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.

Influenza A/H3N2 virus infection in immunocompromised ferrets and emergence of antiviral resistance

“…RNA was extracted from these samples and virus load was determined using the semi-quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) targeting the matrix gene segment. Oseltamivir resistance substitutions at amino acid positions 292 (R/K) and 119 (E/V) of NA were monitored using a mutation specific RT-PCR (msRT-PCR) as described [ 31 ]. An electron-microscopy-counted influenza A/PR/8/34 virus stock (Advanced Biotechnologies Inc., Maryland, USA) was run in parallel to convert cycle thresholds to virus particle counts (viral RNA copies).…”

Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme-Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

“…Before nucleic acid isolation, 10 l phocine distemper virus (PDV) was added as an internal control. Cycle threshold (C T ) values and H1 and H3 subtypes were determined by real-time reverse transcription-PCR (RT-PCR) on a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) or a LightCycler 480 system (Roche Diagnostics, Almere, The Netherlands), as described previously (11).…”

Influenza-induced thrombocytopenia is dependent on the subtype and sialoglycan receptor and increases with virus pathogenicity

“…34) was excluded from the analyses, because the viral load (1.78 log 10 virus particles per milliliter) of the respiratory sample was below the limit of quantification of a polymerase chain reaction (PCR) assay (lower limit of quantification, 2.1 log 10 viral particles per milliliter). 43 The protocol was approved by the hospital medical ethics board (MEC-2012-463).…”