2.2 Extraction of viral RNAs, cDNA synthesis, and RVA detection
by RT-PCR
For viral RNA extraction, stool samples were prepared as a 10%
suspension (w/v) in distilled water and centrifuged at 7000 rpm for 10
minutes. Viral RNAs were extracted from 200 µl of the supernatant of the
suspension using the Viral Nucleic Acid Extraction Kit II (Geneaid,
Taiwan). For cDNA synthesis, 8 µl of the extracted dsRNAs were first
denatured at 95°C for 5 minutes, the denatured RNAs were mixed with 2 µl
of 5x ReverTra Ace® qPCR-RT Master Mix (Toyobo, Japan), and the mixture
was incubated at 37°C for 1 hour for the reverse transcription (RT)
reaction. And for RVA detection, the synthesized cDNAs were used as
templates for RVA screening by RT-PCR using 5x HOT FIREPol Blend Master
Mix (Solis BioDyne, Estonia) with the specific primers Beg9/VP7-1’ that
amplified a 395-bp amplicon.28 PCR products were
subjected to electrophoresis with a 1.5% agarose gel, stained with
RedSafe™ Nucleic Acid Staining Solution (20000x) (iNtRON Biotechnology,
Korea), and observed under UV light using a Gel Doc 1000 UV
trans-illuminator (BIO-RAD, USA).