Background: The genetically heterogeneous Rhinovirus species C (RV-C) has been associated with severe acute respiratory tract infections. Little is known about the pathophysiological mechanisms associated with patient outcome in RV-C infection. Viral load measurement may assist in elucidating this gap in knowledge.
Objective: The aim of this study was to develop and validate qRT-PCR assays that would enable the accurate and reliable quantification of locally circulating RV-C types in nasopharyngeal aspirate (NPA) specimen.
Study design: The four assays developed were designed to amplify a 290bp region located within the 5’ untranslated region (UTR) of RV-C types. These assays were based on the alignment of 204 RV-C sequences obtained in our laboratory from routine diagnostic samples. Each primer/probe set was designed to provide 100% homology to the corresponding RV-C types. Four plasmids each harboring a conserved area of the 5’UTR were used to generate RNA transcripts that were used as standards for the quantification of RV-C positive samples.
Results: Each assay had a linear dynamic range of eight orders of magnitude (101-108 copies). The limit of detection for each of the assays ranged from 765 copies/mL to 1470 copies/mL. All four assays demonstrated a strong linear relationship (r2=>0.995) between quantification cycle (Cq) values and RNA copy number, and all had amplification efficiencies greater than 95%. Repeatability and reproducibility of the method were shown to be high, with coefficients of variations lower than 8% and 15% respectively.
Conclusion: The assays described in this study utilized standard curves generated by RV-C RNA transcripts to accurately and reliably quantify RV-C RNA in clinical samples.