Development of a species-specific colorimetric-PCR assay for detection and species differentiation of Mycobacterium immunogenum and Mycobacterium chelonae and its comparison with quantitative real-time PCR for field metalworking fluids

Mycobacterium immunogenum and Mycobacterium chelonae are closely related species associated with occupational hypersensitivity pneumonitis (HP) and nosocomial infections. There is a need to develop specific and readily adaptable methods for detection and speciation of these agents. Here we report development of a probe-based colorimetric-PCR assay involving heat shock protein (hsp) gene amplification (228bp) and its detection in an ELISA-like reaction. A quantitative format of this assay was developed and validated on metalworking fluids (MWF). The assay showed a minimum detection limit of 10 fg genomic DNA or 1 mycobacterial cell, albeit with variations depending on type and composition of the MWF matrix. When applied to the field MWF samples, the developed assay was found to be comparable to the real-time PCR assay, and allowed direct speciation of MWF mycobacteria without sequencing and/or restriction pattern analysis. In conclusion, the developed colorimetric PCR allows detection and quantification of MWF mycobacteria without culturing and is the first probe-based assay for unambiguous differentiation between the two phylogenetically closely related species, M. immunogenum and M. chelonae. Considering that the assay offers high throughput format involving relatively simpler instrument infrastructure, it has a potential for applications in routine assessment of MWF mycobacteria in diagnostic and industrial laboratories.