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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

Cell membrane-impermeable PMA dye selectively and covalently modifies DNA from dead bacteria with compromised membrane while leaving DNA from viable cells intact. Because PMA-modified DNA can not be amplified, subsequent lysis of viable cells and qPCR permit selective quantitation of viable bacteria.

The ability to selectively and sensitively detect viable bacteria in the presence of dead bacteria is of vital importance for many practical applications, including safety inspection of food products, drinking water quality control, and medical diagnosis. The traditional method of culturing bacterial is time-consuming and has low sensitivity.

Detection based on PCR is a rapid and highly sensitive alternative method. However, PCR methods cannot distinguish live from dead cells. Biotium designed the novel DNA-modifying dye propidium monoazide (PMA) to overcome this problem. Treating bacteria with PMA prior to PCR analysis permits one to selectively detect only viable bacteria in a highly sensitive and reliable manner.

PMA is a photo-reactive dye with a high affinity for DNA. The dye intercalates into dsDNA and forms a covalent linkage upon exposure to intense visible light, resulting in chemically modified DNA, which cannot be amplified by PCR (Figure 1). Because PMA is designed to be cell membrane-impermeable, when a sample comprising both live and dead bacteria is treated with PMA, only dead bacteria are susceptible to DNA modification due to compromised cell membranes. Thus, subsequent lysis of live bacteria followed by qPCR permits selective detection of the live cells. The PMA-qPCR technology can be applied not only to bacteria but to other cell types as well (see the PMA Reference List). Biotium developed PMA as an improvement on ethidium monoazide (EMA) (Nocker et al. 2006). PMA provides better discrimination between live and dead bacteria because it is excluded from live cells more efficiently than EMA, and has higher affinity for nucleic acids.

PMA amplification curves labeled
Representative real-time PMA-PCR amplification curves from untreated and heat-killed E. coli, using primers against 16S rRNA.
The delta Ct of live and killed E. coli with and without PMA treatment. The Ct value of sample without PMA was subtracted from the corresponding sample with PMA cross-linking (Ct with PMA – Ct without PMA).

 New! PMAxx™ for Viability PCR

PMAxx™ dye is a DNA modifier used for viability PCR, invented by scientists at Biotium. PMAxx™ is a new and improved version of our popular viability dye PMA™ (propidium monoazide). Like PMA, PMAxx™ is a photo-reactive dye that binds to dsDNA with high affinity. Upon photolysis with visible light, PMAxx™ dye becomes covalently attached to dsDNA. The PMAxx-modified dsDNA cannot be amplified by PCR. PMAxx™ dye is designed to be cell membrane-impermeable. Thus, in a population of live and dead cells, only dead cells are susceptible to DNA modification due to compromised cell membranes. This unique feature makes PMAxx™ highly useful in selective detection of live bacteria by qPCR.

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.

Bsub_PMAxx_curves_2_labeled
Differentiation of live and dead bacteria using PMAxx™ vs. PMA™ in untreated and heat-killed B. subtilis.

 

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


 New! 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).


New! PMA Real-Time PCR Bacterial Viability Kits

PMA-PCR kits are designed for selective detection of viable bacteria from a specific strain using 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