Viral genome was prepared from a 10% (w/v) stool suspension with phosphate buffer saline (PBS), centrifuged at 4,000×g for 10 min, and supernatants were collected. Viral RNA was automatically extracted from a 200 µL supernatant sample using a magLEAD 12gC instrument (Precision System Science, Chiba, Japan) with a magLEAD Consumable Kit (Precision System Science, Chiba, Japan) according to the manufacturer’s instructions.
Samples were initially tested for the RVA VP6 gene using the QuantiTect SYBR Green 1-step real-time RT-qPCR Kit (Qiagen, Hilden, Germany). The primers VP6-F (5′ GACGGVGCRACTACATGGT 3′) and VP6-R (5′ GTCCAATTCATNCCTGGTGG 3′) a 379-bp region corresponding to nucleotides 747–1,126 of the VP6 gene. Cycling parameters were reverse transcription at 50 °C for 30 min, initial denaturation at 95 °C for 15 min, 45 cycles of denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. Melting curve analysis was explored from 60 °C to 95 °C with 1 °C increments to determine the specificity of the reactions (Kang et al., 2004 (link); Chansaenroj et al., 2020 (link)).
Samples were initially tested for the RVA VP6 gene using the QuantiTect SYBR Green 1-step real-time RT-qPCR Kit (Qiagen, Hilden, Germany). The primers VP6-F (5′ GACGGVGCRACTACATGGT 3′) and VP6-R (5′ GTCCAATTCATNCCTGGTGG 3′) a 379-bp region corresponding to nucleotides 747–1,126 of the VP6 gene. Cycling parameters were reverse transcription at 50 °C for 30 min, initial denaturation at 95 °C for 15 min, 45 cycles of denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. Melting curve analysis was explored from 60 °C to 95 °C with 1 °C increments to determine the specificity of the reactions (Kang et al., 2004 (link); Chansaenroj et al., 2020 (link)).
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