Development and Validation of the Skimmed Milk Pellet Extraction Protocol for SARS-CoV-2 Wastewater Surveillance

Abstract

Wastewater surveillance for SARS-CoV-2 may serve as a useful source of data for public health departments as the virus is shed in the stool of infected individuals. However, for wastewater data to be actionable, wastewater must be collected, concentrated, and analyzed in a timely manner. This manuscript presents modifications on a skimmed milk concentration protocol to reduce processing time, increase the number of samples that can be processed at once, and enable use in resource-limited settings. Wastewater seeded with Human coronavirus OC43 (OC43) was concentrated using a skimmed milk flocculation protocol, and then pellets were directly extracted with the QIAamp Viral RNA Mini kit. This protocol has a higher average effective volume assayed (6.35 mL) than skimmed milk concentration methods, with and without Vertrel XF™, which involve resuspension of the pellets in PBS extraction prior to nucleic acid extraction (1.28 mL, 1.44 mL, respectively). OC43 was selected as a recovery control organism because both it and SARS-CoV-2 are enveloped respiratory viruses that primarily infect humans resulting in respiratory symptoms. The OC43 percent recovery for the direct extraction protocol (3.4%) is comparable to that of skimmed milk concentration with and without Vertrel XF™ extraction (4.0%, 2.6%, respectively). When comparing SARS-CoV-2 detection using McNemar's chi-square test, the pellet extraction method is not statistically different from skimmed milk concentration, with and without Vertrel XF™ extraction. This suggests that the method performs equally as well as existing methods. Added benefits include reduced time spent per sample and the ability to process more samples at a single time. Direct extraction of skimmed milk pellets is a viable method for quick turnaround of wastewater data for public health interventions.

Keywords: Environmental surveillance; Method development; SARS-CoV-2; Wastewater surveillance.

Fig. 1

Effective volume assayed and percent recovery for each method. a The effective volume assayed is the proportional volume of the original wastewater sample assayed by RT-qPCR. The skimmed milk direct pellet extraction has the highest volume assayed per reaction (6.25 mL). Because it is not dependent on variable resuspension volumes, the effective volume assayed for each sample is the same, unlike skimmed milk flocculation with or without Vertrel XF™ extraction. b The OC43 recovery is the volume adjusted recovery using the RT-qPCR standard curves. The mean recoveries for all three compared methods are comparable

Fig. 2

Control charts of the OC43 RT-qPCR Cq value for a skimmed milk flocculation, b skimmed milk flocculation with Vertrel XF™ extraction, and c direct skimmed milk pellet extraction. Average values were calculated by averaging across treatment plant and time. The upper warning limit (UWL) and lower warning limit (LWL) for each method were calculated by adding or subtracting, respectively, the standard deviation from the average Cq. The upper confidence limit (UCL) and lower confidence limit (LCL) were calculated by adding or subtracting, respectively, three times the standard deviation from the average Cq

Fig. 3

Percent positivity for SARS-CoV-2 by assay in either a non-diluted or b 10⁻¹ diluted RT-qPCR reactions. Darker colors indicate more detection. There is higher percent positivity for the N1 assay compared to N2 across all the methods, suggesting N1 contributes more to SARS-CoV-2 detection. SARS-CoV-2 detection is higher in the 10⁻¹ diluted RT-qPCR reactions than the non-diluted reactions, indicating that there are residual effects of inhibitors on the RT-qPCR reactions