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. 2023 Dec 20;34(4):3fc1f5fe.dfa8d906.
doi: 10.7171/3fc1f5fe.dfa8d906. eCollection 2023 Dec.

Expanding a Wastewater-Based Surveillance Methodology for DNA Isolation from a Workflow Optimized for SARS-CoV-2 RNA Quantification

Kristina M Babler  1 , Mark E Sharkey  2 , Ayaaz Amirali  1 , Melinda M Boone  3 , Samuel Comerford  2 , Benjamin B Currall  3 , George S Grills  3 , Jennifer Laine  4 , Christopher E Mason  5   6   7 , Brian Reding  4 , Stephan Schürer  3   8   9 , Mario Stevenson  2 , Dušica Vidović  8 , Sion L Williams  3 , Helena M Solo-Gabriele  1
Affiliations

Expanding a Wastewater-Based Surveillance Methodology for DNA Isolation from a Workflow Optimized for SARS-CoV-2 RNA Quantification

Kristina M Babler et al. J Biomol Tech. .

Abstract

Wastewater-based surveillance (WBS) is a noninvasive, epidemiological strategy for assessing the spread of COVID-19 in communities. This strategy was based upon wastewater RNA measurements of the viral target, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The utility of WBS for assessing the spread of COVID-19 has motivated research to measure targets beyond SARS-CoV-2, including pathogens containing DNA. The objective of this study was to establish the necessary steps for isolating DNA from wastewater by modifying a long-standing RNA-specific extraction workflow optimized for SARS-CoV-2 detection. Modifications were made to the sample concentration process and included an evaluation of bead bashing prior to the extraction of either DNA or RNA. Results showed that bead bashing reduced detection of RNA from wastewater but improved recovery of DNA as assessed by quantitative polymerase chain reaction (qPCR). Bead bashing is therefore not recommended for the quantification of RNA viruses using qPCR. Whereas for Mycobacterium bacterial DNA isolation, bead bashing was necessary for improving qPCR quantification. Overall, we recommend 2 separate workflows, one for RNA viruses that does not include bead bashing and one for other microbes that use bead bashing for DNA isolation. The experimentation done here shows that current-standing WBS program methodologies optimized for SARS-CoV-2 need to be modified and reoptimized to allow for alternative pathogens to be readily detected and monitored, expanding its utility as a tool for public health assessment.

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Conflict of interest statement

Conflict of Interest: The authors declare no conflict or competing interest.

Figures

Figure 1
Figure 1. Laboratory workflow of sample preparation methodology. Experimentation included the splitting of raw wastewater with subsequent pretreatment specific to ENF or VF. BB and the exclusion of BB were performed for each condition evaluated followed by separate RNA and DNA extractions of either bashed or un-bashed concentrates. Image created with BioRender.com.
Figure 2
Figure 2. Quantitation of molecular targets by qPCR from BB and nBB wastewater concentrates. (A) SARS-CoV-2, (B) OC43, and (C) Mycobacterium smegmatis. HAWP membranes illustrated alongside GN-6 membranes quantified from 4 µL nucleic acid input.
See this image and copyright information in PMC

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