Novel Workflows for Separate Isolation of Pathogen RNA or DNA from Wastewater: Detection by Innovative and Conventional qPCR.

Abstract

Wastewater-based surveillance (WBS) can provide a wealth of information regarding the health status of communities from measurements of nucleic acids found in wastewater. Processing workflows for WBS typically include sample collection, a primary concentration step, and lysis of the microbes to release nucleic acids, followed by nucleic acid purification and molecular-based quantification. This manuscript provides workflows from beginning to end with an emphasis on filtration-based concentration approaches coupled with specific lysis and nucleic acid extraction processes. Here, two WBS processing approaches are presented, one focusing on RNA-specific pathogens and the other focused on DNA-specific pathogens found within wastewater: 1) The RNA-specific approach, employed for analyzing RNA viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) couples electronegative filtration of wastewater with the placement of the filter within a lysis buffer followed by direct RNA extraction. 2) The DNA-specific approach, employed for analyzing DNA pathogens like Candida auris, uses size selection membranes during filtration, subsequently followed by a lysis buffer, bead-beating, and DNA extraction. Separate workflows for RNA versus DNA isolations have the advantage of improving the detection of the target pathogen. A novel aspect of the RNA-specific workflow is the direct extraction of nucleic acids from filter lysates, which shows enhanced recoveries, whereas the DNA-specific approach requires bead beating prior to extraction. Novelty is also provided in a new qPCR approach called Volcano 2nd Generation (V2G), which uses a polymerase capable of using RNA as a template, bypassing the reverse transcriptase step normally required for qPCR. Key features • Membrane filtration approaches for concentrating suspended solids from wastewater. After concentration, workflows are optimized for separate recovery of RNA and DNA. • Unique polymerase utilized to perform qPCR analysis, foregoing reverse transcription, for RNA. • Sample products for use with other molecular techniques (e.g., sequencing) as workflow approaches generate high-quality, concentrated nucleic acid extracts with minimal inhibitors. • Validated through COVID-19 surveillance where >1,000 samples of wastewater and >3,000 filter concentrates produced from these samples have been created and analyzed, with published results. This complete protocol was used in: J Biomol Tech (2023), DOI: 10.7171/3fc1f5fe.dfa8d906.