Establishment And Preliminary Application Of Diagnostic Technique For Bovine Tuberculosis

Bovine tuberculosis is a zoonotic disease of humans and animals caused by Mycobacterium bovis.OIE classifies BT as a Class B zoonotic disease,or a class II zoonotic disease in China.The annual economic loss of bovine tuberculosis to the animal husbandry is immeasurable.Not only that,bovine tuberculosis brings major hidden dangers to human safety,so it is very important for the prevention and control of bovine tuberculosis.This study hopes to use exploration as a target for rapid diagnosis.Genes and antigen proteins,based on immunoenzyme-linked adsorption test(ELISA)and real-time fluorescent quantitative PCR technology,establish a diagnostic method for bovine tuberculosis,provide new technical means for tuberculosis prevention and control,and conduct the following research:(1)By means of bioinformatics analysis,the 16 kinds of tuberculosis-specific proteins studied in the early stage of our laboratory were analyzed,and the physical and chemical properties,instability index,hydrophilicity,protein secondary structure,transmembrane domain and B cell were analyzed.The epitope distribution prediction provides theoretical data support for subsequent experiments;16 strains of expressing bacteria were expressed and purified,and verified by Western-Blotting that all of them can react with bovine tuberculosis-positive bovine serum,which has a good source of reaction In the end,the IDEXX bovine tuberculosis antibody detection kit was used to initially screen all the proteins involved in this test.The results showed that MPT70 and MPT83 have excellent diagnostic performance in the indirect ELISA detection of bovine tuberculosis and can be used as diagnostic antigens for further research.(2)MPT70 protein,MPT83 protein,MPT70-83 fusion protein,epitope peptide predicted by MPT70(M1),epitope peptide predicted by MPT83(M2)were used as coating antigens to establish an indirect ELISA detection method.Perform burst titration to determine the optimal coating concentration,serum dilution,enzyme-labeled secondary antibody dilution,and the optimal reaction time of serum,secondary antibody and color development.Under various optimal reaction conditions,the serum and cattle that are positive for bovine tuberculosis The tuberculosis-negative serum is tested to determine its positive and negative coincidence rates.The results showed that the negative and positive coincidence rates of M1 and MPT70 and the negative and positive coincidence rates of M2 and MPT83 were similar.Antigen peptides can be used as diagnostic antigens instead of whole proteins.(3)Establish a real-time fluorescent quantitative PCR-based nucleic acid detection method for Mycobacterium tuberculosis based on the Mpt83 gene,design a pair of specific primers based on the Mpt83 gene,and use the cultured H37 RV standard strain as a template to amplify the target gene and connect the T vector,Construct a standard recombinant plasmid to test the effectiveness of the established method.The SYBR Green I real-time fluorescence quantitative detection method was established after optimization of reaction conditions.The results showed that the established method has a good linear relationship,single melting curve peak,strong specificity and no cross-reaction with common pathogens,and the lowest detection limit is 10copies/reaction is 100 times that of conventional PCR.It can detect 10 CFU/10 m L on simulated milk samples,which can be used for pathogen detection and quantitative analysis.In this experiment,16 kinds of tuberculosis-specific proteins were initially screened through bioinformatics analysis and enzyme-linked immunosorbent assay,and two antigen proteins of MPT70 and MPT83 were screened out for the serological diagnosis of bovine tuberculosis;MPT70 and MPT83 were selected respectively.The two proteins and the epitope peptide predicted by MPT70(M1),the epitope peptide predicted by MPT83(M2)and the MPT70-83 fusion protein were established to establish an indirect ELISA detection method.The results of clinical sample testing showed the dominant antigen of the two proteins The epitope segment can replace the whole protein of the two proteins as the diagnostic antigen;the detection method established by fluorescence quantitative PCR with MPT83 as the target gene has good specificity,and the detection limit can reach 10copies/ reaction,the detection of simulated milk samples can reach 10 CFU/10 m L,which can be used for quantitative analysis.Provide basic test data for rapid diagnosis of bovine tuberculosis.