Critical Analysis of Rhinovirus RNA Load Quantification by Real-Time Reverse Transcription-PCR

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Published inJournal of Clinical Microbiology Vol. 50; no. 9; pp. 2868 - 2872
Main Authors SCHIBLER, Manuel, YERLY, Sabine, VIEILLE, Gaël, DOCQUIER, Mylène, TURIN, Lara, KAISER, Laurent, TAPPAREL, Caroline
Format Journal Article
LanguageEnglish
Published Washington, DC American Society for Microbiology 01.09.2012
Subjects
5' Untranslated Regions
Biological and medical sciences
Fundamental and applied biological sciences. Psychology
Genetic Variation
Humans
Microbiology
Miscellaneous
Picornaviridae Infections - virology
Real-Time Polymerase Chain Reaction - methods
Real-Time Polymerase Chain Reaction - standards
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction - methods
Reverse Transcriptase Polymerase Chain Reaction - standards
Rhinovirus - genetics
Rhinovirus - isolation & purification
RNA, Viral - genetics
RNA, Viral - isolation & purification
Viral Load - methods
Virology
Virus
Real time
Picornaviridae
Reverse transcription polymerase chain reaction
Quantitative analysis
Rhinovirus
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Abstract Article Usage Stats Services JCM Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter current issue JCM About JCM Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JCM RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0095-1137 Online ISSN: 1098-660X Copyright © 2014 by the American Society for Microbiology.   For an alternate route to JCM .asm.org, visit: JCM       
AbstractList Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro -transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5′-untranslated regions (5′UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5′UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.
Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5'-untranslated regions (5'UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5'UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.
ABSTRACT Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro -transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5′-untranslated regions (5′UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5′UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.
Article Usage Stats Services JCM Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter current issue JCM About JCM Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JCM RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0095-1137 Online ISSN: 1098-660X Copyright © 2014 by the American Society for Microbiology.   For an alternate route to JCM .asm.org, visit: JCM       
Author Manuel Schibler
Lara Turin
Gaël Vieille
Caroline Tapparel
Sabine Yerly
Mylène Docquier
Laurent Kaiser
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  fullname: TAPPAREL, Caroline
  organization: Laboratory of Virology, Division of Infectious Diseases and Division of Laboratory Medicine, University of Geneva Hospitals, Geneva, Switzerland
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Snippet Article Usage Stats Services JCM Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley...
Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations....
ABSTRACT Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical...
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SubjectTerms 5' Untranslated Regions
Biological and medical sciences
Fundamental and applied biological sciences. Psychology
Genetic Variation
Humans
Microbiology
Miscellaneous
Picornaviridae Infections - virology
Real-Time Polymerase Chain Reaction - methods
Real-Time Polymerase Chain Reaction - standards
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction - methods
Reverse Transcriptase Polymerase Chain Reaction - standards
Rhinovirus - genetics
Rhinovirus - isolation & purification
RNA, Viral - genetics
RNA, Viral - isolation & purification
Viral Load - methods
Virology
Title Critical Analysis of Rhinovirus RNA Load Quantification by Real-Time Reverse Transcription-PCR
URI http://jcm.asm.org/content/50/9/2868.abstract
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