期刊
MOLECULAR AND CELLULAR PROBES
卷 22, 期 5-6, 页码 273-280出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.mcp.2008.06.002
关键词
Mycobacterium immunogenum; PNA-FISH; Pseudomonads; Metalworking fluids; Sputum; Hypersensitivity pneumonitis
类别
资金
- National Institute of Occupational Safety and Health, Centers for Disease Control and Prevention
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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