Evaluation of viral concentration and extraction methods for SARS-CoV-2 recovery from wastewater using droplet digital and quantitative RT-PCR

Abstract

The ongoing pandemic caused by the emergence of SARS-CoV-2 has resulted in millions of deaths worldwide despite the various measures announced by the authorities. Wastewater-based epidemiology has the ability to provide a day-to-day estimation of the number of infected people in a fast and cost-effective manner. However, owing to the complex nature of wastewater, wastewater monitoring for viral genome copies is affected by the extensive viral fragmentation that takes place all the way to the sewage and the analytical lab. The aim of this study was to evaluate different methodologies for the concentration and extraction of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. We compare 5 different concentration methods and 4 commercially available kits for the RNA extraction. To evaluate the performance and the recovery of these, SARS-CoV-2 isolated from patients was used as a spike control. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and N3). Using spiked samples, recoveries were estimated 2.1-37.6% using different extraction kits and 0.1-2.1% using different concentration kits. It was found that a direct capture-based method, evaluated against a variety of concentration methods, is the best in terms of recovery, time and cost. Interestingly, we noticed a good agreement between the results provided by RT-qPCR and RT-ddPCR in terms of recovery. This evaluation can serve as a guide for laboratories establishing a protocol to perform wastewater monitoring of SARS-CoV-2. Overall, data presented here reinforces the validity of WBE for SARS-CoV-2 surveillance, uncovers potential caveats in the selection of concentration and extraction protocols and points towards optimal solutions to maximize its potential.

Keywords: Droplet digital PCR (ddPCR); Quantitative reverse transcription-polymerase chain reaction (RT-qPCR); SARS-CoV-2; Viral RNA extraction; Viral concentration; Wastewater-based epidemiology.

Graphical abstract

Fig. 1

A. Description of the five selected concentration methods based on PEG precipitation with salt addition, PEG-Glycine, Direct capture - Promega, Ultrafiltration and Adsorption-extraction with electronegative membranes with the same extraction protocol (Manual Enviro Wastewater TNA kit, Promega). B. The selected concentration method (direct capture) was used as the first step followed by extraction based on four different commercially available kits one from IDEXX, two from Qiagen and one from Promega. All the replicates from all concentration and extraction methods were analyzed with both RT-qPCR and RT-ddPCR.

Fig. 2

N1 & N2 SARS-CoV-2 log copies per liter of initial raw wastewater measured by a) RT-qPCR and b) RT-ddPCR on three different days using the five different concentration methods.

Fig. 3

a) % Recovery of the SARS-CoV-2 viral standard as determined by RT-qPCR with the five different concentration methods & b) % Recovery of the SARS-CoV-2 viral standard as determined by RT-qPCR with the four different extraction methods.

Fig. 4

a) % Recovery of the SARS-CoV-2 viral standard as determined by RT-ddPCR with the five different concentration methods & b) % Recovery of the SARS-CoV-2 viral standard as determined by RT-ddPCR with the four different extraction methods.