Key result
Real-time RT-PCR assays showed high sensitivities for enterovirus and human parechovirus transcripts approaching single copies, but more variable detection for human rhinoviruses.
The developed RNA transcripts provide stable reagents for evaluating and improving the sensitivity of real-time PCR diagnostics for enteroviruses and rhinoviruses.
These assays may aid enterovirus reagent validation; leaves open clinical reliability for rhinoviruses pending human studies.
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.
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McLeish et al. (2012) studied Viral infections (enteroviruses, human rhinoviruses, human parechovirus). Real-time reverse transcriptase PCR assays was evaluated on Assay sensitivity and specificity. Real-time RT-PCR assays showed high sensitivities for enterovirus and human parechovirus transcripts approaching single copies, but more variable detection for human rhinoviruses.
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