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PMA Enhancer for Gram Negative Bacteria, 5X Solution

PMA Enhancer for Gram Negative Bacteria was designed to improve PMA- and PMAxx™-mediated discrimination between live and dead gram-negative bacteria.

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Product Description

PMA is a high affinity photoreactive DNA binding dye developed by Biotium for viability PCR (40019). PMAxx™ is our new and improved viability PCR dye (40069). The dyes are weakly fluorescent by themselves but become highly fluorescent upon binding to nucleic acids. Upon photolysis, the dye forms a stable covalent bond with the DNA, thus resulting in permanent DNA modification. The dye is cell membrane-impermeable and thus can be used to selectively modify DNA from dead cells with compromised membrane integrity, while leaving DNA from viable cells intact. PMA and PMAxx™ inhibit PCR amplification of modified DNA templates. Consequently the dye is useful in the selective detection of viable pathogenic cells by quantitative real-time PCR.

PMA Enhancer for Gram Negative Bacteria was designed to improve PMA- and PMAxx™-mediated discrimination between live and dead gram-negative bacteria. PMA Enhancer is provided as a 5X solution, and is added to a sample before the addition of dye. 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. Thus, PMA or PMAxx™ plus Enhancer is the optimal way to perform viability PCR on gram-negative bacteria.

The mechanism by which Enhancer improves live/dead discrimination has not been determined. It may improve passive permeability of dead cell walls or membranes to the dye, and/or improve access of the dye to the dead cell DNA.

E. coli were killed with mild heat treatment (56°C for 3 hrs) and treated with PMAxx™ or PMAxx™ + Enhancer, followed by light exposure using PMA-Lite™, DNA purification, and qPCR with Fast EvaGreen® qPCR Master Mix. dCt values were calculated by subtracting the Ct without dye from the Ct with dye. Only dead cells treated with PMAxx™ + Enhancer showed a large dCt, indicating that the dye successfully inhibited PCR of dead cell DNA.

Note: PMA Enhancer is not intended for use when gram-positive bacteria are to be detected. PMA Enhancer may adversely affect the amplification of live cell DNA from gram-positive bacteria.

For photoactivation of PMA, PMAxx™ or EMA dye, we recommend the use of Biotium’s PMA-Lite™ LED Photolysis Device (E90002), which is designed to conduct photolysis under controlled conditions.

Also see our PMA Real-Time Bacterial Viability Kits.

 PMA diagram

Materials from Biotium are sold for research use only.

References

1. Nocker, A., et al. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbiol. Meth. 67(2), 310-320 (2006).

2. Nocker, A., et al. Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR. J. Microbiol. Meth. 70(2), 252-260 (2007).

3. Fittipaldi, M., et al. Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification. J. Microbiol. Meth. 91(2), 276-289 (2012).

Find a full reference list under the Downloads section on this page

 

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