Toward efficient surveillance: a proof-of-concept comparison of methods for detecting DNA and RNA viruses in wastewater

Wastewater-based epidemiology (WBE) represents a promising method for early detection and ongoing monitoring of various pathogens. This epidemiological approach has recently attracted significant research interest. During the SARS-CoV-2 pandemic, WBE was estimated as a complementary surveillance tool for mapping the spread of the virus within the population. Although this method has great potential for pathogen monitoring at both local and national levels, there are some limitations, notably the presence of high concentrations of inhibitors that contaminate extracted nucleic acids from wastewater, which can lead to false negative results. This proof-of-concept study focuses on detecting RNA and DNA viruses, specifically SARS-CoV-2 and adenovirus, in wastewater by droplet digital PCR (ddPCR) and compares the results with those obtained with the routinely used RT-qPCR and qPCR methods. The results of our proof-of-concept study indicate that WBE represents a promising approach for monitoring pathogens such as SARS-CoV-2 and adenovirus; however, challenges such as contaminants in wastewater need to be addressed to increase detection accuracy. Due to this limitation, novel technologies such as ddPCR offer new opportunities to improve epidemiological monitoring of various infectious agents in the population.

Pandemics caused by viruses and other pathogens have a significant impact on society worldwide. The latest example of such a phenomenon is the recent global COVID-19 pandemic caused by the SARS-CoV-2 virus (1–4). Nevertheless, the occurrence of the next viral outbreaks, both local and global, is extremely difficult to predict (5, 6). The main limitation of the classical epidemiology approach is based on the fact that most input data are obtained via testing samples from patients exhibiting clinical symptoms. This is not optimal for detecting viral epidemics in their early stages, especially in the case of diseases with longer incubation times or those with an inapparent course, during which an asymptomatic yet infectious person can spread the disease before the onset of clinical symptoms and subsequent diagnosis. This was also the main reason why individual clinical testing failed to control the SARS-CoV-2 epidemic in its early stages and allowed it to develop into a global pandemic (7, 8). An alternative means for sampling the population needs to be considered to overcome such a drawback.

Municipal wastewater represents an important reservoir of biological material that reflects comprehensive health information about the entire community and is an invaluable source of information on the state of public health. Unfiltered wastewater is a universal sample, as it consists of urban and industrial wastewater, as well as rainwater. This water contains not only various human secretions such as urine, feces, vomit, and of course water from washing and dental hygiene, but also various pathogens and even human cells. Wastewater, therefore, represents a significant source of various biomarkers that can be used in public health monitoring. Indeed, a wastewater-based epidemiology (WBE) has been proven to be a valuable and effective alternative to individual testing during the COVID-19 pandemic (9) as it aims to determine exposure to pathogens at the population level (10).

However, in addition to matters of biological origin, wastewater also contains a large number of chemical inhibitors that interfere with diagnostic approaches based on PCR reactions (11). The concentration and diversity of these inhibitors are influenced by several factors, including population size, the presence of surrounding industry and agriculture, as well as the impact of climate. These aspects can affect the quality of data produced by WBE and potentially lead to erroneous epidemiological conclusions (7). Therefore, the need for reliable isolation and especially robust detection protocols is obvious. Digital droplet PCR (ddPCR) has proven to be a tool meeting such criteria (12). It is a relatively novel variation of classical PCR that allows absolute quantification of target nucleic acids. The principle of this method lies in its ability to divide samples into small bioreactors (so-called droplets) in which separate PCR reactions occur (13). ddPCR technology exhibits increased tolerance to inhibitory substances due to the distribution and separation of individual micro-reactions, which mitigates the influence of inhibitors on PCR amplification (14). This results in maintaining a recognizable positive signal even in the presence of slight inhibition that may be present in the droplet. Given its robustness and specificity, this method represents an optimal approach for detecting viral particles in wastewater and thus optimizing the monitoring of the epidemiological situation at both local and national levels. Moreover, the simultaneous isolation of RNA and DNA from wastewater in a single step offers significant advantages in terms of efficiency, cost-effectiveness, and comprehensiveness for public health surveillance. This approach enables the concurrent detection of diverse types of pathogens—RNA-based (e.g., viruses) as well as DNA-based (e.g., viruses, bacteria, and archaea)—from a single, limited-volume sample (15). As a reference methodology, we used standard qPCR or RT-qPCR, respectively.

