TY - JOUR
T1 - Rapid quantification of single-nucleotide mutations in mixed influenza A viral populations using allele-specific mixture analysis
AU - Liu, Cindy M.
AU - Driebe, Elizabeth M.
AU - Schupp, James
AU - Kelley, Erin
AU - Nguyen, Jack T.
AU - McSharry, James J.
AU - Weng, Qingmei
AU - Engelthaler, David M.
AU - Keim, Paul S.
PY - 2010/1
Y1 - 2010/1
N2 - 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 ΔCt 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 ΔCt coefficient of variance (CoV) at ≤2% and inter-run ΔCt CoV at ≤5%. Results from the current study demonstrate that FluASMA is a highly sensitive and quantitative SNP analysis method, even for minor mutant components (<1%).
AB - 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 ΔCt 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 ΔCt coefficient of variance (CoV) at ≤2% and inter-run ΔCt CoV at ≤5%. Results from the current study demonstrate that FluASMA is a highly sensitive and quantitative SNP analysis method, even for minor mutant components (<1%).
KW - Allele-specific PCR
KW - Antiviral resistance
KW - Influenza resistance
KW - Mixture analysis
KW - Quantitative genotyping
KW - Single-nucleotide polymorphism (SNP) analysis
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U2 - 10.1016/j.jviromet.2009.09.007
DO - 10.1016/j.jviromet.2009.09.007
M3 - Article
C2 - 19761797
AN - SCOPUS:70649093850
SN - 0166-0934
VL - 163
SP - 109
EP - 115
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 1
ER -