title: Rapid quantification of single-nucleotide mutations in mixed influenza A viral populations using allele-specific mixture analysis.
abstract: Monitoring antiviral resistance in influenza is critical to public health epidemiology and pandemic preparedness activities. Effective monitoring requires methods to detect low-level resistance and to monitor the change in resistance as a function of time and drug treatment. Resistance-conferring single-nucleotide mutations in influenza virus are ideal targets for such methods. In the present study, fives sets of paired TaqMan allele-specific PCR (ASPCR) assays were developed and validated for quantitative single-nucleotide polymorphism (SNP) analysis. This novel method using Delta Ct is termed allele-specific mixture analysis (ASMA) or FluASMA. The FluASMA assays target L26F, V27A, A30T, and S31N mutations in the A/Albany/1/98 (H3N2) M2 gene and H275Y mutation in the A/New Caledonia/20/99 (H1N1) NA gene and have a limit of quantification of 0.25-0.50% mutant. The error for % mutant estimation was less than 10% in all FluASMA assays, with intra-run Delta Ct coefficient of variance (CoV) at <or=2% and inter-run Delta Ct CoV at <or=5%. Results from the current study demonstrate that FluASMA is a highly sensitive and quantitative SNP analysis method, even for minor mutant components (<1%).
authors.affiliation: Division of Pathogen Genomics, Translational Genomics Research Institute (TGen), Flagstaff, AZ 86001, USA.
authors.forename: Cindy M
authors.initials: CM
authors.lastname: Liu
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Driebe
authors.forename: James
authors.initials: J
authors.lastname: Schupp
authors.forename: Erin
authors.initials: E
authors.lastname: Kelley
authors.forename: Jack T
authors.initials: JT
authors.lastname: Nguyen
authors.forename: James J
authors.initials: JJ
authors.lastname: McSharry
authors.forename: Qingmei
authors.initials: Q
authors.lastname: Weng
authors.forename: David M
authors.initials: DM
authors.lastname: Engelthaler
authors.forename: Paul S
authors.initials: PS
authors.lastname: Keim
mesh_terms: D000483:Alleles; D024882:Drug Resistance, Viral / Q000235:genetics*; 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*; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*