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

Abstract

Dynabeads® M-280 Tosylactivated (untargeted magnetic beads) were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Untargeted magnetic beads added to the spiked samples were slightly mixed for 1 hour and separated in a magnetic rack for further detection. Beads recovered more M. smegmatis cells from PBS suspension that the centrifugation method; these results were confirmed by the recovery of 96.31% of 1.68 x 104 CFU/mL viable M. smegmatis by beads and 0% by centrifugation. Likewise, the F57-qPCR detection of MAP cells, after being recovered by beads and centrifugation, were different; cycle threshold (Ct) was lower (p<0.05) for the detection of MAP cells recovered by beads than centrifugation. Magnetic separation of MAP cells from milk, urine, and feces specimens were detected by amplifying 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 of MAP cells from milk and urine suspensions. Milk proteins were denatured by Proteinase k before capturing MAP cells by magnetic beads. M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29; plaque former units were observed clearly from urine containing 2 x 105 and 2 x 103 CFU/mL M. smegmatis in 24 hours. The results of this study encourage further effort to rule out the use of untargeted beads as a simple tool for diagnosis of Johne′s disease and other mycobacterial diseases such as tuberculosis.