title: Reliable quantification of rhinovirus species C using real-time PCR.
abstract: 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.
  
  
  
  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).
  
  
  
  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.
  
  
  
  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.
authors.affiliation: School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia; Department of Microbiology, PathWest Laboratory Medicine WA, Perth, Australia. Electronic address: chisha.sikazwe@research.uwa.edu.au.
authors.forename: Chisha T
authors.initials: CT
authors.lastname: Sikazwe
authors.affiliation: Department of Microbiology, PathWest Laboratory Medicine WA, Perth, Australia.
authors.forename: Glenys R
authors.initials: GR
authors.lastname: Chidlow
authors.affiliation: School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia; Department of Microbiology, PathWest Laboratory Medicine WA, Perth, Australia.
authors.forename: Allison
authors.initials: A
authors.lastname: Imrie
authors.affiliation: School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia; Department of Microbiology, PathWest Laboratory Medicine WA, Perth, Australia.
authors.forename: David W
authors.initials: DW
authors.lastname: Smith
mesh_terms: D020121:5' Untranslated Regions; D002648:Child; D017931:DNA Primers; D018076:DNA, Complementary; D005260:Female; D005838:Genotype; D006801:Humans; D057230:Limit of Detection; D008297:Male; D009666:Nose / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012229:Rhinovirus / Q000235:genetics / Q000302:isolation & purification*; D019562:Viral Load
chemical_list: D020121:5' Untranslated Regions; D017931:DNA Primers; D018076:DNA, Complementary