Table 2 Nucleic acid amplification assays conditions.
From: Complementary methods for SARS-CoV-2 diagnosis in times of material shortage
Assay | Reaction mix | Template volume (µl) | Total reaction volume (µl) | Reverse transcription | Initial melting | Cycle melting | Cycle annealing | # Cycles | Melting curve | |
|---|---|---|---|---|---|---|---|---|---|---|
Hydrolysis probe assaysa11 | RNAse P | 0.4 mM each primer 0.1 mM probe | 3 | 15 | 50 °C, 15 min | 95 °C, 2 min | 95 °C, 15 s | 58 °C, 45 s | × 45 | – |
SARS-CoV-2 E gene | 0.4 mM each primer 0.2 mM probe | 3 | 15 | 50 °C, 15 min | 95 °C, 2 min | 95 °C, 15 s | 58 °C, 45 s | × 45 | – | |
SARS-CoV-2 RdRp gene | 0.6 mM forward primer 0.8 mM reverse primer 0.2 mM probe | 3 | 15 | 50 °C, 15 min | 95 °C, 2 min | 95 °C, 15 s | 58 °C, 45 s | × 45 | – | |
Intercalating dye assaysb | RNAse P | 0.4 mM each primer | 2 | 10 | 45 °C, 10 min | 95 °C, 2 min | 95 °C, 5 s | 60 °C, 30 s | × 45 | 60–95 °C (5 acquisitions/°C) |
SARS-CoV-2 E gene | 0.4 mM each primer | 2 | 10 | 45 °C, 10 min | 95 °C, 2 min | 95 °C, 5 s | 60 °C, 30 s | × 45 | 60–95 °C (5 acquisitions/°C) | |
SARS-CoV-2 RdRp gene | 0.6 mM forward primer 0.8 mM reverse primer | 2 | 10 | 45 °C, 10 min | 95 °C, 2 min | 95 °C, 5 s | 60 °C, 30 s | × 45 | 60–95 °C (5 acquisitions/°C) |
