title: Development and comparison of molecular assays for the rapid detection of the pandemic influenza A (H1N1) 2009 virus.
abstract: Human infection with the novel pandemic influenza A (H1N1) virus was first identified in April 2009. Two months later, the World Health Organization (WHO) had raised the pandemic level to phase 6. Rapid case identification is essential for prompt patient management and public health actions. This study developed real-time and conventional reverse transcription-polymerase chain reaction (rRT-PCR and cRT-PCR) assays for pandemic H1N1 detection, and compared their sensitivities with protocols developed by WHO reference centres. Altogether, three rRT-PCR and one cRT-PCR targeting the matrix gene for universal detection of influenza A; three rRT-PCR, one cRT-PCR targeting the hemagglutinin (HA) gene for specific detection of pandemic H1N1; and one multiplex cRT-PCR for differentiating co-circulating seasonal H1N1, H3N2, and pandemic H1N1 were examined. The lower detection limit ranged from 1.252 to 125.2 copy equivalents. In general, rRT-PCR assays were more sensitive than cRT-PCR assays. All assays showed 100% sensitivity for "optimal" specimens (nasopharyngeal samples collected within 4 days after illness onset). For the other 36 samples, cRT-PCR assays were less sensitive except that the new Protocol I-cRT-pdmH1 still retained 100% sensitivity. The new Protocol F-rRT-PCR-pdmH1 was the only pandemic virus-specific rRT-PCR assay with 100% sensitivity across all specimen categories. In conclusion, rRT-PCR assays are 10-fold more sensitive than cRT-PCR assays. The newly developed cRT-PCR assay targeting the HA gene allows rapid, specific, and sensitive screening of this novel agent, which can serve as an alternative for laboratories where a real-time PCR machine is not available.
authors.affiliation: Faculty of Medicine, Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, People's Republic of China.
authors.forename: Wai-Yip
authors.initials: WY
authors.lastname: Lam
authors.forename: Ting-Fan
authors.initials: TF
authors.lastname: Leung
authors.forename: Nelson
authors.initials: N
authors.lastname: Lee
authors.forename: Jo L K
authors.initials: JL
authors.lastname: Cheung
authors.forename: Apple C M
authors.initials: AC
authors.lastname: Yeung
authors.forename: Yolanda I I
authors.initials: YI
authors.lastname: Ho
authors.forename: Rickjason C W
authors.initials: RC
authors.lastname: Chan
authors.forename: Kitty S C
authors.initials: KS
authors.lastname: Fung
authors.forename: Ian G
authors.initials: IG
authors.lastname: Barr
authors.forename: David S C
authors.initials: DS
authors.lastname: Hui
authors.forename: Joseph J Y
authors.initials: JJ
authors.lastname: Sung
authors.forename: Paul K S
authors.initials: PK
authors.lastname: Chan
mesh_terms: D006389:Hemagglutinins, Viral / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009305:Nasopharynx / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014763:Viral Matrix Proteins / Q000235:genetics
chemical_list: D006389:Hemagglutinins, Viral; C488936:M1 protein, Influenza A virus; D014763:Viral Matrix Proteins; C458881:hemagglutinin fusogenic peptide, influenza virus