PMA-PCR kits are designed for selective detection of viable bacteria from a specific strain using PMA or PMAxx dye and real-time PCR. The kits contain PMA or PMAxx dye, Fast EvaGreen 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 E. coli uidA gene, with 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 Protocol-31050-31050-X and PMA Reference List for more information.
- PMA dye or PMAxx dye, 20 mM in water, 100 uL
- 5X PMA Enhancer for Gram Negative Bacteria, 16 mL
- 2X Fast EvaGreen qPCR Master Mix, 2 x 1 mL (200 reactions)
- 10X ROX reference dye, 1 mL
- uidA 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 (see PMA Reference List). 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.
Fast EvaGreen® Master Mix is a ready-to-use hot-start mix for qPCR and DNA melt curve analysis of PCR amplicons. 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. Fast EvaGreen Master Mix contains Cheetah™ Taq, Biotium’s fast-activating chemically-modified hot-start Taq polymerase, which is particularly suitable for fast PCR cycling protocols.
Escherichia coli is a species of bacteria that is commonly used in laboratories. Some strains of E. coli are food-borne pathogens and can cause digestive illness. For specific detection of the pathogenic E. coli strain O157:H7, please see kit number 31037. PCR to amplify the gene uidA has been published and shown to be highly specific for E. coli (see Reference 2 under the references tab). The primers provided in the kit have been validated at Biotium for real-time qPCR using EvaGreen Master Mix (see product protocol under downloads for details).
Also see our other PMA-PCR kits for detection of Mycobacterium tuberculosis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Listeria monocytogenes, Legionella pneumophila, and Salmonella enterica.
31050 (kit containing PMA) – 200 assays
31050-X (kit containing PMAxx) – 200 assays