Bigdye terminator v3.1 cycle sequencing reaction kit

Ten percent suspensions (1 mL) of each stool sample were prepared in physiological saline solution; alternatively, swab samples were immersed in 200 μL of physiological saline solution.^{15 (link)} Suspensions were clarified by centrifugation for 20 min at 4000 g, and 140 μL of the supernatant was used for RNA extraction. RNA was extracted from samples using the QIAamp viral RNA Mini kit (QIAGEN, Chatsworth, CA, USA). In order to determine the G, P, and E genotypes, we performed reverse transcription–polymerase chain reaction (RT-PCR) using the SuperScript III One-Step RT-PCR System with Platinum Taq High-Fidelity DNA Polymerase Kit (Invitrogen, Carlsbad, CA, USA). Primers for the target genes were designed to amplify the common sequences of RVA strains.^{16–18 (link)} The BigDye Terminator V3.1 Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA, USA) was used for Sanger sequencing of each PCR product. Sequencing products were analyzed on an ABI 3130 Genetic Analyzer (Applied Biosystems). Finally, G, P, and E genotyping of the obtained sequences was performed using the Rota C v2.0 automated genotyping tool (http://rotac.regatools.be/). It is out of order. The ViPR-tool ((https://www.viprbrc.org/brc/rvaGenotyper.spg?method=ShowCleanInputPage&decorator=reo) can be used as an alternative.).^{19 (link)}