Establisment And Evaluation Of Taqman PCR Detect Method Of Mycobacterium Avium Subsp.Paratuberculosis Complex And Seroprevalence Of Paratuberculosis In Sika Deer In Jilin Province

Paratuberculosis is an infectious disease, which caused by Mycobacterium avium subsp Paratuberculosis(MAP), and the character are chronic hyperplastec, intractable enteritis and progressive emaciation. The account of sika deer was about90,0000or so until2011. Paratuberculosis is widely distributed in the world and incidence rate showed an upward trend, which is a huge threat to sika deer farming industry, caused economic losses and infected human health in a potential style.The purpose of the study is to establish the quickly determine method of MAP. Selected the specific conserved sequences of F57gene of MAP according the genome sequence ATCC43015of the standard strain MAP login in GeneBank database, accorded which to design PCR primer and probe and construct the recombinant plasmid successfully, and to establish the standard curve of Tapman PCR. The result showed that the standard curve was eligible and effective, which can quantitatively detect the samples with the concentration of103～107Copies/μl. The experiment showed that the specific was100%by the determine means of Tapman PCR and the reaction were negative to other samples, and the detection sensitivity to MAP could reach5Copies/μl. The results of this study show that Taqman PCR detection method proposed in this study was specifically and sensitively and has important significance in rapid detection and early diagnosis of MAP.Collected1356serum samples in17sika deer farming industries at4representative areas of Jilin province, and research them with imported Mycobacterium paratuberculosis antibody diagnostic kit for enzyme linked immunosorbent assay. Investigated the Paratuberculosis epidemic situation of sika deer from different regions, different gender and different age in Jilin province, then the result was the overall positive rate was19.62%. The positive rate of Paratuberculosis in Liaoyuan was highest and the lowest was Siping in the4regions with significantly prevalence rate. The Paratuberculosis infection rate of deer in different regions was not significantly different, but there were significant different in different gender and different age in different regions, and the difference were significant among young deer, incubation deer and adult deer. Detected the positive sike deer with the method of Taqman PCR and collected197blood samples. Selected bacteria in the culture media and isolated77strains of acid-fast Gram-positive bacteria, and there were6in which were negative through the determine method of ELISA.