Sensitive detection of SARS-CoV-2 molecular markers in urban community sewersheds using automated viral RNA purification and digital droplet PCR

Improving wastewater-based epidemiology performance through streamlined automation

“…Table 3 compares the cost for some of the conventional and automated methods. The details for the cost analysis of ESP methods are presented in Tables S4, S5 and S6 , while details on the cost calculation for the other methods can be found in references [46] , [47] . The automated methods have higher startup costs due to the need for the automated instrument.…”

“…The reagent and consumable cost from raw wastewater to extracted RNA ready to be quantified with RT-qPCR or ddPCR. Method Startup Cost ($) Reagent and Consumable Cost ($ per replicate/sample) Manual Electronegative Membrane with Bead Beating [46] 15,368 7.02 Electronegative Membrane with Elution [46] 11,160 11.32 PEG [46] 20,288 16.54 Ultrafiltration [46] 9,000 17.62 Manual ESP 2,874 3.81 Automated Promega Maxwell RSC [47] N/A ~ 40.00 King Fisher Flex [47] N/A ~ 40.00 Automated ESP 25,939 3.81 …”

Passive swab versus grab sampling for detection of SARS-CoV-2 markers in wastewater

“…Six sites across the City of Detroit were selected for paired methodological comparison, based on results obtained in previous studies ( West et al, 2022 ). The six sites include three dormitory building sewersheds (AS, UC, and WH, with residents numbering <500 each during most of the sampling period), two sewersheds that include a long-term care facility (LTCF) and surrounding neighborhood (CS and JV, with estimated sewershed populations of 2000 and 500, respectively), and SG (“ZIP Code”-sampling sewershed with an estimated population of 12,000 people).…”

“…Grab samples were processed as described in previous publications ( Vasquez et al, 2021 ; West et al, 2022 ). Briefly, 45 mL of the collected sample in a 50 mL polypropylene tube was centrifuged at 3996 rpm (5000 × g ) at 4 °C for 15 min; 10 mL of supernatant was transferred to another tube for purification; samples were “spiked” with phage Phi6 (internal RNA virus recovery control, added as 10 μL of 10 6 pfu) and then treated with lysis buffer and Proteinase K at 55 °C for 30 min; and then processed via an automated magnetic bead-based nucleic acid extraction system (Chemagic 360™) to yield 85 μL of purified nucleic acids in a PCR-compatible elution buffer.…”

Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

“…In comparison with RT-PCR, RT-dPCR is a more efficient and sensitive method for SARS-CoV-2 detection. 95,96 In recent years, RT-dPCR has been developed rapidly for nucleic acid detection and widely used in clinical microbiology. [97][98][99] The principle of RT-dPCR is to split a sample into tens to tens of thousands of micro-drop units, each containing one or more copies of the nucleic acid molecule (i.e., DNA template).…”