A rapid, single-step multiplex reverse transcription-PCR assay for the detection of human H1N1, H3N2, and B influenza viruses.

ABSTRACT

BACKGROUND: Influenza is a viral respiratory pathogen responsible for frequent seasonal epidemics. There are currently three major human influenza viruses in global circulation, H1N1, H3N2 and B. OBJECTIVES: A one-step multiplex reverse transcription (RT)-polymerase chain reaction (PCR) assay targeting the HA1 segment of the human hemagglutinin gene was developed as a rapid surveillance method. STUDY DESIGN: A researcher-blind study was performed using 112 randomly selected, culture-positive clinical samples collected through the Department of Defense (Global Emerging Infectious Surveillance (DOD-GEIS) influenza network during the 2000-2001 influenza season. Three subtype specific primer sets capable of producing PCR products with base-pair lengths of 585, 402 and 290 corresponding to influenza H1, H3, and B subtypes, respectively, were utilized together in a one step, one tube, reaction. RESULTS: Multiplex primers were able to simultaneously type, and subtype 100% (112/112) of positive cultures. CONCLUSIONS: The results confirm that this assay is a highly sensitive and timely surveillance tool for rapid detection and simultaneous subtyping of clinical influenza specimens isolated worldwide.