Wastewater surveillance of SARS-CoV-2 has been shown to be a useful predictor of potential outbreaks. However, for meaningful interpretation of SARS-Cov2 data, both quantification accuracy and data normalization are critical. Moreover, reverse transcription qPCR (RT-qPCR) – the most commonly used method – reports a threshold cycle that requires a standard curve to provide quantitative information. RT-qPCR’s reliance on standard curves means the accuracy of the measurements depends directly on the accuracy and reproducibility of the reference materials used. These factors combined make interpretation of data on a broad scale extremely challenging.
Digital PCR (dPCR) provides absolute quantification, and when combined with multi-colored multiplexing, can incorporate controls in a single reaction to provide normalized results for multiple targets. These results enable more accurate comparisons between samples with varying upstream sample preparation methodologies and more robust longitudinal monitoring.
Benefits of the Absolute Q for SARS-CoV-2 Wastewater Monitoring
- Quantification of three wastewater-specific genomic targets in a single dPCR reaction
- Integration of Human Fecal and Process Controls allows normalization and recovery efficiency to be calculated without additional reactions
- Single instrument qPCR-like workflow in under 2 hours
The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay was designed to detect the N1 and N2 SARS-CoV-2 viral RNA targets alongside the human fecal control, Pepper Mild Mottle virus (PMMoV). In addition to these three targets, the assay also integrates the process control Bovine Coronavirus (BCoV). This inactivated virus, which is not generally present in community sewer systems, is spiked into the initial raw sewage sample before downstream processing. Its similarity to human SARS-CoV-2 allows it to be used as a surrogate to monitor the overall efficiency of sample processing.
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The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay combines four sets of primers/probes into a single multiplexed assay. The assay is composed of two genetic targets N1 and N2 on the SARS-COV-2 N gene, a matrix recovery target (aka. process control) on Bovine coronavirus genome (BCoV), and a Human fecal normalization control target on Pepper Mild Mottle virus (PMMoV). N1 and N2 have been reported to be sensitive and specific for quantifying SARS-CoV-2 RNA in wastewater. Bovine coronavirus is an enveloped virus with a single stranded RNA genome similar to SARS-CoV-2, but not usually present in wastewater. The plant pathogen Pepper Mild Mottle Virus (PMMoV), an indicator of human fecal pollution, is widespread and abundant in wastewater from the United States.
N1, N2, BCoV and PMMoV targets were labeled with FAM, HEX, TAMRA, and TYE665, respectively (Figure 1). All primers were checked for target sequence specificity using NCBI Primer-BLAST1. Primers and probes were also evaluated for primer dimers and cross primer interactions using Multiple Primer Analyzer (Thermo Fisher Scientific, Waltham, MA).
Absolute Q Workflow and Experiment Materials
After preparing the dPCR mix, 9µL of the reaction mixture was loaded into the MAP16 plate followed by an overlay of 15µL of isolation buffer (Figure 1). The prepared MAP16 plate was then loaded on the Absolute Q. Figure 2 details the thermal cycling and reagent preparation protocols for RT-dPCR on the Absolute Q.
Figure 1. Workflow for the SARS-CoV-2 Wastewater Surveillance 4-plex assay
Assay Performance: Sensitivity
Accurate quantification of SARS-CoV-2 RNA is critical when comparing results between locations or performing longitudinal surveillance. We prepared and quantified a serial dilution of the commercially available SARS-CoV-2 control material (Exact Diagnostics, SKU: COV019), which contains both the N1 and N2 targets to demonstrate the quantification accuracy of the assay.
The serial dilutions consisted of three 4-fold dilutions to simulate a range of viral RNA concentrations. These RNA dilutions were spiked into a constant background of BCoV and PMMoV control materials at approximately 500 and 1000 copies per reaction respectively. Figure 3 summarizes the results of this N gene assay sensitivity dilution series.
Figure 3. Results from serial 4-fold dilutions of Exact Diagnostic SARS-CoV-2 control material into a background of BCoV and PMMoV control material at approximately 500 and 1000 copies per reaction respectively. A) The X-axis represents the targeted copies/reaction and Y-axis represents the observed concentrations of the N gene targets, N1 (purple) and N2 (orange). Two-dimensional partition scatters for the N1 (FAM) and N2 (HEX) targets across the RNA control material dilution using 2µL each of the (B) stock control material, (C) 4X dilution (D) 16X dilution and (E) 64X dilution.
The targeted concentrations of the N1 and N2 genes across the dilution series were 550, 137.5, 34.4 and 8.5 copies per reaction with the observed concentrations reported in Table 1. The concentrations are significantly correlated for both N1 and N2 with Pearson R2 values of 1.0 (p<0.001). Across each SARS-CoV-2 dilution point, both BCoV and PMMoV remained constant at 471.8 (±32.4) and 1194.5 (±77.3) copies per reaction respectively.
