title: Quantitative detection and differentiation of free-living amoeba species using SYBR green-based real-time PCR melting curve analysis.
abstract: Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.
authors.affiliation: Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven (KUL), Charles Deberiotstraat 32, 3000, Leuven, Belgium. Jonas.behets@bio.kuleuven.be
authors.forename: Jonas
authors.initials: J
authors.lastname: Behets
authors.forename: Priscilla
authors.initials: P
authors.lastname: Declerck
authors.forename: Yasmine
authors.initials: Y
authors.lastname: Delaedt
authors.forename: Lieve
authors.initials: L
authors.lastname: Verelst
authors.forename: Frans
authors.initials: F
authors.lastname: Ollevier
mesh_terms: D048248:Acanthamoeba castellanii / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016841:Amoebida / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D006249:Hartmannella / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D009253:Naegleria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity