title: A real-time PCR diagnostic method for detection of Naegleria fowleri.
abstract: Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence. Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri.
authors.affiliation: Regional Authority of Public Health Banská Bystrica, Department of Molecular Biology, Cesta k nemocnici 1, Banská Bystrica 975 56, Slovakia. lucia.madarova@vzbb.sk
authors.forename: Lucia
authors.initials: L
authors.lastname: Madarová
authors.forename: Katarína
authors.initials: K
authors.lastname: Trnková
authors.forename: Sona
authors.initials: S
authors.lastname: Feiková
authors.forename: Cyril
authors.initials: C
authors.lastname: Klement
authors.forename: Margita
authors.initials: M
authors.lastname: Obernauerová
mesh_terms: D020808:Central Nervous System Protozoal Infections / Q000175:diagnosis / Q000469:parasitology; D003199:Computer Systems; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis* / Q000134:cerebrospinal fluid / Q000737:chemistry; D005456:Fluorescent Dyes; D005618:Fresh Water / Q000469:parasitology; D057230:Limit of Detection; D008565:Membrane Proteins / Q000235:genetics; D016848:Naegleria fowleri / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015800:Protozoan Proteins / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012984:Software; D013551:Swimming Pools; D013997:Time Factors
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D005456:Fluorescent Dyes; D008565:Membrane Proteins; C460543:Mp2CL5 protein, Naegleria fowleri; D015800:Protozoan Proteins