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Journal of Clinical Microbiology, January 2004, p. 45-51, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.45-51.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Multiplex Real-Time PCR Assay for Detection of Influenza and Human Respiratory Syncytial Viruses

Guy Boivin,1,2* Stéphanie Côté,1,2 Pierre Déry,1,3 Gaston De Serres,4 and Michel G. Bergeron1,2

Research Center in Infectious Diseases of the CHUQ-CHUL,1 Department of Pediatrics,3 Department of Medical Biology,2 Department of Preventive Medicine,6 Laval University, Québec City, Canada4

A multiplex real-time PCR assay was developed with a LightCycler instrument for detection of influenza viruses A and B and the human respiratory syncytial virus (HRSV). Detection of each viral product and of an internal control was based on determination of specific melting temperatures by the LightCycler software. The lower limit of detection in the multiplex PCR assay was found to be 50 copies for each viral target. In an evaluation of nasopharyngeal samples collected from hospitalized children (ages, 0 to 3 years) with acute respiratory tract infections during the winter of 2001 to 2002, a viral pathogen was detected by the multiplex PCR test in 139 (66.8%) of 208 cases, including 45 (21.6%) influenza A virus infections, no (0%) influenza B virus infections, 106 (51%) HRSV infections, and 12 (5.8%) coinfections. The multiplex PCR test was compared to rapid antigen detection assays for influenza viruses A and B (Directigen; Becton Dickinson, Sparks, Md.) and HRSV (RSV TestPack; Abbott Laboratories, Abbott Park, Ill.) in 172 and 204 samples, respectively. After resolution of discrepant test results by use of additional PCR assays targeting other viral genes, the sensitivity (Se) and specificity (Sp) of the multiplex PCR assay for influenza A virus were 100 and 97.7% compared to 43.6 and 98.5% for the antigenic test. Similarly, the Se and Sp of the multiplex PCR assay for HRSV were 94.5 and 98.9% compared to 81.6 and 94.7% for the antigenic test. In conclusion, our multiplex real-time PCR assay combines both rapidity and sensitivity for detecting the most important respiratory viral pathogens in children.

* Corresponding author. Mailing address: Centre de Recherche en Infectiologie, CHUQ, Pavillon CHUL, Room RC-709, 2705 Blvd. Laurier, Sainte-Foy, Québec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: Guy.Boivin{at}crchul.ulaval.ca.

Journal of Clinical Microbiology, January 2004, p. 45-51, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.45-51.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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