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REAL-TIME QUANTITATIVE PCR DETECTION OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS IN DRINKING WATER
KING, D. N., A. BEUMER, AND S. L. PFALLER. REAL-TIME QUANTITATIVE PCR DETECTION OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS IN DRINKING WATER. Presented at American Society for Microbiology 107th General Meeting, Toronto, ON, CANADA, May 21 - 25, 2007.
Develop a real-time PCR method for the rapid detection and quantification of Mycobacterium avium and M. intracellulare in drinking water.
Determine the subspecies of those M. avium complex bacteria in EPA culture collection.
The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and others. MAC are listed on the U.S. Environmental Protection Agency's Contaminant Candidate List (CCL) due to their association with human disease and occurrence in public drinking water systems. Current methods for detecting MAC organisms in drinking water are culture-based. However evidence suggests that culture-based methods have severe limitations including long incubation periods, loss of target due to overgrowth of background organisms, up to 70% loss of target due to harsh decontamination techniques, and inability to recover MAC in a viable-but-non-culturable state. Because of these drawbacks and the need for more accurate and comprehensive occurrence data, we have developed real-time QPCR assays for the detection and quantification of MA, MI, and M. avium subspecies paratuberculosis (MAP) in drinking water. Real-time QPCR assays were developed using primers and TaqMan® probes designed to amplify a region of the 16S rDNA in MA and MI, and regions of IS900 and Target 251 in MAP. Primer/probe sets were found to be highly specific when compared to
sequences in nucleotide databases and confirmed experimentally by screening 104 MAC strains. No false negatives occurred when each species was tested with its own primer/probe set, 2.3% (1/42) MA strains were false positive with the MI prober/probe set, and 2.5% (1/40) MI strains were false positive with the MA primer/probe set. No false positives were obtained when nine non-MAC species were screened with all primer/probe sets. Quantification is linear over a minimum range of six logs of target
concentration in all four assays. Additionally, a control has been developed to measure PCR inhibition due to compounds in the water matrix. We are currently evaluating the QPCR assays for use on actual drinking water samples as a rapid alternative to culture methods in order to generate a more complete understanding of MAC occurrence in drinking water.