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 the methicillin resistance gene mecA, which confers methicillin-resistance in Staphyloccoccus aureus and other bacterial species. The kit includes 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 – 31036 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
- mecA primer mix, 5 uM, 200 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 mecA gene confers methicillin-resistance in S. aureus and other methicillin-resistant bacterial species. The mecA primers included in the kit have been used for detection of methicillin-resistant S. aureus (PMA Reference List), and have been validated for qPCR using Biotium’s Fast EvaGreen Master Mix. Note that the mecA primers are not specific to S. aureus, because mecA is expressed by other methicillin-resistant bacteria. Biotium also offers the Staphylococcus aureus (nuc) PMA-PCR kit, which includes primers against the S. aureus-specific nuc gene, expressed by methicillin-susceptible and methicillin-resistant S. aureus strains.
Cat # 31036 – 1 kit (80 bacterial cultures / 200 PCr reactions)