Bovine Rotavirus Genotyping Protocol

Infected MDBK cells were scraped off the plates and homogenised using three cycles of freezing and thawing. Viral RNA was extracted from 250 μL of supernatant using the QIAsymphony™ SP Instrument (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Oligonucleotide primers for BRV3 detection and identification described by Maidana et al. (2012) [24 (link)] were used to amplify a 328 bp segment of the consensus BRV3 Matrix (M) gene by RT-PCR using the commercial Qiagen One-Step RT-PCR kit (Qiagen). The PCR products were purified using the Qiaquick PCR Purification Kit (Qiagen). Sequencing reactions were performed with BigDye Terminator v3.0 kit (Applied Biosystems, Lennik, Belgium) and analysed with an ABI Prism 3730 DNA Analyser (Applied Biosystems).