Methods
Mutation Assay Development for Alpha and Delta Variants
We developed assays in silico to target mutations present in Alpha (HV69–70) and Delta (Del156–157/R158G). We screened primers and probe sequences (Appendix Table, https://wwwnc.cdc.gov/EID/article/28/5/21-2488-App1.pdf) for specificity using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and then tested them in vitro against a wide range of viral genomes, including wild-type SARS-CoV-2 and SARS-CoV-2 VOCs, including Alpha and Delta. We further tested the sensitivity and specificity of the assays by diluting variant gRNA containing the mutations in no (0 copies), low (100 copies), and high (10,000 copies) background of wild-type gRNA (Appendix).
Wastewater Sample Collection
This study used samples from 2 publicly owned treatment works (POTWs) that serve ≈1.5 million residents of Santa Clara County, California, USA (San Jose), and Sacramento County, California, USA (Sacramento). Details of collection processes have been described.[14]
We collected samples from the POTWs to span the period before and including the presumed emergence of Alpha and Delta variants in the communities. Before presumed emergence, sampling was 1–4 times per month; during the periods of suspected emergence, sampling was 3–7 times per week. At San Jose, 133 (HV69–70) and 48 (del156–157/R158G ) samples and at Sacramento, 64 (HV69–70) and 48 (del156–157/R158G) samples were included for analyses of each mutation.
