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Title: Effect of SARS-CoV-2 digital droplet RT-PCR assay sensitivity on COVID-19 wastewater based epidemiology
We developed and implemented a framework for examining how molecular assay sensitivity for a viral RNA genome target affects its utility for wastewater-based epidemiology. We applied this framework to digital droplet RT-PCR measurements of SARS-CoV-2 and Pepper Mild Mottle Virus genes in wastewater. Measurements were made using 10 replicate wells which allowed for high assay sensitivity, and therefore enabled detection of SARS-CoV-2 RNA even when COVID-19 incidence rates were relatively low (~10 −5 ). We then used a computational downsampling approach to determine how using fewer replicate wells to measure the wastewater concentration reduced assay sensitivity and how the resultant reduction affected the ability to detect SARS-CoV-2 RNA at various COVID-19 incidence rates. When percent of positive droplets was between 0.024% and 0.5% (as was the case for SARS-CoV-2 genes during the Delta surge), measurements obtained with 3 or more wells were similar to those obtained using 10. When percent of positive droplets was less than 0.024% (as was the case prior to the Delta surge), then 6 or more wells were needed to obtain similar results as those obtained using 10 wells. When COVID-19 incidence rate is low (~ 10 −5 ), as it was before the Delta surge and SARS-CoV-2 gene concentrations are <10 4 cp/g, using 6 wells will yield a detectable concentration 90% of the time. Overall, results support an adaptive approach where assay sensitivity is increased by running 6 or more wells during periods of low SARS-CoV-2 gene concentrations, and 3 or more wells during periods of high SARS-CoV-2 gene concentrations.  more » « less
Award ID(s):
2022877
PAR ID:
10402195
Author(s) / Creator(s):
; ; ; ; ; ;
Editor(s):
Santos, Ricardo
Date Published:
Journal Name:
PLOS Water
Volume:
1
Issue:
11
ISSN:
2767-3219
Page Range / eLocation ID:
e0000066
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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