Environmental monitoring of wastewater samples for the presence of SARS-CoV-2 has demonstrated the potential to provide population level estimates of COVID-19 disease burden. 1 While routine monitoring of community wastewater samples for the presence of SARS-CoV-2 viral targets can be complimentary to clinical testing of individuals, quantifying dynamic changes in the number of viral RNA molecules can help identify trends in potential COVID-19 cases within the community. It’s important to recognize the variability arising from differences in sample preparation methods to better track the changes in viral target concentration over time. Process controls are widely used to address sources of variability. Spike in controls, such as Bovine Coronavirus, is one of the most commonly employed targets to provide insight into this variability in wastewater surveillance efforts.
Digital PCR provides more accurate quantitative results
Since dPCR provides standard curve-free absolute quantification of targets, the resulting quantitative data is more accurate and more reproducible than typical qPCR methods. Multiplexing provides additional quantitative data that can be leveraged for normalization of downstream results and help limit the impact of upstream variability. The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay was specifically designed to quantity 2 SARS-CoV-2 viral targets (N1 and N2) alongside a human fecal normalization control (PMMoV) and the sample process control (BCoV) in a single digital PCR reaction. This enables 4x the amount of data to be collected per sample run than many traditional qPCR reactions. Using four separate color channels enables simplified detection of each target without the need for multiple standard curves or tedious manual gating of multiple positive clusters per channel.
This technical note highlights how the sample process control Bovine Coronavirus (BCoV) can be utilized to provide information regarding the success and efficiency of nucleic acid extraction without impacting the sensitivity or accuracy of SARS-CoV-2 detection and quantification.
Using a process control to evaluate sample extraction efficiency
A spike in process control is intended to provide quantitative information about sample loss during sample processing workflow. Typically, a process control is spiked into the initial sample before any processing takes place, and the presence of that control at the end of the workflow serves as an indicator of successful sample recovery.
For the SARS-CoV-2 Wastewater Surveillance 4-plex assay, the endpoint readout of the Bovine Coronavirus target (BCoV) is an absolute quantification of the total number of molecules, determined through digital PCR. By quantifying the control material both prior to spike in and after the processing workflow is complete, an independent measure of the overall yield of the workflow can be calculated. This is enables accurate evaluation of workflows without depending on endogenous targets that could naturally vary from sample to sample.
Interpreting Digital PCR Data
Results reported by the Absolute Q Analysis software are the concentration of each target in copies per microliter (cp/µL) in each dPCR reaction. To calculate the concentration of targets in the original input sample, first calculate the total number of molecules in the dPCR reaction by multiplying the concentration by the 9µL total volume of the dPCR reaction (Equation 1).
Next, to calculate the concentration of the sample used as input into the dPCR reaction, divide the quantity calculated using Equation 1 by the volume of sample used as input. For example, if 5µL of extracted RNA from wastewater was used per dPCR reaction, divide the quantity by 5µL to obtain the original extracted RNA concentration.
Evaluating workflow efficiency
Comparing the concentration of BCoV spiked into the original wastewater sample to the amount of BCoV recovered post processing can provide insight into the overall yield of the workflow. For specific suggestions on how to quantify and use BCoV as a wastewater sample process control consult the SARS-CoV-2 Wastewater Surveillance Kit Instructions for use.
Using Bovine Coronavirus as a process control for wastewater SARS-CoV-2 monitoring
Here we highlight the consistent quantification of BCoV using an example sample extraction workflow. After reconstituting the BCoV control material (materials and methods), 5 serial 10-fold dilutions were prepared, extracted and quantified using the SARS-CoV-2 Wastewater Surveillance Kit. In addition to the BCoV serial dilutions, a SARS-CoV-2 positive reference material and water-only conditions were included as positive and negative extraction controls.
All samples were tested using the SARS-CoV-2 Wastewater Surveillance 4-plex Kit. Figure 1 highlights the consistency of quantification between replicates as well as linearity of the serial dilutions of BCoV (p<0.001). The results suggest that the RNA extraction was successful and the quantification was consistent across the range of input material (Figure 1A). The positive and negative process controls as well as PCR control, which included controls for all 4 assay targets, behaved as expected (Figure 1B-D).
