Multisegment FLUAV 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.