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Viability PCR

PMAxx™ and PMA viability PCR dyes

PMAxx™ and PMA
Dead cell-specific dyes covalently attach to DNA after photolysis
PMAxx™ for Viability PCR
Better than PMA at eliminating dead cell DNA from qPCR
PMA Enhancer for Gram Negative Bacteria
Improve discrimination between live and dead gram-negative bacteria during viability PCR

Principle of viability PCR using PMA or PMAxx 

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 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 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

PMA Real-Time PCR Bacterial Viability Kits

Product NameSKUBacteria StrainGene NameSizePurchase
PMA Real-Time PCR Bacterial Viability Kit – E. coli (uidA)31050E. coliuidA200 assaysPurchase 31050
PMA Real-Time PCR Bacterial Viability Kit – E. coli O157:H7 (Z3276)31037E. coli O157:H7Z3276200 assaysPurchase 31037
PMA Real-Time PCR Bacterial Viability Kit – Listeria monocytogenes (hly)31051Listeria monocytogeneshly200 assaysPurchase 31051
PMA Real-Time PCR Bacterial Viability Kit – Salmonella enterica (invA)31033Salmonella entericainvA200 assaysPurchase 31033
PMA Real-Time PCR Bacterial Viability Kit – E. coli (uidA)31050-XE. coliuidA200 assaysPurchase 31050-X
PMA Real-Time PCR Bacterial Viability Kit – E. coli O157:H7 (Z3276)31037-XE. coli O157:H7Z3276200 assaysPurchase 31037-X
PMA Real-Time PCR Bacterial Viability Kit – Listeria monocytogenes (hly)31051-XListeria monocytogeneshly200 assaysPurchase 31051-X
PMA Real-Time PCR Bacterial Viability Kit – Salmonella enterica (invA)31033-XSalmonella entericainvA200 assaysPurchase 31033-X
PMA Real-Time PCR Bacterial Viability Kit – Legionella pneumophila (mip)31053Legionella pneumophilamip200 assaysPurchase 31053
PMA Real-Time PCR Bacterial Viability Kit – Mycobacterium tuberculosis (groEL2)31034Mycobacterium tuberculosisgroEL2200 assaysPurchase 31034
PMA Real-Time PCR Bacterial Viability Kit – Staphylococcus aureus (mecA)31036Staphylococcus aureusmecA200 assaysPurchase 31036
PMA Real-Time PCR Bacterial Viability Kit – Staphylococcus aureus (nuc)31035Staphylococcus aureusnuc200 assaysPurchase 31035

 

See product information for PMA Real-Time PCR Kits.

* Don’t see your favorite bug? Let us know at techsupport@biotium.com


PMA-Lite™ and Glo-Plate™ Blue for photoactivation of PMAxx™ and PMA

Biotium offers the PMA-Lite™ LED photolysis device for time- and temperature-controlled light treatment of PMAxx- or PMA-treated samples.

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 PMAxx™, PMA, EMA or similar dyes

Biotium offers the Glo-Plate™ Blue LED Illuminator for even light treatment of PMAxx™- or PMA-treated samples.

Glo-Plate™ Blue LED Illuminator

Features:

  • Provides even illumination across a flat surface, ideal for multi-well plates.
  • Four timer settings for 10, 15, 20 or 30 minutes of light exposure.
  • Long-lasting LED lights with blue emission.
  • For efficient activation of PMAxx™, PMA, EMA or similar dyes

Bacterial Viability and Gram Stain Kit

From: $210 Sizes: 200 assaysCatalog #: 32001

Live Bacterial Gram Stain Kit

From: $126 Sizes: 200 assays, 800 assaysCatalog #: 32000, - 32000-1View allHide

Wheat Germ Agglutinin (WGA) Conjugates

From: $63 Sizes: 1 mg, 5 x 1 mgCatalog #: 29021, 29024-1, 29025-1, 29026-1, 29027-1, 29028-1, 29029-1, 29059-1, 29064-1, 29077-1, 29077, 29076-1, 29023-1, 29022-1, 29021-1, 29022, 29023, 29024, 29025, 29026, 29027, 29028, 29029, 29059, 29064, 29073, - 29076View allHide