A new real-time RT-qPCR assay for the detection, subtyping and quantification of human respiratory syncytial viruses positive- and negative-sense RNAs

Abstract

a b s t r a c tHuman respiratory syncytial virus (RSV) is a major health problem and the main cause of hospitalization due to bronchiolitis. RSV is divided into two antigenic subgroups, RSV-A and -B that co-circulate worldwide. Rapid and sensitive detection is desirable for proper patient handling while assessment of viral load may help to evaluate disease severity and progression. Finally RSV subtyping is needed to determine the prevalence and pathogenicity of each RSV subgroup, as well as their sensitivity to treatment. In this study, we took into account the most recent circulating RSV variants and designed two quantitative TaqMan one-step RT-PCR assays to detect and quantify both RSV subgroups separately. Standard dilutions of transcripts of positive and negative polarities were included in the assay validation to assess potential differences in sensitivity on negative-sense genomes and positive-sense RNAs. In addition, RSV detection in respiratory specimens of different types and sampled in different populations was compared to commercially available RSV diagnostic tools. Altogether, the RSV-A and -B assays revealed sensitive and quantitative over a wide range of viral loads, with a slight improved sensitivity of the RSV-B assay on positive sense transcripts, and allowed accurate RSV subtyping. We thus provide a useful tool for both RSV diagnostics and research.

Modified cyclodextrins as broad-spectrum antivirals

“…RSV RNA extracted with NucliSENS easyMAG (bioMérieux) was quantified by qPCR with the QuantiTect Kit (Qiagen, 204443) in a StepOne thermocycler (Applied Biosystems). The primer and probes were previously described (31,33). HCV RNA was extracted from cells or from culture supernatants using Trizol of the QIAamp Viral RNA Mini Kit (Qiagen).…”

Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures

“…RV-B48 (RV from species B), RV-C15 (RV from species C), RV-C8 (RV from species C, isolated from a patient's serum with disseminated infection), 26 EV-D68 (respiratory EV from species D), influenza A H3N2 Victoria like, RSV subgroup B, and HCoV-OC43 (Table I) 16,[26][27][28][29] directly in reconstituted airway epithelia to prevent acquisition of cell adaptation mutations. Viral RNA copies (10 6 ) corresponding to the average load found in clinical samples [30][31][32] were used in each round of infection.…”

The specific features of the developing T cell compartment of the neonatal lung are a determinant of respiratory syncytial virus immunopathogenesis

“…RNA expression levels of RSV and 18S were determined by absolute quantification by the serial dilution of plasmids containing the sequence of interest. For the comparison between RSV A2 and RSV-ON1-H1, the following panRSV primers were used: FW: 5 0 -TGCTAAGA CYCCCCACCGTAAC-3 0 , RV: 5 0 -GGATTTTTGCAGGATTGTTTATGA-3 0 , Probe: 5 0 -C5CT 6GC7CT87W7CA-BHQ1-3 0 [66]. For the quantification of IFN-β, primers were designed as follow: FW: 5 0 -TGGTTCTCCTGCTGTGTTTCTC-3 0 ; RV: 5 0 -CGTTGTTGGAATCGAAGCA A-3 0 ; Probe: 5 0 -FAM-ACCACAGCTCTTTCCAGGAGCTACA-BHQ1-3 0 [67].…”