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Viability PCR is a powerful technology for the sensitive and rapid detection of viable microorganisms. Unlike time-consuming culturing methods, qPCR is a fast and sensitive method of detection. However, normal PCR does not distinguish between live and dead cells. With v-PCR using PMAxx™ or PMA, you get the speed, sensitivity and specificity of PCR, plus quantifiable viability. And because no culturing is required, you can even detect viable but not culturable (VBNC) bacteria.The v-PCR technology can be applied not only to bacteria but to other organisms like yeast and viruses.
PMAxx™ and PMA are photoreactive dyes with high affinity for DNA. The dyes intercalate into dsDNA and form a covalent linkage upon exposure to intense visible light. PMAxx™ and PMA inhibit PCR amplification of modified DNA templates by a combination of removal of modified DNA during purification and inhibition of template amplification by DNA polymerases. Because PMAxx™ and PMA are cell membrane-impermeable, when a sample containing both live and dead bacteria is treated with dye, only dead bacteria with compromised cell membranes are susceptible to DNA modification. In a real-time PCR reaction, dead cell DNA will show delayed amplification and higher Ct than live cells. In a mixed population, v-PCR permits quantitation of cell viability.
Example of viability qPCR of untreated or heat-killed <em>E. coli</em>with PMA dye. PMA treatment eliminates dead cell DNA, resulting in a large delay in Ct compared to live or untreated cells. NTC: no template control.
The principal of viability PCR with PMA or PMAxx™ dyes is based on these membrane impermeable dyes selectively entering dead cells and modifying their DNA. Modified DNA is not amplified, allowing quantitation of viable cell DNA.
PMA is the original dye developed at Biotium for v-PCR. It has been validated in a wide variety of bacterial strains, as well as yeast and viruses. However, while PMA is generally effective at differentiating between live and dead bacteria by qPCR, it does not completely eliminate PCR products from dead cell DNA. This could potentially give false positive results.
PMAxx™ was designed by Biotium scientists to be a superior alternative to PMA. PMAxx™ is much more effective at eliminating PCR amplification of dead cell DNA, and therefore provides the best discrimination between live and dead bacteria.
Live or dead bacteria treated with either PMA or PMAxx™ prior to qPCR. Dead bacteria treated with either PMA or PMAxx™ show delays in amplification compared to either live cells, or untreated dead cells. Bacteria treated with PMAxx™ show a larger delay than those treated with PMA. Therefore PMAxx™ is better able to differentiate live vs dead bacteria by viability PCR.
PMA Enhancer for Gram Negative Bacteria was designed to improve discrimination between live and dead gram-negative bacteria during viability PCR. PMA Enhancer is provided as a 5X solution, and is added to a sample before the addition of viability 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.
PMA Enhancer is compatible with PMAxx™ as well as PMA. PMAxx™ plus Enhancer is the optimal way to perform viability PCR on gram-negative bacteria. PMA Enhancer is available as a stand-alone product, and is also a component of Biotium's PMA Bacterial Viability Kits (gram-negative strains only).
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.
The PMA-Lite™ LED photolysis device allows for time- and temperature-controlled light treatment of PMAxx- or PMA-treated samples in microcentrifuge tubes.