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Viability PCR Starter Kits

Viability PCR starter kits contain the materials that you need for selective detection of viable cells using either PMA or PMAxx™ viability dye and real-time PCR. All of the kits contain Forget-Me-Not™ qPCR Master Mix, and your choice of PMA or PMAxx™ dye.

Product Attributes

Viability dye

PMA, PMAxx™

Bacteria type

All species, Gram negative bacteria

Viability dye
Bacteria type
Size
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200 assays
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Product Description

Viability PCR starter kits contain the materials that you need for selective detection of viable cells using either PMA or PMAxx™ viability dye and real-time PCR. All of the kits contain Forget-Me-Not™ EvaGreen® qPCR Master Mix, and your choice of PMA or PMAxx™ dye. The user will need to supply their own primers to amplify their species of interest (see this blog post for primer design considerations).

This kit contains reagents sufficient to treat 80 bacterial cultures with PMA or PMAxx™, and perform 200 PCR reactions. There is an option to choose either PMA or PMAxx™ for the viability dye (see below for more information about these dyes). The number of samples that can be treated with PMA or PMAxx™ using the kit may vary depending on sample type. See the product protocol under the downloads tab and references for more information.

Principal of viability PCR with PMA or PMAxx™ dyes.

Kit contents:

Storage and Handling

Store kit at -20 °C. After first thaw, Enhancer should be stored at 4°C. Protect PMA, PMAxx™ and Forget-Me-Not™ EvaGreen® qPCR Master Mix from light. Protect PMA and PMAxx™ from light during use. Components are stable for at least 6 months when stored as recommended. Components are stable for several freeze/thaw cycles.

PMA and PMAxx™ viability dyes

PMA is a high affinity photoreactive DNA binding dye developed by Biotium. The dye is weakly fluorescent by itself but becomes highly fluorescent upon binding to nucleic acids. It preferentially binds to dsDNA with high affinity. Upon photolysis, the photoreactive azido group on the dye is converted to a highly reactive nitrene radical, which readily reacts with any hydrocarbon moiety at the binding site to form a stable covalent nitrogen-carbon bond, 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 inhibits PCR amplification of modified DNA templates by a combination of removal of modified DNA during purification and inhibition of template amplification by DNA polymerases. Consequently the dye is useful in the selective detection of viable pathogenic cells by quantitative real-time PCR.

PMAxx™ is a new and improved version of PMA 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.

Forget-Me-Not™ EvaGreen® qPCR Master Mix

Forget-Me-Not™ qPCR Master Mix is a hot-start EvaGreen® dye-based master mix for use in real time PCR applications and DNA melt curve analysis. Forget-Me-Not™ master mix contains a low concentration of blue dye which allows you to see at a glance whether you forgot to add master mix to any of your tubes, so you can catch pipetting mistakes and avoid wasting time, reagents, and your precious DNA samples. It is formulated for qPCR using a fast cycling protocol, but also can be used for qPCR using regular cycling protocols. EvaGreen® dye is a unique DNA-binding dye with features ideal for both qPCR and melt curve analysis. EvaGreen® dye binds to dsDNA via a novel “release-on-demand” mechanism, which permits the use of a relatively high dye concentration in qPCR without PCR inhibition. Forget-Me-Not™ Master Mix contains Cheetah™ Taq, Biotium’s fast-activating chemically-modified hot-start Taq polymerase, which is particularly suitable for fast PCR cycling protocols.

Biotium also offers strain-specific PMA-PCR kits containing validated primer sets for detection of E. coli, E. coli strain 0157:H7, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Salmonella entericaListeria monocytogenes, Legionella pneumophila and Mycobacterium tuburculosis. Don’t see your favorite strain? Let us know at techsupport@biotium.com.

LED Photolysis Devices

Biotium sells two devices for the photolysis step of viability PCR. The PMA-Lite™ LED photolysis device is designed for PMAxx™- or PMA-treated samples in microcentrifuge tubes. The Glo-Plate™ Blue is a flat LED box ideal for samples in microplates, filters, or other formats.

PMA-Lite LED Photolysis Device
PMA-Lite™ LED Photolysis Device, for photoactivation of PMAxx™, PMA or EMA
Glo-Plate™ Blue LED Illuminator for photoactivation of PMAxx™, PMA and EMA

Materials from Biotium are sold for research use only.

References

 

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