Mycobacteriophage lysin-mediated capture of cells for the PCR detection of Mycobacterium avium subspecies paratuberculosis

摘要

Recombinant lysin Gp10 from the mycobacteriophage L5 was coupled to magnetic Dynabeads 280 and these beads were used to capture Mycobacterium avium subsp. paratuberculosis (MAP) cells from complex media. Skim cow milk spiked with MAP cells, skim milk spiked with both MAP and Escherichia coli cells and Middlebrook 7H9 medium spiked with MAP cells were used to model the contaminated food matrices. The beads bearing immobilized Gp10 were incubated with the samples, separated, washed, subjected to DNA extraction and the solution was analyzed by real time PCR. The entire process was completed within 24 hours, demonstrated high specificity towards the MAP cells and increased the sensitivity of detection. The Ct values observed from the pre-concentrated samples were close to those obtained from clean buffer, whereas they were significantly worse without such bead-based pre-concentration. The protocol was successfully tested with two MAP strains (ATCC 19698 and 19851) and two target sequences (IS900 and F57). The suggested method eliminates the need for lengthy culturing steps used in traditional protocols and allows the pre-concentration of MAP cells to get rid of the various PCR inhibitors that may be present in the food matrices. As such, it offers an overall reduction of the time usually required to test milk for MAP. The developed protocol may be instrumental for the prevention, diagnosis and monitoring of Johne's and Crohn's diseases in cattle and humans, respectively a€“ two gastrointestinal diseases having huge economic and public health impact.