Quantitect sybr green 1 step real time rt qpcr kit

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)).

Viral RNA was extracted from 10% (v/v) stool suspension in phosphate buffer saline. RNA was automatically extracted from 200 μL supernatant stool samples after centrifugation at 4,000 x g for 10 minutes 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. Amplification of the partial VP6 gene was performed in a 25 μL reaction volume comprised 10 μM forward primer VP6-F (5’- GACGGVGCRACTACATGGT-3’) and reverse primer VP6-R (5’-GTCCAATTCATNCCTGGTGG -3’), based on the study of Kang G. et al. [21 (link)] and 2 μL RNA template using the QuantiTect SYBR Green 1-step real-time RT-qPCR Kit (Qiagen, Hilden, Germany). Reverse transcription was conducted at 50°C for 30 min, followed by heat denaturation at 95°C for 15 min. In total, 45 cycles of amplification were performed comprising denaturation at 94°C for 15 sec, annealing at 50°C for 30 sec, and extension at 72°C for 30 sec. Melting curve analysis was performed from 60°C to 95°C with 1°C increments. Amplified products were purified and sequenced for I genotype identification.