments.

[0063] The standard ...

Embodiment 3

[0084] Embodiment 3 is to the performance evaluation of the established method specificity, sensitivity and coincidence rate

[0085] ① Specificity evaluation

[0086] With the optimal sensitizing concentration, the optimized reaction conditions were used for Escherichia coli (E.Coli) multifactor serum, Salmonella (SE) multifactor serum, Pasteurella multocida (Pm), Staphylococcus aureus (SA) It was tested with Mycoplasma mycoplasma goat subspecies serum, Mannella haemolytica serum and Mycoplasma hyopneumoniae serum. When the agglutination intensity of "++" and above appeared, it was judged as positive, and the reaction result was recorded.

[0087] Escherichia coli (E.Coli) multifactorial serum, Salmonella (SE) multifactorial serum and Pasteurella multocida (Pm), Staphylococcus aureus (SA) and Mycoplasma filamentous subspecies, Mycoplasma filamentous goat The detection results of the subspecies serum, M. hemolytica serum and Mycoplasma hyopneumoniae serum are shown in Table 4 b...

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Abstract

The invention discloses an establishment method of a mycoplasma ovipneumoniae indirect hemagglutination detection method. According to the method, recombinant protein obtained by cloning and expressing a C terminal of a mycoplasma ovipneumoniae adhesin gene P113 is used as an antigen, healthy sheep erythrocytes are sensitized by the recombinant protein antigen, the optimal sensitization concentration and the optimal reaction conditions are screened for IHA titers of the same batch of serum, and the mycoplasma ovipneumoniae indirect hemagglutination detection method which is high in specificity, sensitivity and coincidence rate and easy and convenient to operate is established. The detection method can be applied to rapid detection of mycoplasma ovipneumoniae in the veterinary field, and provides a powerful tool for disease prevention, diagnosis, prevention and control.

Description

technical field [0001] The invention belongs to the technical field of detection of veterinary biological products, and in particular relates to a method for establishing an indirect hemagglutination detection method for Mycoplasma pneumoniae. Background technique [0002] Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae) is an important pathogen that causes respiratory diseases and can cause atypical pneumonia in goats and sheep. Mycoplasma ovipneumoniae was first isolated in 1963, and has since been found in many other countries and regions. It is currently widely distributed around the world. Mycoplasma ovis pneumonia, caused by Mycoplasma ovis pneumoniae, causes very large economic losses in the sheep industry worldwide. It is able to bind to the cilia of tracheal epithelial cells and cause ciliary dysfunction, resulting in impaired ciliary clearance. In addition, the susceptibility of sheep to other pathogens such as Pasteurella multocida, Mannella hemolyticus and i...

Claims

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Application Information

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IPC IPC(8): G01N33/86
CPCG01N33/86
Inventor 杨发龙王远微刀筱芳
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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