title: Rapid and inexpensive species differentiation using a multiplex real-time polymerase chain reaction high-resolution melt assay.
abstract: We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers.
authors.affiliation: Department of Chemistry, Towson University, Towson, MD 21252, USA. Electronic address: kmelkins@towson.edu.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Elkins
authors.affiliation: Department of Chemistry, Towson University, Towson, MD 21252, USA.
authors.forename: Anjelica C U
authors.initials: AC
authors.lastname: Perez
authors.affiliation: Department of Chemistry, Towson University, Towson, MD 21252, USA.
authors.forename: Katherine C
authors.initials: KC
authors.lastname: Sweetin
mesh_terms: D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity; D013696:Temperature
keywords: High-resolution melt; LCGreen plus; Real-time PCR; SYBR Green I