Giuseppe
Emeritus
J Virol Methods. 2009 Sep 14. [Epub ahead of print]
Rapid quantification of single-nucleotide mutations in mixed Influenza A viral populations using Allele Specific Mixture Analysis.
Liu CM, Driebe EM, Schupp J, Kelley E, Nguyen JT, McSharry JJ, Weng Q, Engelthaler DM, Keim PS. - Division of Pathogen Genomics, Translational Genomics Research Institute (TGen). Flagstaff, AZ, USA.; The Center for Microbial Genetics and Genomics (MGGen), Northern Arizona University, Flagstaff, AZ 86001.
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(R) allele-specific PCR (ASPCR) assays were developed and validated for quantitative single-nucleotide polymorphism (SNP) analysis. This novel method using DeltaCt is termed Allele Specific Mixture Analysis (ASMA) or FluASMA. The FluASMA assays target L26F, V27A, A30T, and S31N mutations in the A/Albany/1/1998 (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 DeltaCt coefficient of variance (CoV) at less than or =2% and inter-run DeltaCt CoV at less than 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%).
PMID: 19761797 [PubMed - as supplied by publisher]
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Rapid quantification of single-nucleotide mutations in mixed Influenza A viral populations using Allele Specific Mixture Analysis.
Liu CM, Driebe EM, Schupp J, Kelley E, Nguyen JT, McSharry JJ, Weng Q, Engelthaler DM, Keim PS. - Division of Pathogen Genomics, Translational Genomics Research Institute (TGen). Flagstaff, AZ, USA.; The Center for Microbial Genetics and Genomics (MGGen), Northern Arizona University, Flagstaff, AZ 86001.
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(R) allele-specific PCR (ASPCR) assays were developed and validated for quantitative single-nucleotide polymorphism (SNP) analysis. This novel method using DeltaCt is termed Allele Specific Mixture Analysis (ASMA) or FluASMA. The FluASMA assays target L26F, V27A, A30T, and S31N mutations in the A/Albany/1/1998 (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 DeltaCt coefficient of variance (CoV) at less than or =2% and inter-run DeltaCt CoV at less than 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%).
PMID: 19761797 [PubMed - as supplied by publisher]
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