Molecular detection of rotavirus
Details of nucleic acid extractions, reverse transcription, and
rotavirus molecular detection were described previously (19). Briefly,
total nucleic acid was extracted from 30% rectal swab suspension or
10% stool suspension, prepared using NucliSENS® lysis buffer
(BioMérieux, Laval, QC, Canada) by NucliSENS® easyMAG® (BioMérieux,
Laval, QC, Canada) according to manufacturer’s instructions. All
resultant nucleic acid extracts were immediately stored at -70°C until
testing. cDNA was synthesized from viral RNA using
SuperScriptTM II reverse transcriptase (Invitrogen,
MA, USA). Group A rotaviruses in stool and swabs were identified by an
in-house reverse transcription real-time PCR (RT-qPCR) assay, the
Gastroenteritis Viral Panel (GVP), and a multi-target bead-based
molecular assay, the Luminex Gastrointestinal Pathogen Panel (GPP)
(Luminex Molecular Diagnostics, ON, Canada), as described previously
(28).