title: Molecular assays for quantitative and qualitative detection of influenza virus and oseltamivir resistance mutations.
abstract: Sensitive and reproducible molecular assays are essential for influenza virus diagnostics. This manuscript describes the design, validation, and evaluation of a set of real-time RT-PCR assays for quantification and subtyping of human influenza viruses from patient respiratory material. Four assays are included for detection of oseltamivir resistance mutations H275Y in prepandemic and pandemic influenza A/H1N1 and E119V and R292K in influenza A/H3N2 neuraminidase. The lower limits of detection of the quantification assay were determined to be 1.7 log(10) virus particles per milliliter (vp/mL) for influenza A and 2.2 log(10) vp/mL for influenza B virus. The lower limits of quantification were 2.1 and 2.3 log(10) vp/mL, respectively. The RT-PCR efficiencies and lower limits of detection of the quantification assays were only marginally affected when tested on the most dissimilar target sequences found in the GenBank database. Finally, the resistance RT-PCR assays detected at least 5% mutant viruses present in mixtures containing both wild-type and mutant viruses with approximated limits of detection of 2.4 log(10) vp/mL. Overall, this set of RT-PCR assays is a powerful tool for enhanced influenza virus surveillance.
authors.affiliation: Department of Virology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Erhard
authors.initials: E
authors.lastname: van der Vries
authors.forename: Jeer
authors.initials: J
authors.lastname: Anber
authors.forename: Anne
authors.initials: A
authors.lastname: van der Linden
authors.forename: Yingbin
authors.initials: Y
authors.lastname: Wu
authors.forename: Jolanda
authors.initials: J
authors.lastname: Maaskant
authors.forename: Ralph
authors.initials: R
authors.lastname: Stadhouders
authors.forename: Ruud
authors.initials: R
authors.lastname: van Beek
authors.forename: Guus
authors.initials: G
authors.lastname: Rimmelzwaan
authors.forename: Albert
authors.initials: A
authors.lastname: Osterhaus
authors.forename: Charles
authors.initials: C
authors.lastname: Boucher
authors.forename: Martin
authors.initials: M
authors.lastname: Schutten
mesh_terms: D024882:Drug Resistance, Viral / Q000235:genetics*; D005069:Evaluation Studies as Topic; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000821:virology; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics; D053139:Oseltamivir / Q000627:therapeutic use*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral; D053139:Oseltamivir; D009439:Neuraminidase