Virus identification and genome sequencing
All experiments were conducted under biosafety level (BSL)-2 conditions.
The swab tubes were swirled, and the supernatants were collected after
centrifugation. RNAs were extracted by referring to the manual of
MagMAXTM Pathogen RNA/DNA Kit (Applied Biosystems,
USA). The extraction process was completed on the Magmax-96 Express
instrument
(Applied
Biosystems). After extraction, positive samples were screened using a
real-time reverse transcription PCR system with primers specific for the
matrix gene primer and probe set (WHO, 2009) on a 7500 real-time PCR
instrument (Applied Biosystems), and then positive samples were
transcribed into cDNA using the Uni12 primer (5’-AGC AAA AGC AGG-3’) and
PrimScript™ II 1st Strand cDNA Synthesis Kit
(Takara,
Japan).
The
subtypes were determined using specific primers for HA and NA
(Huang et al., 2013;
Kim et al., 2019) and the eight segments
of these H10-H12 subtypes were amplified using the universal primers
(Hoffmann et al., 2001). The PCR reaction
contained 1 μL of cDNA, 1 μL of forward and reverse primers, 12.5 μL of
Taq HS Perfect Mix (Takara, Japan) and 10.5 μL Rnase-free water, with a
final volume of 25 μL. All sequences
were confirmed using a BigDye termination kit (Applied Biosystems,
Foster City, CA, USA) on an ABI 3730 sequence analyzer.