Program: CFX96 Deep PCR machine
Channels:
-
SARS-CoV-2: FAM
-
PMMoV: HEX
-
BRSV: Cy5
| Step | T (°C) | Time | Cycles |
|---|---|---|---|
| RT | 55 | 10 min | 1 |
| Initial denaturation | 95 | 5 min* | 1 |
| Denaturation | 95 | 10 s | 40 |
| Annealing and extension | 60 | 30 s** |
*To complete RT denaturation
**For Applied Biosystems (ABI), use 60 s
| Virus | Target gene | Oligonucleotide Sequence (5’>3’) | Length (pb) | Reference | |
|---|---|---|---|---|---|
| Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) | Nucleocapsid (N) | Forward | GAC CCC AAA ATC AGC GAA AT | 72 | CDC, 2020 |
| Reverse | TCT GGT TAC TGC CAG TTG AAT CTG | ||||
| Probe | FAM ACC CCG CAT /ZEN/ TAC GTT TGG TGG ACC IABkFQ | ||||
| Bovine Respiratory Syncytial Virus (BRSV) | Nucleocapsid (N) | Forward | GCA ATG CTG CAG GAC TAG GTA TAA T | 124 | Boxus et al., 2005 |
| Reverse | ACA CTG TAA TTG ATG ACC CCA TTC T | ||||
| Probe | Cy5 ACC AAG ACT /ZEN/ TGT ATG ATG CTG CCA AAG CA IABkFQ | ||||
| Pepper Mild Mottle Virus (PMMoV) | Coat protein (CP) | Forward | TAC TTC GGC GTT AGG CAA TCA G | 122 | McGill design |
| Reverse | TGA AAC CAG TAG CAG GAA ATC TAA C | ||||
| Probe | HEX CAG CAG TTC TCT GAT GTG TGG IABkFQ |