| Contagious bovine pleuropneumonia(CBPP)is a serious contact infectious disease caused by Mycoplasma mycoides subsp.Mycoides(Mmm),which seriously endangers animal health.World Organisation for Animal Health(OIE)has listed infectious diseases as needed.In May 2011,China was declared as a country free of bovine pneumonia by the World Organisation for Animal Health(OIE).In order to maintain China’s disease-free status,CBPP serological surveillance will be carried out nationwide in accordance with OIE requirements every year.In the CBPP serological test,there is no domestically produced kit,so only imported competitive ELISA(cELISA)kits can be used for testing.The testing cost is high and the delivery period is long,which cannot meet the large-scale monitoring and effective prevention and control of the disease Requirements.Although no cases of CBPP infection have been found in China,the disease is still prevalent in the neighboring countries of China,and the risk of introduction into China is very high.Therefore,this study focuses on the serological diagnosis of CBPP,with a view to providing a cost-effective detection method for large-scale monitoring of CBPP in China,and also to provide an effective technical reserve for the risk assessment and emergency prevention and control of CBPP introduction into China.Since Mycoplasma does not have a cell wall,its surface lipoproteins anchored outside the plasma membrane interact directly with the host,stimμLating the body to produce a strong immune response.Surface lipoproteins have drawn great attention in both mycoplasma diagnosis and pathogenicity,and have been reported to have the potential to diagnose antigens.Therefore,in the laboratory,12 lipoproteins were initially selected for subsequent research through bioinformatics analysis.Recombinant proteins of each candidate lipoprotein were obtained using the prokaryotic expression system.Through preliminary ELISA and Western Blot analysis,the recombinant protein rP0308 with better specificity and antigenicity was screened.The purified rP0308 was used as the coating antigen,and an indirect ELISA method for rP0308 was established by optimize the reaction conditions and determine the critical values.The optimal working conditions of the ELISA were determined through experiments.The resμLts showed that the optimal coating concentration of the antigen was 2.5 ug/mL;the serum samples were diluted 1:80 at37℃ for 1 h;the optimal blocking conditions were 1% fish gelatin blocked at 4℃ overnight;The optimal working conditions for the secondary antibody are 1: 5000 dilution of the enzyme-labeled secondary antibody at 37℃ for 45 minutes;the optimal color development time is TMB at 37℃ for 10minutes;the critical value is 0.36.In order to further evaluate its application value,the sensitivity,specificity,accuracy,cross-reactivity,repeatability,and compliance with commercial kits of the method were analyzed.The resμLts showed that the sensitivity of the method was 92%,the minimum detection limit was 1280-fold dilution of the serum sample;the specificity was 96%,and there was no cross-reaction with the positive serum of several other common bovine respiratory diseases.the CV ofinter and intra assay were 2.41%~6.03% and 2.94%~6.59%,less than 10%,and the repeatability was good.Using this method and the competitive ELISA kit recommended by OIE to detect 1648 clinical samples stored in the laboratory,the total coincidence rate of the two methods was 88.7%.The assembled kit was sealed and stored at 4 ℃ for 8 months with good performance.The test resμLts showed that the performance of each reagent was good,and the CV of the detection res μ Lts of sensitivity and cross-reaction were less than 10%.The above resμLts show that the method has good sensitivity,specificity and stability,and has certain application value,which lays a foundation for the further development of CBPP detection kit. |
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