While the COVID-19 pandemic has shown that the WBE approach is reliable and useful for its epidemiological management, surveillance of other pathogens shed into wastewaters might be beneficial as well (16). In this proof-of-concept study, we aimed to compare and optimize the protocols for the identification of both DNA and RNA viral pathogens.

Sample processing

Samples of wastewater for SARS-CoV-2 analysis were obtained from the sewage plant Vrútky, which collects wastewater from the majority of the Turiec region with approximately 110,000 inhabitants. Sewage water was processed according to protocol steps, which include transporting samples to the laboratory on ice and keeping them at 4 °C, and subsequently centrifuging them at 4,000 g for 30 minutes at 4 °C to remove debris. Twelve independent wastewater samples were collected for this study to support method optimization and further detection. One liter of wastewater was collected per sample. To detect adenoviral DNA in sewage, we processed wastewater obtained from the Žilina Public Health Office, which collects wastewater from the Horne Považie region (approximately 260,000 inhabitants). Three independent wastewater samples were collected for this study to support method optimization and subsequent detection. One liter of wastewater was collected per sample. The protocol steps were the same as the SARS-CoV-2 transport and processing workflow.

Isolation of nucleic acids

Viral RNA isolation

A commercially available Zymo Environ (Zymo Research, Irvine, CA, USA) kit was used to extract RNA from wastewater samples, which enables virus concentration, sample homogenization, and RNA purification in a single protocol. 5 mL of wastewater was used for extraction. Subsequently, 70 μl of Water Concentrating Buffer was then added to the tube. The remaining steps were carried out according to the manufacturer’s recommendations.

Viral DNA isolation

A modified Zymo Environ kit (Zymo Research) was used to extract DNA from wastewater samples, allowing virus concentration, sample homogenization, and RNA and DNA purification within a single protocol. Briefly, 1 mL of wastewater was used for extraction. 70 μl of Water Concentrating Buffer was then added to the tube. The next steps involved incubation for 10 min at room temperature, followed by centrifugation at 3,000g for 15 min. The supernatant was then removed, leaving 500 μl of liquid with the pellet. Sample homogenization consisted of adding 750 μl DNA/RNA Shield into a tube with liquid fraction with pellet and supernatant, followed by transport of the solution into a ZR BashingBead Lysis Tube and subsequent vortexing for 5 min, centrifugation at 12,000 g for 2 min, and transferring 400 μl of solution into an RNase-free tube. The final purification of DNA consisted of adding 800 μl DNA/RNA Buffer into 400 μl of solution in an RNase-free tube, followed by vortexing and transferring the solution into the Zymo-Spin IIC-XLR Column and subsequent centrifugation at 10,000 g for 2 min. In the following steps, we performed two rounds of washing with 500 μl Viral Wash Buffer and centrifugation at 10,000 g for 30 s. Then we added 500 μl of ethanol (95 – 100%) followed by centrifugation at 10,000 g for 60 s. The last step consisted of transporting the spin column to the new 1.5 mL tube, adding 50 μl of DNase/RNase-free water on the spin column, and final centrifugation at 10,000 g for 30 s.

SARS-CoV-2 RNA detection

As a surrogate virus representing pathogens with RNA genomes, we used SARS-CoV-2. At the time of conducting this study, SARS-CoV-2 was still abundant in sewer waters.

Detection using RT-ddPCR

Reverse transcription digital droplet PCR (RT-ddPCR) was performed in a 20 μl reaction volume consisting of 17 μl of mastermix and 3 μl of sample. The mastermix contained 5 μl of supermix, 2 μl of reverse transcriptase (RT), and 1 μl of 300 mM dithiothreitol (DTT) solution, all included in the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad Laboratories, Hercules, California, USA). Primers and probes (Table 1) (Generi Biotech, Hradec Králové, Czech Republic) were added at a final concentration of 500 and 250 nM, respectively. Droplets were generated by an automated droplet generator (Bio-Rad Laboratories) according to the manufacturer’s instructions. PCR was performed using a T100 thermal cycler (Bio-Rad Laboratories) with the following cycling conditions: enzyme activation at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s, followed by annealing/extension at 54 °C for 1 min, and droplet stabilization at 98 °C for 10 min. Samples were then analyzed using a QX200 Droplet Reader (Bio-Rad Laboratories), which allows detection of both fluorescence signals for FAM and HEX in one reaction.

Detection using RT-qPCR

Detection of SARS-CoV-2 RNA was performed using an IVD-certified gb OneStep RT-qPCR Kit, SARS-CoV-2 Multiplex (Generi Biotech). This kit detects viral genes E and RdRp. To eliminate false negative results, we used external positive controls (EPC) added to the reaction. We performed the PCR reaction step-by-step according to the manufacturer´s recommendation. The PCR reaction was carried out in a total volume of 20 μl, which included 10 μl of Master Mix OneStep Multi, 5 μl of the multiplexed Assay CoV-2 E-RdRp, 0.25 μl of EPC Template RNA, and 5 μl of extracted RNA. The PCR conditions were established as follows: reverse transcription was conducted at 42 °C for 30 min, followed by an initial denaturation step at 95 °C for 3 min. This was followed by 50 cycles comprising two phases: denaturation at 95 °C for 10 s and annealing plus elongation at 60 °C for 30 s. Final analysis was performed using the RT-qPCR system qTOWER (Analytic Jena, Jena, Germany).

Detection of adenoviral DNA

We used adenoviruses to represent DNA viruses, as they are ubiquitous, present in relatively high numbers, and detected throughout all seasons.

Detection using ddPCR

RT-ddPCR was performed in a 20 μl reaction volume, consisting of 10 μl mastermix and 10 μl sample. The mastermix contained 5 μl of supermix, 2 μl of reverse transcriptase, and 1 μl of 300 mM dithiothreitol (DTT) solution, all included in the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad Laboratories), primers and probes (Table 1) (Generi Biotech) at a final concentration of 500 and 250 nM, respectively. Droplets were generated by an automatic droplet generator (Bio-Rad Laboratories) according to the manufacturer’s instructions. PCR was performed using a T100 cycler (Bio-Rad Laboratories) with the following cycling conditions: enzyme activation at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s, followed by annealing/extension at 55 °C for 1 min, and droplet stabilization at 98 °C for 10 min. Samples were then analyzed using a QX200 Droplet Reader (Bio-Rad Laboratories), which allows detection of both fluorescence signals for FAM and HEX in one reaction.

Detection using qPCR

Adenoviral DNA was detected using the same primer and probe set utilized in ddPCR analysis (Table 1). For the qPCR analysis, we used a CFX96 Touch™ real-time PCR detection system (Bio-Rad Laboratories). The PCR reaction was performed in a total volume of 10 μl, which included 5 μl of TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Waltham, MA, USA), 0.125 μl of probe (250 nM), 0.225 μl of each primer (500 nM both), and 4.4 μl of extracted DNA. The PCR conditions were established as follows. A holding step was conducted at 50 °C for 2 min, followed by an enzyme activation at 95 °C for 20 s. This was followed by 40 cycles consisting of two phases: denaturation at 94 °C for 3 s and annealing and extension at 55 °C for 30 s.

