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Detection of Mycobacterium avium subspecies paratuberculosis in Drinking Water and Biofilms Using Quantitative PCR
Beumer, A., D. N. KING, M. J. DONOHUE, J. Mistry, T. C. COVERT, AND S. L. PFALLER. Detection of Mycobacterium avium subspecies paratuberculosis in Drinking Water and Biofilms Using Quantitative PCR. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 76(21):7367-7370, (2010).
The objectives of this Task are the following: 1) Develop a integrated method to collect, concentrate, purify (if needed), extract nucleic acid and real-time PCR for the quantification of Mycobacterium avium and M. intracellulare in drinking water. 2) Develop a method for detection and enumeration of M. paratuberculosis in environmental samples. 3) Determine the subspecies of M. avium complex bacteria in EPA culture collection. 4) To determine passive binding of Microcystin to Dynal magnetic beads to natural organic matters 5) To characterize covalently coupled monoclonal antibodies against MC-LR with a solid phase matrix and determine reuse capability 6) To optimize binding and determine recovery rates of MC-LR in spiked water samples using different samples volumes 7) To determine loading capacity of individual variants of MC and mixture of MCs 8) To compare recovery rates of immunomagnetic beads (IMB) and traditional solid phase extraction (SPE) in drinking and surface waters by HPLC with PDA and MS detection 9) Develop rapid methods, e.g. QPCR, microarray analysis, etc to measure the occurrence of and quantify potential risk of exposure to waterborne pathogens, especially those on the CCL and PCCL.
Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease in domestic animals and has been implicated in Crohn’s disease in humans. This bacterium is a slow growing, gram-positive, acid-fast organism which can be difficult to culture from the environment. For this reason, quantitative PCR (qPCR) assays have been developed to detect and quantify MAP in various matrices. In the current study, a qPCR assay targeting the IS900 insertion element was incorporated into a complete method for analyzing environmental samples. Positive results were confirmed with a second, MAP-specific Target 251 qPCR assay. The method was used to analyze drinking water and biofilm samples collected from the Midwestern United States during spring and summer of 2007, followed in 2009 by an analysis of drinking water samples from geographically scattered sites in the U.S. MAP was detected by both IS900 and Target 251 assays in 88% (29/33) of drinking water and 77% (25/33) of biofilm samples from the Midwest in 2007. However water and biofilm samples collected from ten sites in the Midwest during spring 2009, including five sites sampled in 2007, were negative for MAP. Not one water sample collected during the national survey in 2009 was positive for MAP using both assays, though 2.1% (5/238) of samples were positive for the IS900 qPCR assay. Our results suggest that MAP may be common in drinking water and faucet biofilms at times, which could be a source of human exposure to MAP. Abiotic and biotic factors affecting the occurrence of MAP in drinking water require further elucidation.