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, 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?strain O157:H7 Z3276 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. See the product protocol under the downloads tab and references for more information.
PMA 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
Z3276 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 Reference 1 under the references tab). Consequently the dye is useful in the selective detection of viable pathogenic cells by quantitative real-time PCR.
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.
E. coli O157:H7 is a pathogenic strain of Escherichia coli bacteria. Often a food-borne pathogen, it can cause digestive illness.?PMA-based viability PCR to detect E. coli O157:H7 has been reported using the primers provided in the kit, and ORF Z3276 was shown to be specific for strain O157:H7 ?(see Reference 2 under the references tab). For pan-E. coli primers to detect other strains of E. coli besides O157:H7, please see kit 31050. In addition, these primers 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 Listeria monocytogenes,?Mycobacterium tuberculosis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Salmonella enterica. Don’t see your favorite strain? Let us know at firstname.lastname@example.org.
Materials from Biotium are sold for research use only.