COPMAN: A novel high-throughput and highly sensitive method to detect viral nucleic acids including SARS-CoV-2 RNA in wastewater

Abstract

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater-based epidemiology (WBE) attracted attention as an objective and comprehensive indicator of community infection that does not require individual inspection. Although several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection methods from wastewater have been developed, there are obstacles to their social implementation. In this study, we developed the COPMAN (Coagulation and Proteolysis method using Magnetic beads for detection of Nucleic acids in wastewater), an automatable method that can concentrate and detect multiple types of viruses from a limited volume (∼10 mL) of wastewater. The COPMAN consists of a high basicity polyaluminum chloride (PAC) coagulation process, magnetic bead-based RNA purification, and RT-preamplification, followed by qPCR. A series of enzymes exhibiting a high tolerance to PCR inhibitors derived from wastewater was identified and employed in the molecular detection steps in the COPMAN. We compared the detectability of viral RNA from 10-mL samples of virus-spiked (heat-inactivated SARS-CoV-2 and intact RSV) or unspiked wastewater by the COPMAN and other methods (PEG-qPCR, UF-qPCR, and EPISENS-S). The COPMAN was the most efficient for detecting spiked viruses from wastewater, detecting the highest level of pepper mild mottle virus (PMMoV), a typical intrinsic virus in human stool, from wastewater samples. The COPMAN also successfully detected indigenous SARS-CoV-2 RNA from 12 samples of wastewater at concentrations of 2.2 × 10⁴ to 5.4 × 10⁵ copies/L, during initial stages of an infection wave in the right and the left bank of the Sagami River in Japan (0.65 to 11.45 daily reported cases per 100,000 people). These results indicate that the COPMAN is suitable for detection of multiple pathogens from small volume of wastewater in automated stations.

Keywords: Automation; COVID-19; Environmental surveillance; PCR; Sewage; Wastewater-based epidemiology.

Graphical abstract

Fig. 1

Workflow of the COPMAN: coagulation and proteolysis method using magnetic beads for detection of nucleic acids in wastewater.

Fig. 2

Comparative analysis of four methods. The diagram shows four methods with different strategies for quantification of viral RNA from wastewater. Each method was used to detect viral RNA in 10 mL of wastewater. The COPMAN uses both liquid and solid fractions by employing a PAC coagulation procedure. PEG-qPCR uses the liquid fraction by removing the solid fraction before the assay. Ultrafiltration collects molecules larger than 100 kDa from both fractions. The EPISENS-S collects molecules in the solid fraction by removing the liquid fraction.

Fig. 3

Detection of viral RNA from wastewater spiked with various amounts of HI-SARS-CoV-2 and RSV. Heat-inactivated (HI) SARS-CoV-2 and intact RSV were spiked and recovered from 10-mL wastewater. Recovery rates are shown in each cell with standard deviations when viral RNA was detected in 3/3 samples. 1/3 and 2/3 represents RNA detection in one and two samples out of three, respectively. N.D. represents no RNA detection in the samples (0/3). *One of three samples showed a Ct value of 44.94.

Fig. 4

Comparison of detection abilities of viral RNA from wastewater. Each method was used to quantify viral RNA from 10 mL of wastewater during the initial stage of a SARS-CoV-2 outbreak. The COPMAN was compared with the EPISENS-S, PEG-qPCR, and UF-qPCR. (A) Total RNA concentration of the purified RNA extracts. Note that for PEG-qPCR, 5.6 μg carrier RNA was added during the RNA extraction with the QIAamp Viral RNA Mini kit according to the manufacturer's instructions. (B, C) Detection of SARS-CoV-2 (B) and PMMoV (C). The black bar is the average of 12 samples (calculated by substituting N.D. to 0). L and R represent the WWTP in the left and the right bank of the Sagami river in Japan, respectively. N.D., not detected. Pairwise comparison was performed using the Tukey test (p < 0.01).