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J. Clin. Microbiol. doi:10.1128/JCM.01739-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Real-time RT-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Gastroenteritis and Respiratory Virus Laboratory Branch, Division of Viral Diseases, Biotechnology Core Facility Branch, Centers for Disease Control and Prevention, Atlanta, GA; Department of Laboratory Medicine, University of Washington, Seattle, WA; Viral and Rickettsial Disease Laboratory, California Department of Health Services, Richmond, CA; Departments of Pediatrics and Microbiology and Immunology, Vanderbilt School of Medicine, Nashville, TN; Department of Infectious Diseases, University of Rochester School of Medicine and Dentistry, Rochester, NY

^* To whom correspondence should be addressed. Email: dde1{at}cdc.gov.

	Abstract

Human rhinoviruses (HRVs) are important contributors to respiratory illness, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time RT-PCR assay targeting the viral 5'-noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus (HEV) 68 that showed the greatest sequence homology with the HRV primers and probe set was not detected below a concentration of 5 x 10⁵ copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories using different in-house HRV/HEV RT-PCR assays. Eighty-seven HRV culture positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEV positive samples were positive for HRV. HRV-specific sequences were subsequently identified in these 4 specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff.