Reliable quantification of rhinovirus species C using real-time PCR

Chisha T. Sikazwe, G.R. Chidlow, Allison Imrie, David William Smith

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

© 2016 The Authors.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 296 bp 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 (104-1011 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 (r2 = >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.
Original languageEnglish
Pages (from-to)65-72
Number of pages8
JournalJournal of Virological Methods
Volume235
DOIs
Publication statusPublished - 2016

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Rhinovirus
Real-Time Polymerase Chain Reaction
Genotype
Polymerase Chain Reaction
5' Untranslated Regions
Viral Load
Computer Simulation
Reverse Transcription
Limit of Detection

Cite this

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title = "Reliable quantification of rhinovirus species C using real-time PCR",
abstract = "{\circledC} 2016 The Authors.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 296 bp 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 (104-1011 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 (r2 = >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.",
author = "Sikazwe, {Chisha T.} and G.R. Chidlow and Allison Imrie and Smith, {David William}",
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Reliable quantification of rhinovirus species C using real-time PCR. / Sikazwe, Chisha T.; Chidlow, G.R.; Imrie, Allison; Smith, David William.

In: Journal of Virological Methods, Vol. 235, 2016, p. 65-72.

Research output: Contribution to journalArticle

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AU - Chidlow, G.R.

AU - Imrie, Allison

AU - Smith, David William

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N2 - © 2016 The Authors.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 296 bp 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 (104-1011 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 (r2 = >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.

AB - © 2016 The Authors.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 296 bp 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 (104-1011 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 (r2 = >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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