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PMAxx™, PMA, PMA-Lite™, GLo-Plate™ Blue & strain-specific kits for viability PCR

Principle of viability PCR using PMA or PMAxx

Viability PCR (v-PCR)
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.

How does v-PCR work?
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. The v-PCR technology can be applied not only to bacteria but to other cell types as well.

PMA amplification curves labeled
Representative real-time PMA-PCR amplification curves from untreated and heat-killed E. coli, using primers against 16S rRNA.
Heat-killed E. coli treated with PMA show a large reduction in cycle (dCt) in qPCR. Live E. coli treated with PMA do not show a change in Ct.

PMAxx™ for Viability PCR

PMAxx™ was designed by Biotium scientists to be a superior alternative to PMA. 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. Biotium’s new dye PMAxx™ is much more effective at eliminating PCR amplification of dead cell DNA, and therefore provides the best discrimination between live and dead bacteria.

Download the PMA™ and PMAxx™ flyer to learn more, or see PMAxx™ product information.


PMA Enhancer for Gram-Negative Bacteria

PMA Enhancer for Gram Negative Bacteria was designed to improve PMA-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 PMA. 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. Thus, PMA 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 a component of our PMA Bacterial Viability Kits (gram-negative strains only) (see below). See product information for PMA Enhancer for Gram Negative Bacteria.
E. coli that were killed by mild heat shock (56C for 3 hours) were treated with 25 uM PMA and/or 1X PMA Enhancer, followed by exposure with PMA-Lite and DNA isolation. Real-time PCR was performed using Fast EvaGreen Master Mix and primers that amplify a 377-bp fragment of E. coli DNA. Dead cells treated with PMA and Enhancer showed a significant further delay in amplification than dead cells treated with PMA alone. Enhancer treatment without PMA had no effect on dead cell DNA (or live cell DNA, data not shown).
E. coli that were killed by mild heat shock (56C for 3 hours) were treated with 25 uM PMA and/or 1X PMA Enhancer, followed by exposure with PMA-Lite and DNA isolation. Real-time PCR was performed using Fast EvaGreen Master Mix and primers that amplify a 377-bp fragment of E. coli DNA. Dead cells treated with PMA and Enhancer showed a significant further delay in amplification than dead cells treated with PMA alone. Enhancer treatment without PMA had no effect on dead cell DNA (or live cell DNA, data not shown).


Real-Time strain-specific PCR Bacterial Viability Kits

Strain-specific v-PCR kits are designed for selective detection of viable bacteria from a specific strain using PMAxx or PMA dye and real-time PCR. The kits contain PMA dye OR PMAxx™ dye, Forget-Me-Not™ qPCR Master Mix, and validated PCR primers for detection of selected strains of bacteria that are of widespread interest to food safety, public health, and/or antibacterial research.

Kits include:

• PMA dye OR PMAxx dye
• Forget-Me-Not qPCR Master Mix
• ROX reference dye
• PMA Enhancer (gram-negative strains only)
• Validated PCR primers for specific bacterial strain

PMA Real-Time PCR Bacterial Viability Kits are available for:
Salmonella enterica
Mycobacterium tuberculosis
Staphylococcus aureus
Staphylococcus aureus (methicillin-resistant)
Listeria monocytogenes
E. coli
E. coli O157:H7
Legionella pneumophila

See product information for PMA Real-Time PCR Kits.
* Don’t see your favorite bug? Let us know at techsupport@biotium.com


PMA-Lite™ for photoactivation of PMA

Biotium offers the PMA-Lite™ LED photolysis device for time- and temperature-controlled light treatment of PMA and other photoreactive dyes.

PMA-Lite™ LED photolysis device.
PMA-Lite™ LED Photolysis Device.

Features:

  • Provides even illumination to up to 18 microcentrifuge tubes.
  • Internal fan keeps temperature < 37°C.
  • Four timer settings for 10, 15, 20 or 30 minutes of light exposure.
  • Long-lasting LED lights with 465-475 nm emission
  • For efficient activation of PMA, PMAxx™, EMA or similar dyes

See product information for PMA-Lite™ LED Photolysis Device.

 


View products associated with this technology…

PMAxx™, PMA, and other microbiology products