Reliable quantification of rhinovirus species C using real-time PCR
Chisha T. Sikazwea b c✉, Glenys R. Chidlowc, Allison A. Imriea c, David W. Smitha c
- aSchool of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia
- bDepartment of Microbiology, PathWest Laboratory Medicine WA, Perth, Australia. Electronic address: chisha.sikazwe@research.uwa.edu.au
- cDepartment of Microbiology, PathWest Laboratory Medicine WA, Perth, Australia
- ✉Corresponding author
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Abstract
BACKGROUND: Rhinovirus C (RV-C) is an important respiratory pathogen of children, but little is known about its contribution to disease severity, though viral load appears to be important. Difficulty in RV-C cultivation and target sequence variation has precluded the development of a PCR based quantification method. OBJECTIVE: The aim of this study was to develop and validate reverse transcription quantitative PCR (RT-qPCR) assays for a broad range of circulating RV-C genotypes in nasopharyngeal aspirates (NPAs). STUDY DESIGN: Four assays were designed to quantify a 296bp region located within the 5' untranslated region (UTR) of RV-C types. These assays were based on in silico analysis of available RV-C sequences. Probes were designed to provide 100% homology to the corresponding RV-C genotypes. RESULTS: The linear dynamic range of each of the four assays spanned eight orders of magnitude (10(4)-10(11) copies/mL). The limit of detection for assays 1-4 was estimated to be 1147 copies/mL, 765 copies/mL, 1138 copies/mL and 1470 copies/mL respectively. Each assay demonstrated a strong linear relationship (r(2)=>0.995) and amplification efficiency greater than 95%. Repeatability and reproducibility of the method were shown to be high, with coefficients of variations lower than 8% and 15% respectively.
TL;DRFour in-house assays are developed and validated to reliably determine viral load of several different RV-C genotypes in nasal samples and facilitate accurate investigations into the role of RV-C load in disease outcome. (Semantic Scholar)