Rapid quantification of single-nucleotide mutations in mixed influenza A viral populations using allele-specific mixture analysis.
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%).
Liu
Cindy M
CM
Division of Pathogen Genomics, Translational Genomics Research Institute (TGen), Flagstaff, AZ 86001, USA.
Driebe
Elizabeth M
EM
Schupp
James
J
Kelley
Erin
E
Nguyen
Jack T
JT
McSharry
James J
JJ
Weng
Qingmei
Q
Engelthaler
David M
DM
Keim
Paul S
PS
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*