title: Design and clinical application of a molecular method for detection and typing of the influenza A/H1N1pdm virus.
abstract: In March/April 2009, Mexico experienced an outbreak of respiratory illness, due to a new influenza of swine origin virus, which spread rapidly via human-to-human transmission, and became pandemic (A/H1N1pdm). Because of its unique genome composition, which includes gene segments of swine, avian and human origin, and to the considerable differences to the human influenza A viruses that have circulated so far, the currently used molecular methods proved inadequate. Based on published sequences, a primer set targeting the nucleoprotein gene was designed, which provided enhanced sensitivity for the new strain and proved suitable for sequence-based strain identification. The novel nucleoprotein reverse-transcription-PCR showed higher sensitivity for A/H1N1pdm than a commercial test for influenza A, and was comparable to the real-time-based method developed by the Centers for Disease Control and Prevention. It was used to screen 177 clinical samples referred to the laboratory for suspected A/H1N1pdm infection, detecting 17 (9.6%) infections that were confirmed by sequence analysis (100% sensitivity as compared to the real-time kit). The novel method is suitable for the diagnosis of A/H1N1pdm, and is also suitable, at least in the screening phase, for laboratories not equipped with the real-time PCR technology.
authors.affiliation: National Institute for Infectious Diseases L. Spallanzani, Rome, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Lalle
authors.forename: Licia
authors.initials: L
authors.lastname: Bordi
authors.forename: Concetta
authors.initials: C
authors.lastname: Castilletti
authors.forename: Silvia
authors.initials: S
authors.lastname: Meschi
authors.forename: Marina
authors.initials: M
authors.lastname: Selleri
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Carletti
authors.forename: Daniele
authors.initials: D
authors.lastname: Lapa
authors.forename: Damiano
authors.initials: D
authors.lastname: Travaglini
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ippolito
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Capobianchi
authors.forename: Antonino
authors.initials: A
authors.lastname: Di Caro
mesh_terms: D017931:DNA Primers / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers