title: Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.
abstract: Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening.
authors.affiliation: Molecular and Cell Biology Team, LGC, Teddington, United Kingdom alexandra.whale@lgcgroup.com.
authors.forename: Alexandra S
authors.initials: AS
authors.lastname: Whale
authors.affiliation: Molecular and Cell Biology Team, LGC, Teddington, United Kingdom.
authors.forename: Claire A
authors.initials: CA
authors.lastname: Bushell
authors.affiliation: Virology Laboratory, Clinical Microbiology and Virology, University College London Hospital NHS Foundation Trust, London, United Kingdom.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Grant
authors.affiliation: Statistics Team, LGC, Teddington, United Kingdom.
authors.forename: Simon
authors.initials: S
authors.lastname: Cowen
authors.affiliation: National Institute of Biology, Ljubljana, Slovenia.
authors.forename: Ion
authors.initials: I
authors.lastname: Gutierrez-Aguirre
authors.affiliation: Molecular and Cell Biology Team, LGC, Teddington, United Kingdom.
authors.forename: Denise M
authors.initials: DM
authors.lastname: O'Sullivan
authors.affiliation: National Institute of Biology, Ljubljana, Slovenia.
authors.forename: Jana
authors.initials: J
authors.lastname: Žel
authors.affiliation: National Institute of Biology, Ljubljana, Slovenia.
authors.forename: Mojca
authors.initials: M
authors.lastname: Milavec
authors.affiliation: Molecular and Cell Biology Team, LGC, Teddington, United Kingdom.
authors.forename: Carole A
authors.initials: CA
authors.lastname: Foy
authors.affiliation: Virology Laboratory, Clinical Microbiology and Virology, University College London Hospital NHS Foundation Trust, London, United Kingdom.
authors.forename: Eleni
authors.initials: E
authors.lastname: Nastouli
authors.affiliation: Department of Infection, Division of Infection and Immunity, University College London, London, United Kingdom.
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Garson
authors.affiliation: Molecular and Cell Biology Team, LGC, Teddington, United Kingdom Department of Infection, Division of Infection and Immunity, University College London, London, United Kingdom.
authors.forename: Jim F
authors.initials: JF
authors.lastname: Huggett
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology* / Q000627:therapeutic use; D024882:Drug Resistance, Viral; D005814:Genes, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000235:genetics; D009980:Influenza A virus / Q000187:drug effects* / Q000235:genetics*; D007251:Influenza, Human / Q000175:diagnosis* / Q000188:drug therapy / Q000821:virology*; D058889:Molecular Typing; D009154:Mutation; D053139:Oseltamivir / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D000998:Antiviral Agents; D053139:Oseltamivir