| Ovine pulmonary adenomatosis(OPA)was caused by Jaagsiekte sheep retrovirus(JSRV).In order to diagnose and prevent the disease early,the detection method should be established by us.In order to establish the JSRV dot blot hybridization assay,this study was designed based on the sequence of exogenous Jaagsiekte sheep retrovirus(exJSRV)and endogenous Jaagsiekte sheep rctrovirus(enJSRV)reported in GenBank.Pairs of specific-primers of the exJSRV env gene and U3 sequences.Using the total RNA of lung tissue infected with OPA as templates,env(164bp)and U3(176bp)fragments were amplified by RT-PCR and the PCR products was sequenced.The sequence analysis showed that the two sequences were associated with the pathogen that caused the disease.The homology of the gene sequence of exogenous Jaagsiekte sheep retrovirus was 98.3%and 99.4%respectively.Using the purified PCR product as template and the digoxigenin marker was used to prepare pairs of specific-probes.Env,U3 probe sensitivity were 0.24 ng/μL and 0.21 ng/μL,respectively.To establish a JSRV spot blot detection method.The optimum concentration of the probe in the hybridization solution was determined to be 50 ng/mL.5 cases of lung tissues and 93 cases of blood samples that were infected by OPA were clinically tested.The results showed that the positive samples were compared with the nested RT-PCR results of the laboratory,and the coincidence rate of the peripheral blood samples was 92%,and the OPA disease lung compliance rate of 100%.The results of specificity test showed that the probes labeled in this study did not get the positive spots with the genomic DNA of pathogens such as peripheral blood of healthy sheep,mycoplasma,pneumococcus,Escherichia coli and Pasteurella.The sensitivity test results show that the method can detect 1.335ng of standard template DNA at a minimum.The reproducibility test showed that the method had good reproducibility and stability.This shows that the method established in this study,for the detection of OPA has good clinical application value,great specificity and high sensitivity.Dot blot hybridization has the advantages of simple operation,which provides a convenient,reliable and economical and practical method for the early detection of OPA. |
