Establishment Of PCR Detection Technique For Ovine Pulmonary Adenocarcinoma And Analysis Of Correlation Between ExJSRV And EnJSRV

Ovine pulmonary adenocarcinoma(OPA),is an important infectious disease of sheep caused by jaagsiekte sheep retrovirus(JSRV).Since the discovery of the OPA in the 19th century,there has been no reliable way to control its spread and affect the development of sheep industry in many countries.The disease has an important feature is in the OPA sheep blood has not been detected in circulating antibodies,it can not use conventional serological methods for diagnosis.In order to establish an early detection method for the disease and to explore the relative expression of exogenous jaagsiekte sheep retrovirus(exJSRV)and endogenous Jaagsiekte sheep retrovious(enJSRV),the establishment of high sensitivity,specificity and can quickly detect exJSRV method.In this study,nested RT-PCR and exJSRV Taqman real-time quantitative PCR were used to detect the early and rapid detection of OPA.Based on the genome sequence of exJSRV available in GenBank(accession number JQ837489.1 and AF105220.1),the nested RT-PCR primers,Taqman real-time qPCR primers and complete sequence of exJSRV primers were designed and synthesized in this study the relationship between exJSRV and enJSRV in each sample was analyzed by using the method of double standard curve.The positive blood samples were detected by the two methods,and the histopathological examination was carried out.Nested RT-PCR,Taqman real-time qPCR method were used to detect 9.72 fg of viral RNA and 10-1 copies of the standard plasmid.The specificity test results showed that the two-methods only specifically amplified exJSRV.The positive samples(1 nasal discharge sample and 2 blood samples)were tested and sequenced,The results showed that the sequences of the nested outer and inner RT-PCR amplified products of the nasal discharge samples and the blood samples with JQ837489.1 homology was as high as 98.9%or more,the amino acid sequence of the amplified gene sequence contained pathogenic sequence "YXXM" peculiar to exJSRV.The established curve of qPCR had a good linear relationship with the template concentration.the relationship between exJSRV and enJSRV expression in the same sample was also analyzed.In the natural infected sheep lung,the laboratory long-term follow-up sheep lung tissues and suspected OPA sheep nasal fluid,the expression of exJSRV was higher than enJSRV,however blood samples were in the opposite.lt was explained that the exJSRV-env gene was amplified in all samples.The amplification products of the sheep lung and nasal fluid had high homology with exJSRV;the amplified products of blood samples had high homology with enJSRV.The method that established in this study has a high clinical practice value with a higher sensitivity and a better specificity,which provided a convenient,reliable,economical and practical diagnostic method for early clinical detection of OPA.This study laid the foundation for the early rapid diagnosis and quantitative analysis of exJSRV infection.