ISSN: 1546-962X
Published Online: 4 April 2005
Page Count: 9

Recovery and Quantification of Mycobacterium Immunogenum DNA from Metalworking Fluids Using Dual-Labeled Probes

Veillette, M
Research Assistant,Laval Hospital Research Center,

Pagé, G
Undergraduate Microbiology Student,Laval University,

Thorne, PS
Director, EHSRC,The University of Iowa,IA,

Duchaine, C
ProfessorMember of the GESER,Laval University,

(Received 7 October 2004; accepted 12 November 2004)

Abstract

Mycobacteria in metalworking fluids (MWF) are associated with hypersensitivity pneumonitis but are difficult to recover using culture. Quantitative PCR is a promising approach to quantify mycobacteria, but three challenges exist: mycobacterial cell lysis, high-yield DNA extraction, and removal of PCR inhibitors. We used Mycobacterium spp. primers to amplify polymorphic regions of 16S-rDNA flanked with highly conserved regions. A standard curve was constructed by cloning M. immunogenum amplification product. We developed single tube DNA extraction employing mixer mill cell disruption, enzymatic digestions (lysozyme, proteinase K) followed by a mechanical disruption, and column purification. MWF was spiked with M. immunogenum, and DNA was successfully extracted. Mycobacterial 16S-RNA genes were quantified by comparing PCR amplification detection (Cycle Threshold) from our samples with that obtained from the standard curve. Recovery and quantification of mycobacterial DNA from spiked samples approached 100 %. A rapid method for quantification of mycobacteria in MWF was demonstrated.

Keywords:
metalworking fluid, real-time PCR, mycobacteria, fluorescent probes, hypersensitivity pneumonitis

Paper ID: JAI12840
DOI: 10.1520/JAI12840
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