Development of a Respiratory Virus Panel (RVP) Test for the Detection of Twenty Human Respiratory Viruses using Multiplex PCR and a Fluid Microbead-based Assay.
Journal of clinical microbiology 2007 Jun 27; 45(9):2965-70
Mahony J J, Chong S S, Merante F F, Yaghoubian S S, Sinha T T, Lisle C C,
McMaster University, Department of Pathology & Molecular Medicine and St. Joseph's HealthCare, H
Virology laboratories have historically used DFA and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000 there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single five hour test. The assay employs multiplex PCR using 14 virus-specific primer pairs followed by a multiplexed Target Specific Primer Extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified using a fluid microsphere-based array (Universal Array(TM), TmBioscience) and the Luminex x-MAP system. The assay detected Influenza types A and B, Influenza A subtypes H1, H3, and H5 (including the H5N1 Asian lineage), Parainfluenza types 1, 2, 3, and 4, RSV types A and B, Adenovirus, Metapneumovirus, Rhinovirus, Enterovirus, and Coronaviruses OC43, 229E, SARS-CoV, NL63, and HKU1. In a prospective evaluation using 294 NP specimens, DFA/culture detected 119 positives and the RVP test detected 112 for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that were either not detected by DFA/culture or were not tested for by DFA/culture. After resolution of discordants using a second unique PCR assay and using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives for a sensitivity of 98.5% compared to DFA and culture which detected only126 of 183 for a sensitivity of 68.8%. The RVP test should improve the capability of hospital and public health laboratories for diagnosing viral respiratory tract infections and assist Public Health agencies in identifying etiologic agents in respiratory tract outbreaks.