Use of the bead beater for preparation of Mycobacterium paratuberculosis template DNA in milk

Mycobacterium paratuberculosis is a recognized chronic enteric pathogen tha t can affect many different species of animals, including primates. It has been suggested that this organism is associated with Crohn's disease in hum ans, and that milk is a potential source of human exposure to this organism . The limit of the detection of M. paratuberculosis in milk samples by dire ct PCR was 10(5) cfu/mL if the traditional boiling method was used for temp late DNA preparation. In this study, an improved method for template DNA pr eparation was examined. The method involves the use of a bead beater, which breaks up bacterial cell wall mechanically by vibrating bacteria with micr obeads at high speed. The effectiveness of this method for lysing M. paratu berculosis cells was compared to that of the freeze-thaw method, and use of commercial kits such as the InstaGene Matrix and the QIAamp Tissue Kit. Th e bead beater procedure was tested in combination with various cell lysis a nd template DNA preparation procedures to determine which of these steps im proved the limit of detection of PCR assay that amplifies a 413 by fragment of the IS900 gene. Results showed that the use of the bead beater, in comb ination with the use of lysis buffer, boiling, and isopropanol precipitatio n, decreased the limit of detection of M. paratuberculosis in milk by the P CR to 10(2) cfu/mL. The limit of detection was further decreased to 10 cfu/ mL when 0.0037% bovine serum albumin was included in the PCR reaction mixtu res. The improved assay was 10- to 10(4)-fold more sensitive than the PCR a ssays using template DNA prepared by other lysis procedures including boili ng alone, freeze-thaw plus boiling, or use of commercial kits for lysis.