Many users have had success using PMA in viability PCR with environmental samples, but these samples can be more challenging than pure cultures. We’ve outlined some of the challenges below.
Water samples In order to perform viability PCR on water samples, some users have developed methods to concentrate the microorganisms onto a 0.45 um filter, and then treat the filter with PMA. An example protocol can be found in this publication: Ditommasso S., et al. (2015) Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths. Mol. Cell Probes. doi: 10.1016/j.mcp.2015.05.011.
Opaque or complex samples Viability PCR with PMA has been successfully reported in various complex sample types, such as sewage or soil. No reports have been made yet with PMAxx™ in complex samples, but we would expect it to work as well or better than PMA. The main considerations for complex samples are dye concentration and light penetration. Typically a higher dye concentration (100 uM or more) is needed because some dye may bind to contaminants in the sample. Longer light treatment and more mixing during light treatment may be required, since it will be harder for the light to penetrate through the sample. An example protocol can be found in this publication: Guo F. and Zhang T. (2014) Detecting the nonviable and heat-tolerant bacteria in activated sludge by minimizing DNA from dead cells. Microb. Ecol. dio 10.1007/s00248-014-0389-2.
Viability PCR using PMA has been published in hundreds of publications, with dozens of different organisms, and many different types of samples. For more information, please see our PMA publication list or do a literature search for your application of interest.View more FAQs