| Mycoplasma suis is a pathogen that can cause anemia in pigs.It can cause the infection of hemolytic anemia or mild chronic anemia which can obviously threaten life.Mycoplasma suis is a global disease,has a very important economic impact on pig production worldwide.Therefore,it is necessary to establish an effective method for detection of Mycoplasma suis.It is an important method to detect the infection of Mycoplasma suis by detecting the content of antibodies to Mycoplasma suis in pig serum.In this experiment,an indirect ELISA assay based on the water-soluble membrane protein of Mycoplasma suis was established and its preliminary application was carried out.The membrane protein was first extracted by Tritonx-114 extraction method,and the Mycoplasma suis membrane protein was extracted.The extracted membrane protein was analyzed by SDS-PAGE electrophoresis.An indirect ELISA assay was established using the water-soluble membrane protein of Mycoplasma suis as an antigen and its conditions were optimized.Based on the basic steps of indirect ELISA detection method,optimize the coating concentration of the antigen,the coating conditions,the serum dilution and the reaction time,the dilution of the secondary antibody,and the reaction time.30 samples of negative samples from pigs were selected for indirect ELISA test,and OD450nm value was determined.The specificity test was carried out by using pathogenic positive serum such as Toxoplasma gondii,pig Escherichia coli and Mycoplasma hyopneumoniae as samples.The stability of the indirect ELISA detection method was evaluated by in-batch and interbatch repeatability tests.The results show;the molecular weight of the extracted water-soluble membrane protein of Mycoplasma suis is mainly concentrated at 45-180 Ku.The molecular weight of the fat-soluble membrane protein of Mycoplasma suis is mainly concentrated in 25-35 Ku.ELISA results show that the optimum test conditions for indirect ELISA determination of water-soluble membrane proteins based on Mycoplasma suis are:Membrane protein optimal coating concentration was 15 μg/μL,overnight at 4 ℃ for optimal coating conditions,5%skim milk was the best blocking solution,blocking condition was blocked at 37 ℃ for 2 h,serum was diluted 320 times with PBS,37 ℃ Incubate for 1 h,dilute the enzyme-labeled secondary antibody 2 000 times,incubate at 37 ℃ for 1 h,color development time 5 min,add 2M H2SO4 to stop the reaction,determine the OD450nm value.The negative samples of 30 Mycoplasma suis were tested and the critical value was 0.376,and those with an OD450nm value greater than 0.376 were positive and vice versa.All coated antigens did not cross-react with positive sera such as E.coli,Toxoplasma gondii,and Mycoplasma hyopneumoniae.The indirect ELISA method established in this experiment is more specific.Repeatability test results showed that the coefficient of variation was less than 10%.and 40 pigs were tested for anticoagulant.The positive detection rate was 47.5%.This experiment establishes a special and sensitive serological detection method for Mycoplasma suis,which can be applied to the clinical detection of Mycoplasma suis. |
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