IELISA method for screening mycoplasma ovipneumoniae and mycoplasma capricolum subsp. capripneumoniae serological negative sheep

Abstract

The invention discloses an iELISA method for screening mycoplasma ovipneumoniae and mycoplasma capricolum subsp. capripneumoniaeserological negative sheep. The method comprises the following steps: (1) inoculating a mycoplasma capricolum subsp. capripneumoniae1801 strain into an MTB culture medium for expanding culture, centrifuging and sterilizing a bacterial solution, performing PBS ultrasonic resuspension, and separating to obtain an antigen; (2) coating an elisa plate with the antigen, and staying overnight at 4 DEG C after 1 hour in an incubator at 37 DEG C; adding a confining liquid, andsealing at 37 DEG C for 2 hours; adding a serum sample diluted by 200 times, and incubating at 37 DEG C for 1 hour; adding a rabbit anti-goat secondary antibody which is diluted by 5,000 times by using a 5% skim milk powder, and incubating at 37 DEG C for 1 hour; and (3) washing the plate with PBST, adding a TMB color developing solution, and incubating at 37 DEG C for 10 minutes; adding a stop solution to stop areaction; and reading an OD450nm value by a microplate reader. The iELISA method has advantages that sensitivity can reach 98%, operation is simple, a sample treatment amount is large, the method can be used for screening Mccp and Mo pathogen serological negative sheep, and the method can also be used for monitoring a Mccp antibody level in experiments.

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, mycoplasma, and parasite. In my country, there are two main types of mycoplasma that cause ovine pneumonia clinically: Mycoplasma ovipneumoniae (Mo) and Mycoplasma ovipneumoniae (Mo) species (Mycoplasma capricolum subsp. capripneumoniae, Mccp), both of ...

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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