Recovery and quantification of mycobacterium immunogenum DNA from metalworking fluids using dual-labeled probes

Authors: Veillette, MarcPagé, Guillaume; Thorne, Peter S.; Duchaine, Caroline
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.
Document Type: Article de recherche
Issue Date: 4 April 2005
Open Access Date: 20 December 2016
Document version: VoR
This document was published in: Journal of ASTM International, Vol. 2 (4), 1-9 (2005)
ASTM International
Alternative version: 10.1520/JAI12840
Collection:Articles publiés dans des revues avec comité de lecture

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