Real-Time PCR Quantification of Mycobacterium immunogenum in Used Metalworking Fluids

Abstract

Rapid detection and quantification of Mycobacterium immunogenum in field samples of metalworking fluids (MWFs) is important for factory fluid surveillance programs. The applicability of the developed DNA extraction and quantitative real-time PCR (qPCR) methods to detect and quantify M. immunogenum in used MWFs was evaluated. Total DNA from these samples was extracted, and M. immunogenum measured by qPCR by comparison with a standard curve derived from plasmid vectors. PCR counts were compared with bacterial culture counts. PCR counts of M. immunogenum varied from 1.42 × 10³ to 3.68 × 10⁶ cells/mL of MWFs. Recovery of M. immunogenum by bacterial culture varied from 2.5% to 70% of qPCR count in corresponding samples. Quantitative PCR could be used to measure M. immunogenum load in MWF samples with greater sensitivity and shorter processing time than the classic bacterial culture-based approach. The proposed qPCR approach could be routinely used in real-time PCR-equipped laboratories to provide early detection of M. immunogenum and to control proliferation that probably leads to hypersensitivity pneumonitis in exposed workers.

Keywords:

• M. immunogenum
• metalworking fluids
• quantitative PCR

ACKNOWLEDGMENTS

The authors gratefully acknowledge financial support from the Fondation J-D Bégin de la Chaire de pneumologie de l'Université Laval. Caroline Duchaine is the recipient of CIHR/IRSST and FRSQ Junior 2 scholarships, and Peter Thorne acknowledges support from NIEHS P30 ES05605.