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Journal of Clinical Microbiology, February 2008, p. 533-539, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01739-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses{triangledown}

Xiaoyan Lu,1 Brian Holloway,2 Ryan K. Dare,1 Jane Kuypers,3 Shigeo Yagi,4 John V. Williams,5 Caroline B. Hall,6 and Dean D. Erdman1*

Gastroenteritis and Respiratory Virus Laboratory Branch, Division of Viral Diseases,1 Biotechnology Core Facility Branch, Centers for Disease Control and Prevention, Atlanta, Georgia,2 Department of Laboratory Medicine, University of Washington, Seattle, Washington,3 Viral and Rickettsial Disease Laboratory, California Department of Health Services, Richmond, California,4 Departments of Pediatrics and Microbiology and Immunology, Vanderbilt School of Medicine, Nashville, Tennessee,5 Department of Infectious Diseases, University of Rochester School of Medicine and Dentistry, Rochester, New York6

Received 31 August 2007/ Returned for modification 11 October 2007/ Accepted 7 November 2007

Human rhinoviruses (HRVs) are important contributors to respiratory disease, 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 reverse transcription-PCR (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 68 (HEV68) that showed the greatest sequence homology to 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 that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used 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 subsequently were identified in these four 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.

* Corresponding author. Mailing address: 1600 Clifton Road, N.E., Mailstop G04, Atlanta, GA 30333. Phone: (404) 639-3727. Fax: (404) 639-4416. E-mail: dde1{at}cdc.gov

{triangledown} Published ahead of print on 5 December 2007.

Journal of Clinical Microbiology, February 2008, p. 533-539, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01739-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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