| Bovine adenovirus(Bovine adenovirus, BAV) is an family member of(Adenoviridae) mammal genus adenovirus(Mastadenovirus).which Had reported a total of 10 bovine adenovirus serotypes, BAV-1,-2,-3,-9, and-10 bovine adenovirus belonging to the genus mammalian adenovirus(Mastadenovirus), while BAV-4,-5,-6,-7 and-8 thymus gland virus belonging to the genus(Atadenovirus). BAVs infection is widespread worldwide, but the prevalence of BAVs cattle in our country are still unclear. Given the presence of bovine serum cross-reactivity between the different adenovirus serotypes, the use of a serotype BAV can simultaneously detect multiple serotypes BAVs, this research laboratory application of bovine adenovirus type 3(BAV3) for the detection of antigen, establish detection of bovine adenovirus indirect ELISA method, and the establishment of an indirect ELISA for adenovirus epidemiological investigation over 2000 sera from different regions. The main results obtained are as follows: By comparing different methods of ELISA to choose the established ELISA method to detect BAV3, this topic mainly includes the following several aspects of research. 1. The preparation of virus antigen and determination of virus drops degreesinoculated cotton rat lung fibroblasts(VIDO-DT1) by 0.1 MOI of BAV3, for a lot of BAV3 amplification and viral titer. In the measured 106.47 as TCID50 for the initial concentration of coating antigen. 2. The optimization of indirect ELISA detection methodThis study aim at BAV3, by phalanx titration to determine the best antigen package concentration is 1:120 with the method of ELISA, that is 106.47 The best concentration of serum diluent is 1: 80. Sealing fluid is used 5% skimmed milk powder react 90 minutes in 37℃. Ra M IgG-HRP, in which the optimum concentration is 1:1200 and react 90 minutes in 37℃. Add 1 mol/L H2SO4 to stop reaction after the substrate effect 15 minutes. Under the condition of the above to test 30 cattle adenovirus antibody negative serum, the calculation results are as follows: Average of 30 negative serum(X) is 0.1832, the standard deviation(SD) was 0.0657, determine positive critical point(X + 3SD) was 0.3803, the negative point(X + 2SD) was 0.3146. Use the method in detecting bovine FMD positive serum, IBR positive serum, CBPP positive serum, BTB positive serum, bovine ephemeral fever positive serum, BAV3 positive serum and negative serum. Results in this method, repetitive experiments were showed that the coefficient of variation within-run was between 3.8511%~7.5916% and coefficient of variation(CV) < 5%; and the between-run was between 4.0124%~8.7293% and coefficient of variation(CV) < 5%. This shows that the ELISA method repeatability is good. By optimizing and improving the established ELISA method from the above aspects, improve the positive value effectively, and reduce the negative background values, and verify the stability of the established ELISA method. 3. The investigation of BAV3 epidemiological in Xian yangWith this method of established indirect ELISA,it tests 2000 bovine serums from Xian yang eight district 23 different size of zap. The results showed that: positive samples number in the 200 cattle samples is 933, namely the sample average positive rate was 46.65%, the lowest positive rate was 31.96% in positive group, and the highest was 91.74%. This results show that the established indirect ELISA method is sensitive, specific and can be used for clinical detection for BAV3 serum antibody. This has laid a foundation for the construction of ELISA diagnostic kit, provided scientific basis for build rapid diagnostic BAV3 infection method in large-scale bovine farms. |
PDF Full Text Request