Sewage, Salt, Silica and SARS-CoV-2 (4S): An economical kit-free method for direct capture of SARS-CoV-2 RNA from wastewater

Abstract

Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods’ sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled “Sewage, Salt, Silica and SARS-CoV-2” (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol and silica RNA capture matrices to recover 6-fold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, both suitable as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and could be performed in approximately 3 hours. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic.SYNOPSISThe 4S method for measuring SARS-CoV-2 in wastewater is promising for effective, economical, and accessible wastewater-based epidemiology.ABSTRACT ART

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

“…Findings from the limited studies available on pasteurization or cold storage of wastewater samples are somewhat discordant. For example, in Whitney et al (2020), no significant reduction in SARS-CoV-2 RNA signal was observed when wastewater samples were pasteurized at 70°C for 45 min, suggesting that at least some heat pre-treatment conditions may not adversely affect the results. In contrast, the effect of pasteurization was determined at 70ºC for 40 min on SARS-CoV-2 N gene concentrations in triplicate wastewater collected from three WWTPs in Southern California, USA (Fig.…”

“…It is recommended that concentration of wastewater samples should be performed in a BSL-2 or BC2 laboratory with unidirectional airflow and BC3/BSL-3 precautions (CDC, 2019). Heat pre-treatment pasteurization can be used to minimize exposure risk and help ensure safe sample handling, especially in instances of sample manipulations that may generate aerosols (i.e., sample concentration) (Whitney et al, 2020;Wu et al, 2020). However, temperature, in general, can significantly influence microbial decay (Espinosa et al, 2020;Korajkic et al, 2019;Muirhead et al, 2020), with extended survival (minimal degradation) typically observed at lower temperatures (<15°C).…”

SARS-CoV-2 RNA is enriched by orders of magnitude in solid relative to liquid wastewater at publicly owned treatment works

“…Sewage, Salt, Silica, and SARS-CoV-2 (4S) method (OS): Samples were processed using the 4S protocol. 11 In brief, after collection, viruses in the samples were lysed and RNA stabilized by addition of NaCl. After receiving samples in the lab, BCoV (Bovilis Coronavirus Calf Vaccine, Merck Animal Health, NJ) was spiked into the wastewater sample as a positive control and the sample was pasteurized at 70°C for 45 minutes.…”

“…4,5 The COVID-19 pandemic has greatly increased interest in utilizing wastewater-based epidemiology to supplement clinical testing data, which can be limited due to test seeking behavior and test availability. 6 Throughout the pandemic, researchers have successfully detected and monitored SARS-CoV-2 RNA in wastewater [7][8][9][10][11] and programs have been developed to aid public health decision makers in assessing the disease burden of COVID-19 in their communities. 12,13 Sewage consists of liquid and solid fractions, and SARS-CoV-2 RNA has been quantified in both.…”

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples

“…We continue to offer the saliva assay described here as a surveillance test, often in a take-home setting, in order to enable testing of smaller target groups who are difficult to recruit into larger surveillance programs or who have difficulties tolerating swab-based collection. For example, a UC Berkeley surveillance project recently identified SARS-CoV-2 in wastewater draining from university-owned housing units [ 19 , 20 ], which triggered the rapid deployment of saliva-based surveillance testing to residents. In conclusion, we have found detection of SARS-CoV-2 from saliva to be an important alternative sample collection method that complements swab-based diagnostic and surveillance testing.…”