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).