August 1, 2012.
Currently, there are no portable instruments capable of both pathogen viability assessment and microbial identification. There is an urgent need to distinguish between viable and inactivated microorganisms, as well as assess if they are pathogenic. Propidium monoazide (PMA) is a DNA intercalating dye that is cell membraneimpermeable, thus it only comes in contact with the DNA of dead cells. Polymerase chain reaction (PCR) amplifies specific sequences of DNA allowing for the precise identification of organisms, such as pathogens. Through binding of DNA, PMA strongly inhibits PCR amplification of dead cells, which enables the selective DNA‐based detection of viable pathogens. Therefore, a PMA‐PCR combined assay, rather than conventional PCR, has the potential to distinguish between specific viable and nonviable bacteria or viruses. However promising, there is a lack of evidence demonstrating PMA’s use as a true quantitative tool for viability assessment, which will be critical for most real‐world applications. This stems in part from: i) the lack of a rigorous comparison of PMA‐qPCR with gold standards such as culturing, and ii) a lack of information on how PMA enters cells and inhibits PCR. To elucidate the quantitative effectiveness of PMA‐qPCR, mixed fractions of viable (non‐heat –inactivated) and dead (heat‐inactivated) E. coli K12 strain cells were prepared (Viable:Dead, 100:0, 75:25, 50:50, 25:75, 10:90, 1:99, 0:100) and examined through PMA‐qPCR, as well as fluorescence microscopy and cell culturing for comparison against conventional methods. We also assessed the mechanism of PMA permeability through PMA‐qPCR of E. coli inactivated by mechanisms independent of membrane permeation. We found that PMA‐qPCR is only quantitative at 10% and lower viable proportion of cells in a sample, and that the PMA mechanism of action may be part precipitation, as well as an inhibitor of polymerase processivity.
NASA Jet Propulsion Laboratory (JPL)
This material is based upon work supported by the S.D. Bechtel, Jr. Foundation and by the National Science Foundation under Grant No. 0952013 and Grant No. 0833353. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the S.D. Bechtel, Jr. Foundation or the National Science Foundation. This project has also been made possible with support of the National Marine Sanctuary Foundation. The STAR program is administered by the Cal Poly Center for Excellence in Science and Mathematics Education (CESaME) on behalf of the California State University (CSU).