Detection of Mycobacterium avium subsp. paratuberculosis in bovine semen by real-time PCR

摘要

Although semen is not a common way for spread of paratuberculosis, isolation of MAP from semen has been reported. However, as the risk of spread by artificial insemination is not fully investigated, there is a demand for a semen test intended for use in paratuberculosis free herds. The rapidity, sensitivity and specificity of real-time PCR would greatly benefit the detection of MAP. We have applied the following method on artificially infected bovine semen. Samples of diluted MAP free semen were spiked with MAP, counted in burker-chamber since CFU-count usually underestimates the number of detectable genomes. The MAP suspension used was also washed from free DNA to avoid overestimating the sensitivity of the test. Incubation with lysisbuffer and proteinase K followed by mechanical disruption by beadbeating released the DNA, which was recovered with phenol/chloroform extraction.