Table 1. Average observed N1 and N2 copies per reaction using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. At least 3 replicates were run for each condition. In addition, a water control was included. In one of three NTCs, a single false positive partition was identified for N1 and N2.
Assay Performance – Specificity
To demonstrate the high specificity and low cross reactivity of the 4-plex assay, individual materials and mixtures of the target control materials were tested against the 4-plex assay. The 4-target PCR control material demonstrated successful amplification in all four targets (Figure 4a) and the no-template added negative control showed zero false positive amplification events (Figure 4b). Subsequent tests of individual target control materials demonstrated high specificity for the intended target for each assay component (Figure 4c-f).
Figure 4. Partition amplification plots shown for each target N1 (blue channel, FAM), N2 (green channel, HEX), BCoV (yellow channel, TAMRA) and PMMoV (dark red channel, TYE665) by rows are: A) a PCR control 4-plex containing single stranded DNA (ssDNA) N1, N2, and PMMoV controls alongside inactivated BCoV RNA control material; B) water only no template control which yielded no false positives; C) N1 ssDNA control; D) N2 ssDNA control; E) PMMoV ssDNA control; F) BCoV RNA control.
Using Human Fecal and Process Controls for Data Comparisons
Viral load present in wastewater can be impacted by a variety of factors, including differences in preparation methods as well as the total amount of human fecal matter present. Understanding the amount of human fecal matter relative to the quantitative measurement of SARS-CoV-2 enables more accurate data interpretation for community level testing. The SARS-CoV-2 Wastewater Surveillance 4-plex assay incorporates two orthogonal controls in order to help interpret results. Quantification of those controls (BCoV, process control and human fecal control PMMoV) in the same reaction as the SARS-CoV-2 N gene targets enables more precise comparison between samples. The following dataset illustrates one option for how the BCoV and PMMoC controls can be used to interpret SARS-CoV-2 wastewater-based epidemiology data.
We tested four contrived samples using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. All four samples demonstrated similar overall N1 and N2 quantities (Figure 5). However, in the same four samples, the measured PMMoV concentration varies significantly (Figure 6a).
Figure 6. A) Initial concentration in copies per reaction of each contrived sample for the N1 (purple), N2 (orange) and PMMoV (red) targets. B) Chart reflects the concentration of N1 (purple) and N2 (orange) normalized to the median PMMoV value of 5357 copies of PMMoV. Using the SARS-CoV-2 Wastewater Surveillance 4-plex assay, three replicates were tested per contrived sample.
In order to make viral load comparisons, normalization to the human fecal marker PMMoV can be used to account for information such as the size of the community sampled. In this example, the ratio of N1 or N2 quantities to the concentration of PMMoV was normalized to the median PMMoV concentration measured in this dataset (5357 PMMoV copies) as detailed in the Methods section. While Samples B and C demonstrated very similar levels of N1 and N2, their PMMoV concentrations varied substantially (Figure 6a). After normalization to the PMMoV median, Sample B had the highest relative abundance of SARS-CoV-2 targets (Figure 6b).
Finally, the process control (BCoV) levels can be used to verify there are no large discrepancies in sample preparation efficiencies between the samples. As shown in Figure 7 the measured BCoV concentrations are comparable to one another across the four samples. Assuming an equivalent amount of control material was spiked into the native sample at the start of processing, this would indicate processing variations were minimal.
When comparing quantitative data, consistent measurement techniques that introduce as few variables as possible are essential. Digital PCR (dPCR), which provides absolute quantification of targets without standard curves, enables the quantification of all targets (including process and internal controls) to produce more accurate and more broadly comparable wastewater datasets – even when upstream preparation methods vary.
The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay was designed to detect and quantify SARS-CoV-2 viral targets while simultaneously providing normalization and recovery data in a single reaction. Using both human fecal markers and an orthogonal process control, sources of variability such as fecal load variation due to population levels or inconsistencies in sample processing can be accounted for. With high specificity, sensitivity, and best in class sample utilization of 95%, the Absolute Q provides more accurate and consistent quantification of these wastewater relevant targets.
Materials and Methods
Wet-lab validation of the assay has been performed using control materials. For assay specificity evaluation, single stranded DNA controls were used for the N1, N2 and PMMoV targets and Bovilis Coronavirus Calf Vaccine was used as the BCoV positive control. For assay sensitivity evaluation, the Exact Diagnostic SARS-CoV-2 RNA control material was used. For contrived samples, the N1, N2, BCoV, and PMMoV controls were mixed to create varying abundance ratios.
To normalize the concentration of N1 and N2 with respect to PMMoV for the contrived sample experiment, the following steps were performed. First, the concentration of each target (N1, N2, and PMMoV) were multiplied by the reaction volume to calculate the total copies per reaction. Subsequently, the concentration of N1 or N2 was divided by the concentration of PMMoV to obtain a ratio. Finally, the ratio was multiplied by the median PMMoV concentration for the dataset.