Journal
MOLECULAR AND CELLULAR PROBES
Volume 22, Issue 5-6, Pages 273-280Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.mcp.2008.06.002
Keywords
Mycobacterium immunogenum; PNA-FISH; Pseudomonads; Metalworking fluids; Sputum; Hypersensitivity pneumonitis
Categories
Funding
- National Institute of Occupational Safety and Health, Centers for Disease Control and Prevention
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Specific and rapid detection and quanti. cation of mycobacteria in contaminated metalworking fluid (MWF) are problematic due to complexity of the matrix and heavy background co-occurring micro. ora. Furthermore, cross-reactivity among neighboring species of Mycobacterium makes species differentiation difficult for this genus. Here, we report for the first time a species-specific peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for Mycobacterium immunogenum, a non-tuberculous Mycobacterium species prevalent in MWF and implicated in occupational lung disease hypersensitivity pneumonitis and pseudo-outbreaks. A novel species-specific 14-bp PNA probe was designed for M. immunogenum based on its 16S rRNA gene sequence and was validated for specificity, by testing against a panel of other phylogenetically closely related rapidly growing mycobacteria and representative species of gram-positive, gram-negative, and acid fast organisms. In addition, a DNA-FISH protocol was optimized for co-detection of Pseudomonas, the most predominantly co-occurring genus in contaminated MWF. Reliable quanti. cation for both the test organisms was achieved at or above a cell density of 10(3) cells ml(-1), a recognized minimum limit for microscopic quanti. cation. The mycobacterial PNA-FISH assay was successfully adapted to human sputum demonstrating its potential for clinical diagnostic applications in addition to industrial MWF monitoring, to assess MWF-associated exposures and pseudo-outbreaks. (C) 2008 Elsevier Ltd. All rights reserved.
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