Table 1.
Quantitative real-time PCRs used in the study
| Assay and references | Reaction conditions | Cycling conditions |
|---|---|---|
| Lassa virus (Demby et al., 1994, Asper et al., 2004) | 20-μl reaction based on Brilliant single-step quantitative RT-PCR kit (Stratagene): 2 μl RNA, 1× buffer, 2.5 mM MgCl2, 800 μM dNTP, 0.8 μg bovine serum albumin, 5 μM SybrGreen-binding molecule SGS (Drosten, 2003), SybrGreen 1:10,000 (Molecular Probes), 1.25 U StrataScript RT, 1 U SureStart Taq polymerase, 200 nM primer 36E2 (ACC GGG GAT CCT AGG CAT TT), and 300 nM primer 80F2 (ATA TAA TGA TGA CTG TTG TTC TTT GTG CA) | LightCycler (Roche): 20 min at 45 °C; 12 min at 95 °C; 10 precycles with 10 s at 95 °C, 10 s at 60 °C with a decrease of 0.8 °C/cycle, and 20 s at 72 °C; 25 cycles with 5 s at 95 °C, 10 s at 55 °C, 30 s at 65 °C, and 20 s fluorescence read at 80 °C (F1 detection channel); melting curve analysis. |
| SARS-CoV (Drosten et al., 2003) | 25-μl reaction based on Superscript II RT/Platinum Taq polymerase one-step RT-PCR kit (Invitrogen): 5 μl RNA, 1× buffer, 3.6 mM additional MgSO4, 0.6 μl enzyme mixture, 240 nM probe BNITMSARP (FAM-TCG TGC GTG GAT TGG CTT TGA TGT-TAMRA), 200 nM primer BNITMSARS1 (TTA TCA CCC GCG AAG AAG CT), and 200 nM primer BNITMSARAs2 (CTC TAG TTG CAT GAC AGC CCT C) | 7000 SDS machine (Applied Biosystems): 15 min at 45 °C; 3 min at 95 °C; 40 cycles with 15 s at 95 °C and 30 s at 58 °C with fluorescence measured at 58 °C (FAM detection channel without passive reference dye) |
| Ebola virus (Gibb et al., 2001) | 20-μl reaction based on Brilliant single-step quantitative RT-PCR kit (Stratagene): 2 μl RNA, 1× buffer, 2.5 mM MgCl2, 800 μM dNTP, 1.25 U StrataScript RT, 1 U SureStart Taq polymerase, 250 nM probe EBOGP-1DZPrb (FAM-CTA CCA GCA GCG CCA GAC GG-TAMRA), 500 nM primer EBOGP-1D forward (TGG GCT GAA AAY TGC TAC AAT C), and 500 nM primer EBOGP-1D reverse (CTT TGT GMA CAT ASC GGC AC) | 7000 SDS machine (Applied Biosystems): 30 min at 50 °C; 10 min at 95 °C; 45 cycles with 15 s at 95 °C and 30 s at 58 °C with fluorescence measured at 58 °C (FAM detection channel without passive reference dye) |
Abbreviations: FAM, 6-carboxyfluorescein; TAMRA, 6-carboxy-N,N,N′,N′-tetramethylrhodamin; RT, reverse transcriptase.