2.2 RV classification using VP4/2 sequences
Thirty-two RV-positive samples, representing diverse time points, were
randomly selected for genotyping. Genotyping was performed by amplifying
the partial VP4/VP2 region using nested
PCR[19].The primer sequences employed are detailed
in the Supplementary Material (Table S1). To extract total viral RNA,
200 μl of each sample was processed using the QIAamp Viral RNA Mini Kit
(Qiagen, Valencia, CA), following the manufacturer’s guidelines. The
nested PCR was performed using the SuperScript™ III One-Step RT-PCR
System (Invitrogen, USA) and Go Taq™ Colorless Master Mix (Promega,
USA).
The initial PCR cycle entailed a 5-minute activation at 94°C, followed
by 40 cycles of 30 seconds at 95°C for denaturation, 30 seconds at 55°C
for annealing, and 5 minutes at 72°C for extension, concluding with an
additional 10-minute extension at 72°C. The second round of PCR mirrored
the first, with the exception of the annealing temperature, set at 56°C.
Each PCR run included a negative control alongside the samples. The
resulting 540 bp amplification products were visualized on a 1.5%
agarose gel. PCR products from positive samples were forwarded to
Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. Sequence
alignments were conducted using SeqMan software, and the sequences were
verified through database comparison using the BLAST tool at the
National Center for Biotechnology Information (NCBI).