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

Abstract

Mycobacterium paratuberculosis is a recognized chronic enteric pathogen that can affect many different species of animals, including primates. It has been suggested that this organism is associated with Crohn's disease in humans, 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 direct PCR was 10(5) cfu/mL if the traditional boiling method was used for template DNA preparation. In this study, an improved method for template DNA preparation was examined. The method involves the use of a bead beater, which breaks up bacterial cell wall mechanically by vibrating bacteria with microbeads at high speed. The effectiveness of this method for lysing M. paratuberculosis 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. The bead beater procedure was tested in combination with various cell lysis and template DNA preparation procedures to determine which of these steps improved the limit of detection of PCR assay that amplifies a 413 bp fragment of the IS900 gene. Results showed that the use of the bead beater, in combination with the use of lysis buffer, boiling, and isopropanol precipitation, decreased the limit of detection of M. paratuberculosis in milk by the PCR 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 mixtures. The improved assay was 10- to 10(4)-fold more sensitive than the PCR assays using template DNA prepared by other lysis procedures including boiling alone, freeze-thaw plus boiling, or use of commercial kits for lysis.