title: Diagnostic testing for pandemic influenza in Singapore: a novel dual-gene quantitative real-time RT-PCR for the detection of influenza A/H1N1/2009.
abstract: With the relative global lack of immunity to the pandemic influenza A/H1N1/2009 virus that emerged in April 2009 as well as the sustained susceptibility to infection, rapid and accurate diagnostic assays are essential to detect this novel influenza A variant. Among the molecular diagnostic methods that have been developed to date, most are in tandem monoplex assays targeting either different regions of a single viral gene segment or different viral gene segments. We describe a dual-gene (duplex) quantitative real-time RT-PCR method selectively targeting pandemic influenza A/H1N1/2009. The assay design includes a primer-probe set specific to only the hemagglutinin (HA) gene of this novel influenza A variant and a second set capable of detecting the nucleoprotein (NP) gene of all swine-origin influenza A virus. In silico analysis of the specific HA oligonucleotide sequence used in the assay showed that it targeted only the swine-origin pandemic strain; there was also no cross-reactivity against a wide spectrum of noninfluenza respiratory viruses. The assay has a diagnostic sensitivity and specificity of 97.7% and 100%, respectively, a lower detection limit of 50 viral gene copies/PCR, and can be adapted to either a qualitative or quantitative mode. It was first applied to 3512 patients with influenza-like illnesses at a tertiary hospital in Singapore, during the containment phase of the pandemic (May to July 2009).
authors.affiliation: Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore.
authors.forename: Hong Kai
authors.initials: HK
authors.lastname: Lee
authors.forename: Chun Kiat
authors.initials: CK
authors.lastname: Lee
authors.forename: Tze Ping
authors.initials: TP
authors.lastname: Loh
authors.forename: Julian Wei-Tze
authors.initials: JW
authors.lastname: Tang
authors.forename: Lily
authors.initials: L
authors.lastname: Chiu
authors.forename: Paul A
authors.initials: PA
authors.lastname: Tambyah
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Sethi
authors.forename: Evelyn Siew-Chuan
authors.initials: ES
authors.lastname: Koay
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012846:Singapore / Q000453:epidemiology; D019562:Viral Load
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D012333:RNA, Messenger