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 Staphylococcus aureus-specific nuc 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 Protocol-31035 and PMA Reference List for more information.
- PMA dye, 20 mM in water, 100 uL
- 2X Fast EvaGreen qPCR Master Mix, 2 x 1 mL (200 reactions)
- 10X ROX reference dye, 1 mL
- nuc 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.
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.
Staphylococcus aureus is an anaerobic gram negative bacterium that can cause of a variety of infections in humans, and methicillin-resistant S. aureus is a rising cause of hospital-acquired infections. The primers included in this kit have been tested for amplification of the S. aureus-specific gene nuc gene (reference 2) from genomic DNA from both methicillin-susceptible and methicillin-resistant strains of S. aureus (see product protocol under downloads for details). Biotium also offers the Staphylococcus aureus (mecA) PMA-PCR kit, which includes primers against the methicillin-resistance gene mecA.
Cat # 31035 – 1 kit (80 bacterial cultures / 200 PCR reactions).