To verify the BCoV concentration obtained from the multiplex assay, a simplex BCoV assay was used to quantify the same purified RNA samples. The average concentration of each sample from the simplex assay showed high is completely in concordance with the concentration from the multiplex assay with a significant Pearson correlation (R2 = 1.0, P<0.0001) Table 1.
Calculating input concentration by dPCR
Using the dPCR data, concentration of the original BCoV material can be calculated using Equation 1. Since one microliter was used as input into each PCR reaction, concentration of the original material can be calculated by accounting for the initial dilution factor for sample extraction. Based on the results, the concentration of the original BCoV input material prior to extraction is 2.39E+06 (Table 2).
Accuracy and sensitivity for SARS-CoV-2 targets remain high
To demonstrate the sensitivity and accuracy of the 4-plex assay in the presence of spike in process control material, we validated the quantification accuracy of the 4-plex assay using three levels of SARS-CoV-2 target concentrations with backgrounds of 5-log concentration range of BCoV (materials and methods).
Three concentrations of SARS-CoV-2 reference RNA (stock , 10-fold and 100-fold dilutions) were added to each point of the BCoV extracted RNA dilution series along with a constant quantity of PMMoV ssDNA (materials and methods). Each mixed sample was then tested using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. The 3 serial 10-fold SARS-CoV-2 reference RNA control materials were also tested without BCoV and PMMoV to evaluate the effects of the spike in material on quantification.
As shown in Figure 2, while the BCoV concentration decreases with each serial dilution point, the quantity of the N1 and N2 remain constant for the stock concentration (Figure 2a), 10-fold dilution (Figure 2b) and 100-fold dilution (Figure 2c) of SARS-CoV-2 reference material. The level of PMMoV (red-bars) remained constant for all conditions tested and yielded a concentration of 46.37 copies/µL (±4.78cp/µL) across all 47 reactions. The measured concentrations with and without BCoV spike-in RNA in the reactions are highly correlated for both N1 and N2 targets as shown in Figure 2d.
Workflow/Materials and Methods
Nucleic Acid Material
Extracted RNA, synthetic construct and standard reference materials were used in the experiments. The Bovine Coronavirus RNA has been extracted from the modified live virus BOVILIS CORONAVIRUS (Merck Animal Health). A 10-dose was reconstituted in 2 milliliters 1x TE buffer (Thermo Fisher Scientific, Waltham, MA) and serially diluted to 1:10, 1:100, 1:1,000, 1:10,000 and 1:100,000. RNA extraction was performed on Maxwell RSC 16 (Promega, Madison, WI). 50 µL of each dilution were extracted along with extraction controls including nuclease free water as negative control and positive control AccuPlex SARS-CoV-2 Positive Reference Material (SeraCare, Milford, MA). All samples were eluted in 50 µL nuclease free water and stored in -80ºC freezer. Additionally, single stranded synthetic DNA for Pepper Mild Mottle Virus (PMMoV) and Exact Diagnostic SARS-CoV-2 RNA control material (Bio-Rad, Hercules, CA) with three dilutions (stock, 1:10 and 1:100) were used in the digital PCR experiments.
Two hydrolysis probe-based assays were used in the study.
Combinati SARS-CoV-2 Wastewater Surveillance 4-plex Kit
The Combinati 4-plex SARS-CoV-2 Wastewater Surveillance assay specifically detects SARS-CoV-2 N1 (FAM) and N2 (HEX) as well as the targets for BCoV process control (TAMRA) and PMMoV human fecal normalization control (TYE) in a single multiplexed reaction. A simplex assay with a probe in the TAMRA channel was used to measure the extracted BCoV RNA.
After preparing the one step RT-dPCR mix, 9µL of the reaction mixture was loaded into the MAP16 plate followed by an overlay of 15µL of isolation buffer. The prepared MAP16 plate was then loaded on the Absolute Q. Figure 3 details the thermal cycling and reagent preparation protocols for RT-dPCR on the Absolute Q. Following the RT-dPCR, the sample concentrations were determined using the Absolute Q Analysis Software with the following threshold for each target FAM, 5000 fluorescence units, HEX 1000 fluorescence units, TAMRA 1300 fluorescence units). Statistical Analyses were performed using Graphpad Prism 9.1.0 (La Jolla, CA)
Wu, Fuqing, et al. “SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases.” MSystems, vol. 5, no. 4, 2020, doi:10.1128/msystems.00614-20.