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Reliable quantification of rhinovirus species C using real-time PCR.

Reliable quantification of rhinovirus species C using real-time PCR.
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Sikazwe CT, Chidlow GR, Imrie A, Smith DW,


Sikazwe CT, Chidlow GR, Imrie A, Smith DW, (click to view)

Sikazwe CT, Chidlow GR, Imrie A, Smith DW,

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Journal of virological methods 2016 05 20235() 65-72 doi 10.1016/j.jviromet.2016.05.014

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.

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