title: Development and assay of RNA transcripts of enterovirus species A to D, rhinovirus species a to C, and human parechovirus: assessment of assay sensitivity and specificity of real-time screening and typing methods.
abstract: Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and -20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.
authors: Infection and Immunity Division, Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.
authors: Nigel J
authors: NJ
authors: McLeish
authors: Jeroen
authors: J
authors: Witteveldt
authors: Lucy
authors: L
authors: Clasper
authors: Chloe
authors: C
authors: McIntyre
authors: E Carol
authors: EC
authors: McWilliam Leitch
authors: Alison
authors: A
authors: Hardie
authors: Susan
authors: S
authors: Bennett
authors: Rory
authors: R
authors: Gunson
authors: William F
authors: WF
authors: Carman
authors: Susan A
authors: SA
authors: Feeney
authors: Peter V
authors: PV
authors: Coyle
authors: Barry
authors: B
authors: Vipond
authors: Peter
authors: P
authors: Muir
authors: Kimberley
authors: K
authors: Benschop
authors: Katja
authors: K
authors: Wolthers
authors: Matti
authors: M
authors: Waris
authors: Riikka
authors: R
authors: Osterback
authors: Ingo
authors: I
authors: Johannessen
authors: Kate
authors: K
authors: Templeton
authors: Heli
authors: H
authors: Harvala
authors: Peter
authors: P
authors: Simmonds
mesh_terms: D004770:Enterovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008403:Mass Screening / Q000379:methods; D008969:Molecular Sequence Data; D030061:Parechovirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012229:Rhinovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014773:Virology / Q000379:methods
chemical_list: D012367:RNA, Viral