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Implementation of a Quantitative Real-Time PCR Assay for the Detection of Mycobacterium immunogenum in Metalworking Fluids

By G. Rhodes, A. Fluri, A. Ruefenacht and R. Pickup

Abstract

The recently described human pathogen M. immunogenum (Wilson et al., 2001; Loots et al., 2005; Sampaio et al., 2006) has been implicated in the causation of hypersensitivity pneumonitis (HP) in automobile workers and other occupational workers exposed to metal working fluids (MWF; Moore et al., 2000; Falkinham, 2003; Beckett et al., 2005). The bacterium has as closest relatives M. chelonae, M. abscessus and collectively these bacteria form the M. chelonae complex. The early detection of M. immunogenum and other bacteria implicated in the causation of HP may be crucial to reducing its prevalence. As detection by conventional techniques such as cultured is severely limited, an M. immunogenum-specific quantitative real-time PCR system was developed (Rhodes et al., 2008). This technique made use of a 5’ nuclease assay (Taqman) to detect fluorescence in real time upon specific amplification of target DNA. When initially validated laboratory strains and on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4 x101 and 1.9 x 104 cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples; Rhodes et al., 2008). The initial description of the assay gave an indication as to its potential for routine assessment of coolant samples for contamination with this important organism. However, the true value of the technique will become apparent only after more in depth study and application to industrial systems.\ud The present report describes further testing of the assay and direct comparison against culture, with its implementation on a larger scale on a range of different contaminated coolant samples obtained from the USA and Europe over a 19 month period (from December 2006 to July 2008). \u

Topics: Biology and Microbiology, Electronics, Engineering and Technology, Health
Publisher: Taylor & Francis
Year: 2011
DOI identifier: 10.1080/15459624.2011.590737
OAI identifier: oai:nora.nerc.ac.uk:4812
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