COVID-19 RBD amplification protocol 1. Prepare a master mix using the following reagents in a sterile Biosafety cabinet. Reagent 2X Platinum SuperFi RT-PCR Master Mix* SuperScript IV RT Mix* Fw primer – MCJ2395 (50 µM) Rv primer – MCJ2452 (50 µM) Nuclease free water RNA template from wastewater extraction *Invitrogen, Catalog #: 12594100 B. C. D. E. F. G. H. Volume per reaction (µl) 12.5 0.25 0.25 0.25 6.75 5 2. Preform primary amplification in a thermocycler using the steps listed below. A. RT enzyme activation: 25°C for 2 minutes Reverse transcription: 50°C for 20 minutes RT inactivation/Initial denaturation: 95°C for 2 minutes Denaturation: 95°C for 15 seconds Annealing: 55°C for 30 seconds Extension: 72°C for 1 minute Repeat steps D – F for 25 cycles Hold at 4°C 3. Prepare master mix for a nested PCR using the following reagents. Reagent Q5 High-Fidelity DNA Polymerase* Q5 Reaction Buffer* Deoxynucleotide Solution Mix# (10 mM for each dNTP) Fw primer – MCJ2469 (50 µM) Rv primer – MCJ2370 (50 µM) Nuclease free water DNA template from primary reaction *New England Biolabs, Catalog #: M0491L # New England Biolabs, Catalog #: N0447S Volume per reaction (µl) 0.125 4 0.25 0.25 0.25 15.125 5 4. Preform nested amplification in a thermocycler using the steps listed below. A. Initial denaturation: 95°C for 2 Minutes B. Denaturation: 95°C for 15 seconds C. Annealing: 55°C for 30 seconds D. Extension: 72°C for 1 minute E. Repeat steps B – D for 20 cycles F. Hold at 4°C 5. Confirm amplification through gel electrophoresis. Expected amplicon size is ~600bp MCJ2395 CTGCTTTACTAATGTCTATGCAGATTC MCJ2452 NCCTGATAAAGAACAGCAACCT MCJ2469 ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTRATGAAGTCAGMCAAATYGC MCJ2370 gtgactggagttcagacgtgtgctcttccgatctATGTCAAGAATCTCAAGTGTCTG