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


OBJECTIVE
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.


METHODS
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.
Daum
Luke T
LT
Molecular Epidemiology Branch, 2601 West Gate Road, Brooks AFB, San Antonio, TX 78235-5241, USA. luke.daum@brooks.af.mil
Canas
Linda C
LC
Schadler
Cassandra A
CA
Ujimori
Veronica A H
VA
Huff
William B
WB
Barnes
William J
WJ
Lohman
Kenton L
KL
D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype; D053122:Influenza A Virus, H3N2 Subtype; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D019267:Hemagglutinin Glycoproteins, Influenza Virus