Table 4.
Primers and probes used in the in-house assays specifically detecting SARS-CoV-2, mastermix composition and cycling conditions.
| E_ Sarbeco_F1forward primer | ACAGGTACGTTAATAGTTAATAGCGT |
| E_Sarbeco_E2 reverse primer | ATATTGCAGCAGTACGCACACA |
| E_Sarbeco_P1 probe | FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1 |
| RdRp_ SARSr-F forward primer | GTGARATGGTCATGTGTGGCGG |
| RdRp_SARSr_reverse primer | CARATGTTAAASACACTATTAGCATA |
| RdRp_SARS-P2 probe* | FAM-CAGGTGGAACCTCATCAGGAGATGC-BHQ1 |
| N gene forward | CACATTGGCACCCGCAATC |
| N gene reverse | GAGGAACGAGAAGAGGCTTG |
| N gene probe | FAM-ACTTCCTCAAGGAACAACATTGCCA-BHQ1 |
| ORF1ab forward | CCCTGTGGGTTTTACACTTAA |
| ORF1ab reverse | ACGATTGTGCATCAGCTGA |
| ORF1ab probe | FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1 |
| ORF1b forward | CATGGTGGACAGCCTTTGTTAC |
| ORF1b reverse | TCGCGTGGTTTGCCAAGAT |
| ORF1b probe | FAM- AATGTGAATGCGTCATCATCTGAAGCA-BHQ1 |
| M gene forward | CAAGGACCTGCCTAAAGAAATCAC |
| M gene reverse | ACGCTGCGAAGCTCCCAAT |
| M gene probe | FAM- TGTTGCTACATCACGAACGCTTTC-BHQ1 |
| Master Mix | ||
|---|---|---|
| AgPath-ID™ One-Step RT-PCR Kit (Applied Biosystems™ Catalog number: 4387424 m) | ||
| 1x (20 + 5) reaction uL | ||
| AgPath RT mix 2x | 2x | 10 |
| Forward primer | 20 μM | 0.625 |
| Reverse primer | 20 μM | 0.625 |
| Probe (FAM/BHQ1) | 20uM | 0.312 |
| BGL PCO3 | 100uM | 0.0625 |
| BGL PCO4 | 100uM | 0.0625 |
| BGL probe BGL Quasar 670 | 100uM | 0.0312 |
| Water | 7.4828 | |
| AgPath 25x RT enzyme | 20 μM | 0.8 |
| Total 20 | ||
| Sample extract | 5 | |
| Cycling conditions LC480 II | |||
|---|---|---|---|
| Cycles | Temperature | Time | Ramp rate (°C/s) |
| 1x | 45 °C | 15 min | 4.4 |
| 1x | 95 °C | 15 min | 4.4 |
| 45x | 95 °C | 15 s | 4.4 |
| 60 °C | 45 s | 2.2 | |
| 1x | 40 °C | 30 s | 2.2 |
*Specific for SARS CoV-2 that does not detect SARS-CoV.
Reaction volume 20 μL+5 μL template. We run at 16 μL+ 4 u L volumes (economy).