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Originally published online as doi:10.2353/jmoldx.2010.090071 on November 30, 2009

Published online before print November 30, 2009
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Journal of Molecular Diagnostics 2010, Vol. 12, No. 1
Copyright © 2010 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2010.090071

A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

Duc H. Do*, Stella Laus{dagger}, Amy Leber{ddagger}, Mario J. Marcon{ddagger}, Jeanne A. Jordan§, Judith M. Martin{dagger}|| and Robert M. Wadowsky*{dagger}

From the Department of Infectious Diseases and Microbiology, * Graduate School of Public Health, Pittsburgh, Pennsylvania; the Children’s Hospital of Pittsburgh of University of Pittsburgh Medical Center, {dagger} Pittsburgh, Pennsylvania; the Department of Laboratory Medicine, {ddagger} Nationwide Children’s Hospital, Columbus, Ohio; the Magee Women’s Research Institute, § Pittsburgh, Pennsylvania; and the Departments of Pathology and Pediatrics, || School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID50 (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform.






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Copyright © 2010 by the American Society for Investigative Pathology and the Association for Molecular Pathology.