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PMA and PMAxx™ for viability PCR

Viability PCR originally used EMA (ethidium monoazide) to inactivate dead cell DNA. Biotium developed PMA (propidium monoazide) in collaboration with investigators at Montana State University (Nocker et al. 2006). PMA is more selective for dead cells than EMA, and became widely used for selective detection of viable microbes and viruses. PMAxx™ is Biotium’s newest viability PCR dye, designed to be more effective than PMA at eliminating PCR amplification of dead cell DNA. Therefore it provides the best discrimination between live and dead bacteria.

Learn more about PMA and PMAxx™ for viability PCR.

Biotium offers the PMA-Lite™ LED Photolysis Device for light-induced cross-linking of PMAxx™ and PMA to dsDNA. The PMA-Lite™ is designed for samples in microcentrifuge tubes. We also offer the Glo-Plate™ Blue LED Illuminator for photoactivation of larger tubes, 96-well plates, or other plate formats.

Commercial halogen lamps (>600 W) for home use have been employed for photoactivating PMA in some publications, though results have not been consistent due to inevitable variation in the set-up configurations. If you decide to use a halogen lamp, we recommend that you lay tubes on a block of ice set 20 cm from the light source, on a rocking platform to ensure continuous mixing. Set the lamp so that the light source is pointing directly downward onto the samples (up to 45° downward slant is OK). Expose samples to light for 5-15 min.

The LEDs in PMA-Lite have brightness of 600-800 millicandela (mcd). There are three LEDs next to each tube (one bottom, two side) and the wavelength is 465-475 nm.

Both PMA and PMAxx™ are both offered as 20 mM solutions in water. We also offer PMA as a solid, and customers often ask whether it needs to be dissolved in DMSO. While in some publications users have prepared their PMA stock solution in DMSO, this is not necessary. Unlike the older viability dye EMA, which is not soluble in water, PMA and PMAxx™ are very water soluble.

We have compared PMA and PMAxx™ for their ability to differentiate between live and dead cells in viability PCR assays, and in all bacteria strains tested*, PMAxx™ had better activity. Therefore, while we haven’t tested every bacterial strain, we recommend PMAxx™ for bacterial viability PCR.

A recent publication by Randazzo et al. compared several viability PCR dyes in norovirus, and found that PMAxx™ worked the best for that organism.

We have also compared PMA and PMAxx™ in viability PCR assays with the yeast Saccharomyces cerevisiae, and found that PMA was equal to or better than PMAxx™. Therefore we recommend PMA for yeast and fungus viability PCR.

*E. coli, Salmonella enterica, Listeria monocytogenes, Bacillis subtilis, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Lactobacillus casei.

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.

PMA and PMAxx™ dyes are chemically stable as long as they are protected from light (stored in the original amber vial, for example). We have confirmed by HPLC analysis that the dyes are unaffected after being left for 10 days at ambient temperature.

PMA and PMAxx™ dyes are very sensitive to light. Light exposure, even ambient room light, causes a chemical change in the dye molecule. Therefore we recommend that vials of PMA or PMAxx™ dyes only be opened in darkened rooms or very dim light. We always suggest that you perform a small test experiment if you are concerned that a dye may have lost activity.

In our standard viability PCR assay protocols we say that following PMA treatment, DNA should be purified from the cells before being used as a template for PCR. However there have been reports of the successful use of bacterial cell lysates in viability PCR following PMA treatment. The main consideration is to ensure that all of the dye is efficiently photolysed before preparing the lysate.  Unreacted PMA dye in the lysate could potentially inhibit the PCR reaction by binding to the DNA or polymerase. If you are considering trying this, we would suggest pelleting the cells after dye and light treatment to remove the excess free dye from the solution. In addition, control experiments should be performed to determine the ideal PMA and light treatment conditions that result in no PCR inhibition.

PMA and PMAxx™ are membrane-impermeant dyes that selectively modify DNA in dead cells with damaged membranes. Therefore, samples should not be frozen before PMA treatment, because this will permeabilize live cell membranes to the dye, and live/dead discrimination will be lost.

After PMA treatment and photolysis, PMA or PMAxx™ becomes covalently linked to dead cell DNA, and any excess dye in solution should be photolyzed and rendered non-reactive. Cells can be frozen at this point for storage before DNA extraction and PCR. For full cell recovery, we would recommend pelleting the cells and removing the supernatant before freezing.

For some strains of bacteria, we sell complete viability PCR kits, which include strain-specific primer sets that have been validated at Biotium, as well as in publications in most cases. However any validated primer sets may be used to target your species of interest. We and others have found that amplicon length can affect your viability PCR results. Using longer amplicons generally results in better suppression of dead cell PCR products. For an example see this publication: Banihashimi A., et al. (2012) Long-amplicon propidium monoazide-PCR enumeration assay to detect viable Campylobacter and Salmonella. J. Appl. Miobiol. dio 10.1111/j.1365-2672.2012.05382.x.

While you can perform viability PCR with amplicons as short at 100 bp, we advise that you use longer amplicons for better results.

 

No, the PMA Enhancer should only be used when only gram- bacteria will be studied. The PMA Enhancer has a detrimental effect of gram+ bacteria.

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