An ielisa method for screening seronegative sheep of Mycoplasma ovine pneumonia and Mycoplasma capricolum subspecies cape pneumonia

Abstract

The invention discloses an iELISA method for screening serologically negative sheep of Mycoplasma ovis pneumoniae and Mycoplasma capricoides subspecies capneumoniae. , the bacterial solution was centrifuged, sterilized, and ultrasonically resuspended in PBS to separate the antigen; (2) Coat the microtiter plate with the antigen, incubator at 37°C for 1 hour, then overnight at 4°C; add blocking solution, block at 37°C for 2 hours; add Serum samples diluted 200 times were incubated at 37°C for 1 hour; rabbit anti-goat secondary antibody diluted 5000 times with 5% skimmed milk powder was added, and incubated at 37°C for 1 hour; (3) After washing the plate with PBST, add TMB chromogenic solution and incubate at 37°C 10min; add stop solution to terminate the reaction; read OD with microplate reader 450nm value. The sensitivity of the iELISA method of the invention can reach 98%, the operation is simple, and the sample processing capacity is large, which can not only be used for the screening of Mccp and Mo two pathogenic serologically negative sheep, but also be used for monitoring the level of Mccp antibody in the experiment.

Description

technical field [0001] The invention belongs to the technical field of veterinary detection, and in particular relates to an iELISA method for screening serologically negative sheep of Mycoplasma ovis pneumoniae and Mycoplasma capricatus subspecies goat pneumonia. Background technique [0002] my country is the country with the largest number of sheep and goats raised and slaughtered in the world. In 2017, the number of sheep and goats on hand reached 302.317 million. However, sheep are prone to a variety of respiratory diseases during the breeding process, among which sheep pneumonia is the most common clinical disease. The sheep industry has had a huge impact. Ovine pneumonia can be divided into four types: viral, bacterial, mycoplasmal and parasitic. In my country, there are two main types of mycoplasma that cause clinical ovine pneumonia: Mycoplasma ovis pneumoniae ( Mycoplasma ovipneumoniae , Mo) and Mycoplasma capricum subsp. goat pneumonia ( Mycoplasma capricolum su...

AI Technical Summary

Problems solved by technology

At present, alternative common serological methods include: ELISA, IHA and CFT, etc., but the existing ELISA, IHA and CFT have the following defects when detecting Mo or Mccp antibodies: Although the cELISA kit for detecting Mccp antibodies has Good sensitivity and specificity, but expensive, and the detection cost is 40 yuan / sample; IHA needs to sensitize sheep red blood cells each time, which often leads to a decrease in batch-to-batch reproducibility; CFT has poor specificity and sensitivity, and even There will be a phenomenon that the test result of infected sheep is negative

In addition, these methods can only detect serological antibodies to one pathogen, Mo or Mccp, which means that when screening Mccp and Mo-negative sheep, two rounds of detection must be carried out, which costs a lot of manpower and material resources, and is not suitable for screening a large number of samples Work

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