2.2 RVA detection
Viral RNA was automatically extracted from 200 μL supernatant stool
samples using the Nucleic Acid Extraction Kit (Tianlong Biotechnology).
Two-step reverse transcription-polymerase chain reaction (RT-PCR) was
used to detect the presence of RVA in each stool sample, cDNA was
synthesized with a random primer using Prime-Script™ II Reverse
Transcriptase (Takara Biotechnology) with the reaction condition:97°C 5
min, 37°C for 30 min, 85°C for 5 sec and hold at 4°C. After that, 384 bp
of the VP6 gene was then amplified using previously published primers23. The amplification condition was 94°C for 2 min,
followed by 40 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 30
sec, and with a final extension at 72°C for 5 min.