A filter-based propidium monoazide technique to distinguish live from membrane-compromised microorganisms using quantitative PCR

Propidium monoazide (PMA) was used to differentiate live from membrane-compromised bacteria in PCR methods. We have adapted this technique for use on membrane-filtered water samples and determined its efficacy using qPCR. Independent labs at three institutions replicated these findings. ► PMA differ...

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Bibliographic Details
Published in:Journal of microbiological methods 2012-04, Vol.89 (1), p.76-78
Main Authors: Hellein, Kristen N., Kennedy, Elizabeth M., Harwood, Valerie J., Gordon, Katrina V., Wang, Shiao Y., Lepo, Joe Eugene
Format: Article
Language:English
Subjects:
Azides - metabolism
bacteria
Bacteriological Techniques - methods
Biological and medical sciences
Cell Membrane - physiology
Enzyme Inhibitors - metabolism
Filtration - methods
Fundamental and applied biological sciences. Psychology
Membrane filtration
Microbial Viability
Microbiology
propidium
Propidium - analogs & derivatives
Propidium - metabolism
Propidium monoazide
qPCR
quantitative polymerase chain reaction
Real-Time Polymerase Chain Reaction - methods
Reproducibility of Results
Viability
Water Microbiology
Water quality
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Summary:Propidium monoazide (PMA) was used to differentiate live from membrane-compromised bacteria in PCR methods. We have adapted this technique for use on membrane-filtered water samples and determined its efficacy using qPCR. Independent labs at three institutions replicated these findings. ► PMA differentiation of live-dead PCR targets adapted to membrane-filtered waters. ► An array of 16 blue LEDs replaced quartz-halogen activation of PMA on bacteriological filters. ► We compared qPCR Ct of DNA from “live” and “dead” filtered samples. ► PMA treatment inhibited PCR of bacterial DNA from "dead” but not “live” samples. ► Similar results were obtained at three independent laboratories.
ISSN:0167-7012
1872-8359
DOI:10.1016/j.mimet.2012.01.015