title: Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses.
abstract: Emerging infectious diseases have caused a global effort for development of fast and accurate detection techniques. The rapidly mutating nature of viruses presents a major difficulty, highlighting the need for specific detection of genetically diverse strains. One such infectious agent is SARS-associated coronavirus (SARS-CoV), which emerged in 2003. This study aimed to develop a real-time RT-PCR detection assay specific for SARS-CoV, taking into account its intrinsic polymorphic nature due to genetic drift and recombination and the possibility of continuous and multiple introductions of genetically non-identical strains into the human population, by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV S, E, M and N genes. These were applied in simple, reproducible duplex and multiplex real-time PCR assays on 25 post-mortem samples and constructed RNA controls, and they demonstrated high target detection ability and specificity. This assay can readily be adapted for detection of other emerging and rapidly mutating pathogens.
authors.affiliation: Laboratory of Biotechnology and Molecular Virology, Department of Biological Sciences, University of Cyprus, 75 Kallipoleos Avenue, Nicosia, Cyprus.
authors.forename: Andreas V
authors.initials: AV
authors.lastname: Hadjinicolaou
authors.forename: Gabriella A
authors.initials: GA
authors.lastname: Farcas
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Demetriou
authors.forename: Tony
authors.initials: T
authors.lastname: Mazzulli
authors.forename: Susan M
authors.initials: SM
authors.lastname: Poutanen
authors.forename: Barbara M
authors.initials: BM
authors.lastname: Willey
authors.forename: Donald E
authors.initials: DE
authors.lastname: Low
authors.forename: Jagdish
authors.initials: J
authors.lastname: Butany
authors.forename: Sylvia L
authors.initials: SL
authors.lastname: Asa
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Kain
authors.forename: Leondios G
authors.initials: LG
authors.lastname: Kostrikis
mesh_terms: D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis*; D014773:Virology / Q000379:methods*
chemical_list: D015345:Oligonucleotide Probes