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, Forget-Me-Not™ qPCR Master Mix, and PCR primers for detection of selected strains of bacteria that are of widespread interest to food safety, public health, and/or antibacterial research. This kit contains primers for amplification of Listeria monocytogenes hly gene, with reagents sufficient to treat 80 bacterial cultures with PMA, and perform 200 PCR reactions. The number of samples that can be treated with PMA using the kit may vary depending on sample type. Kit contents:
- PMA dye, 20 mM in water, 100 uL
- 2X Forget-Me-Not™ qPCR Master Mix, 2 x 1 mL (200 reactions)
- ROX reference dye, 1 mL
- hly primer mix, 5 uM, 400 uL
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. 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.
Listeria monocytogenes is a food-borne pathogen that can cause digestive illness and fetal harm. The gene hly encodes the Listeria Lysin O (LLO) protein that is specific for
Listeria. PMA-PCR using these primers for viability PCR in
Listeria has been published. In addition, these primers have been validated at Biotium for real-time qPCR using Forget-Me-Not™ Master Mix. We also offer viability PCR kits with PMAxx™, 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. Materials from Biotium are sold for research use only.
