Use of untargeted magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and Real-Time-PCR

Untargeted magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Untargeted magnetic beads recovered more M. smegmatis cells from PBS suspension than the typical centrifugation method; 96.31% of 1.68 x 104 CFU/mL viable M. smegmatis were recovered by beads and 0% by centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p<0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of higher sensitivity. Magnetic separation of MAP cells from milk, urine, and feces specimens were detected by F57 and IS900 sequences. Ct values demonstrated that beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 x 105 and 2 x 103 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of Johnes disease and other mycobacterial diseases such as tuberculosis.