16 capillary 3130xl genetic analyzer

Viral RNA was isolated from clinical specimens using PureLink (Invitrogen/Thermo Fisher, Carlsbad, CA) or QIAamp (Qiagen, Hilde, Germany) RNA extraction kits, according to the manufacturers’ instructions. Quantitative detection of H5N1 RNA was done batch-wise using real-time RT-PCR as described previously [11 (link), 12 (link)]. The presence of mutations in the viral neuraminidase and matrix 2 (M2) genes that confer resistance to oseltamivir and adamantanes, respectively, was analyzed using pyrosequencing or Sanger sequencing [16 (link), 17 (link)]. Viral RNA was reverse transcribed as described previously [8 (link), 11 (link), 12 (link)] and amplified using gene-specific primers. Primer sequences are available upon request. Amplification was performed using Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA) or HotStar Taq (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. The ExoSAP-IT (Affymetrix, Inc., Santa Clara, CA) purification kit was used to purify the PCR products. Pyrosequencing was performed on the PyroMark ID instrument using Pyro Gold Reagents kits (Biotage AB, Uppsala, Sweden). For Sanger sequencing, DNA was sequenced using the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer’s instructions using a 16-capillary 3130xl Genetic Analyzer (Applied Biosystems).