Rapid quantification of single-nucleotide mutations in mixed influenza A viral populations using allele-specific mixture analysis
| Autor: | Qingmei Weng, Paul Keim, David M. Engelthaler, Elizabeth M. Driebe, Erin Kelley, Jack Nguyen, James M. Schupp, James J. McSharry, Cindy M. Liu |
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| Rok vydání: | 2010 |
| Předmět: |
Genetics
Mutation Reverse Transcriptase Polymerase Chain Reaction Influenza A Virus H3N2 Subtype Mutant Single-nucleotide polymorphism Drug resistance Biology medicine.disease_cause Polymorphism Single Nucleotide Virology Influenza A Virus H1N1 Subtype Drug Resistance Viral Influenza Human Genotype TaqMan medicine Humans Point Mutation Variants of PCR Alleles SNP array |
| Zdroj: | Journal of Virological Methods. 163:109-115 |
| ISSN: | 0166-0934 |
| DOI: | 10.1016/j.jviromet.2009.09.007 |
| Popis: | 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) ator=2% and inter-run Delta Ct CoV ator=5%. Results from the current study demonstrate that FluASMA is a highly sensitive and quantitative SNP analysis method, even for minor mutant components (1%). |
| Databáze: | OpenAIRE |
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