Superscript 3 high fidelity rt pcr kit

MS-RTPCR was performed in all FLUAV-positive swabs, as described previously [20 (link)] with minor modifications. Briefly, 2.5 µL of extracted RNA was used as a template in a 25 µL MS-RTPCR reaction (Superscript III high-fidelity RT-PCR kit, ThermoFisher, Waltham, MA, USA), using Opti1-F1 (0.06 µM), Opti1-F2 (0.14 µM), and Opti1-R1 (0.2 µM) primers [14 (link)]. The cycling conditions were: 55 °C for 2 min, 42 °C for 1 h, 5 cycles (94 °C for 30 s, 44 °C for 30 s, 68 °C for 3 min), followed by 31 cycles (94 °C for 30 s, 57 °C for 30 s, 68 °C for 3 min) with a final extension at 68 °C for 10 min. The MS-RTPCR final product was analyzed in 1% agarose gel to corroborate whole genome amplification.

Multisegment amplification of FLUAV genes (MS-RTPCR) was performed from RNA extracted from all FLUAV-positive swabs as described previously (27 (link)) with minor modifications. Briefly, 2.5 μL of extracted RNA was used as a template in a 25-μL MS-RTPCR reaction (Superscript III high-fidelity RT-PCR kit; Thermo Fisher) using Opti1-F1 (0.06 μM), Opti1-F2 (0.14 μM), and Opti1-R1 (0.2 μM) primers (17 (link)). The cycling conditions were as follows: 55°C for 2 min, 42°C for 1 h, 5 cycles (94°C for 30 s, 44°C for 30 s, 68°C for 3 min), followed by 31 cycles (94°C for 30 s, 57°C for 30 s, 68°C for 3 min). Final extension was at 68°C for 10 min. The MS-RTPCR final product was analyzed in 1% agarose gel to corroborate whole-genome amplification.

The whole IAV genome was amplified by a multisegment RT-PCR (mRT-PCR)³⁰ and sequenced by Illumina. The mRT-PCR was performed at Molecular Virology Laboratory, Pontificia Universidad Católica de Chile. Briefly, RNA was subjected to reverse transcription and PCR amplification with the SuperScript III high-fidelity RT-PCR kit (Invitrogen, Carlsbad, CA, USA), using the primers Opti1-F1 (5-GT TA CGC GCC AGC AAA AGC AGG), Opti1-F2 (5-GTT ACG CGC CAG CGA AAG CAG G), y Opti1-R1 (5′-GTT ACG CGC CAG TAG AAA CAA GG). A 50 μL total RT-PCR volume containing 25 μL of buffer, 0.35 μL of Opti1-F1, 0,65 of Opti1-F2 and 1 μL of Opti1-R1, 1 μL of Enzyme Mix, 17 μL of water, and 5 μL of RNA was performed. The thermal cycler program consisted of one cycle of 55 °C for 2 min, 42 °C for 60 min and 94 °C for 2 min, five cycles of 94 °C for 30 s, 44 °C for 30 s and 68 °C for 3,5 min; 26 cycles of 94 °C for 30 s, 57 °C for 30 s and 68 °C for 3.5 min, and one cycle of 68 °C for 10 min. PCR products were purified using Agencourt AMPure XP 5-ml kit (Beckman Coulter, Brea, CA, USA), and those with ≥ 25 ng/μL of DNA concentration were submitted for sequencing. The purified PCR products were submitted to the Center for Research on Influenza Pathogenesis (CRIP), Icahn School of Medicine at Mount Sinai (New York City, NY, USA) for sequencing on an Illumina HiSeq 2000 sequencer.