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.
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.
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.
PMA (propidium monoazide) is a high-affinity photoreactive DNA binding dye invented by scientists at Biotium for viability PCR of bacteria and other organisms. The dye is weakly fluorescent by itself but becomes more fluorescent after binding to nucleic acids. It preferentially binds to dsDNA with high affinity. Upon photolysis, the dye covalently reacts with DNA, thus resulting in permanent DNA modification. The dye is cell membrane-impermeable, and thus can be selectively used to modify only exposed DNA from dead cells while leaving DNA from viable cells intact. This feature makes the dye highly useful in the selective detection of viable pathogenic cells by quantitative real-time PCR in the presence dead cells whose DNA has been PMA-modified and thus can not be amplified.
Since Biotium first developed PMA dye, there have been numerous publications on the use of the dye in pathogenic bacterial detection related to food and water safety, medical diagnosis and biodefense. Check the Downloads section to view the PMA&PMaxx™ Reference List and the PMA&PMAxx™ Validated Bacterial Strains List. PMA dye is also available in H2O (40019), a convenient ready-to-use format.
Abs/Em = 510/610 nm (following photolysis and covalent attachment to DNA/RNA)
Dark red solid soluble in water
Store at 4°C and protect from light at all times
PMAxx™ Dye for Viability PCR
PMAxx™ (40069) is a new and improved dye for viability PCR. It functions in a manner very similar to PMA, but has much greater activity and ability to distinguish between live and dead bacteria. PMAxx™ can be used as a replacement for PMA in any bacterial viability-PCR assay. We recommend that customers new to viability PCR try PMAxx™ instead of PMA, for improved detection of viable bacteria.
PMA Enhancer for Gram-Negative Bacteria
For viability PCR of gram-negative bacteria we highly recommend using PMA Enhancer (31038) in conjunction with PMA. PMA Enhancer for Gram Negative Bacteria was designed to improve PMA-mediated discrimination between live and dead gram-negative bacteria. When a sequence from a gram-negative bacteria is amplified by PCR, samples pre-treated with Enhancer show a decrease in the signal from dead cells, with no change in the signal from live cells.
Viability PCR Kits
To more easily get started with viability PCR, learn about our customizable Viability PCR Starter Kits. You choose which viability PCR dye you prefer, and whether or not you need Enhancer for gram-negative bacteria. All of the starter kits come with an EvaGreen® qPCR master mix and a detail protocol to get you started.
Biotium also provides strain-specific PMA Real-Time PCR Bacterial Viability Kits with validated primers for selected pathogens: M. tuberculosis, S. aureus, MRSA, Salmonella, E. coli, E. coli O157:H7, Legionella and Listeria. These kits provide everything that you need for the selective detection of your favorite species of live bacteria by real-time PCR.
Nocker, A., Cheung, C.Y., and Kamper, A.K. (2006). Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbio Meth. 67(2), 310-320.
The Glo-Plate™ Blue LED Illuminator is a multi-functional blue LED light box. It can be used for photolysis of PMAxx™ or PMA in viability PCR. It is also an excellent way to develop the staining of gels stained with the visible blue DNA gel stain DNAzure®.
Viability PCR starter kits contain the materials that you need for selective detection of viable cells using either PMA or PMAxx™ viability dye and real-time PCR. All of the kits contain Forget-Me-Not™ qPCR Master Mix, and your choice of PMA or PMAxx™ dye.