Analysis of the presence of SARS-CoV-2 in wastewater using ddPCR

Using a commercially available kit to isolate viral RNA from wastewater and subsequent analysis, we detected the presence of viral RNA for the RdRp gene (Figure 1A, blue dots) and the E gene (Figure 1B, blue dots) in the wastewater sample. The presence of GAPDH RNA (Figures 1A, B, green dots) in the analyzed sample shows evidence of the successful PCR reaction.

Analysis of the presence of SARS-CoV-2 in wastewater using RT-qPCR

Using a commercially available IVD-certified SARS-CoV-2 Multiplex kit (Generi Biotech) for the RT-qPCR analysis, we did not detect any amplification of viral RdRp or E gene targets. Amplification of the EPC yielded a Ct value of 32.64, confirming successful PCR reaction. The absence of detectable amplification curves for the RdRp and E genes was most likely caused by the low amount of viral RNA in the sample, with a possible contribution from PCR inhibitors that interfered with efficient target detection.

Analysis of the presence of adenoviral DNA in wastewater using ddPCR

Using a modified kit for the isolation of viral DNA/RNA from wastewater and subsequent analysis, we detected the presence of viral DNA specific for adenoviruses (Figure 2, top) in the wastewater sample. Evidence of the successful PCR reaction is documented by the presence of mitochondrial DNA (mtDNA) (Figure 2, bottom) in the analyzed sample.

Analysis of the presence of adenovirus in wastewater using qPCR

To detect adenoviral DNA in sewage samples, we conducted qPCR analysis using the same primers and probe set that we utilized in ddPCR analysis. No amplification curve was detected for adenoviral DNA, indicating either the absence of adenoviral DNA or significant inhibition of the PCR reaction due to contaminants in the wastewater sample. The presence of an amplification curve for the control mtDNA (Ct value 26.49) confirmed a successful PCR reaction.

The following table briefly summarizes the results obtained from all the detection methods used in our study (Table 2).

Wastewater monitoring involves collecting samples from municipal or institutional sewage systems (17). For community-level surveillance, sewage systems are a universal tool that collects human waste at centralized sites. However, sampling can also be conducted at much more localized scales, including in areas lacking a centralized wastewater collection and treatment system. Sampling from centralized sites can provide valuable insights into the presence of pathogenic viruses, bacteria, and protozoa circulating within the community. Pathogens that are stable in wastewater or continuously excreted in feces or urine represent particularly suitable candidates for monitoring the epidemiological situation at both local and national levels (18).

Although wastewater monitoring of such pathogens is valuable, several factors currently limit the broader application of this approach (19). As demonstrated during the SARS-CoV-2 pandemic, the inherent composition of water and the significant dilution of the viral particles in wastewater posed a considerable challenge in interpreting epidemiological data. Pathogen detection in wastewater currently relies on nucleic acid-based methods, including quantitative polymerase chain reaction (qPCR) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). However, the effectiveness of these detection methods is notably reduced by substances that inhibit the PCR reaction (13, 19). The possible solution is to introduce a method that combines high sensitivity and robustness while minimizing the effects of inhibitors. One promising alternative offers the application of ddPCR technology. In our work, we designed and optimized assays for detecting RNA (SARS-CoV-2) and DNA (Adenovirus) viruses in wastewater using the ddPCR. We then compared the results with data obtained from routinely used RT-qPCR and qPCR. The choice of these two virus types was based on their higher prevalence in the population and their potential to cause severe clinical manifestations in patients (20, 21).

We used a commercially available Zymo Environ isolation kit for RNA extraction from wastewater and subsequently optimized the protocol to enable the isolation of both RNA and DNA viruses. We employed the RT-ddPCR method, allowing the simultaneous detection of DNA and RNA in a single eluate. The reaction mix contained reverse transcriptase to convert RNA to cDNA while leaving DNA intact.

Wastewater often contains high levels of PCR inhibitors, such as humic substances, polysaccharides, lipids, metals, and proteins. These substances interfere with DNA polymerase activity, bind to DNA, or chelate essential ions, resulting in unsuccessful or less efficient PCR reactions and making microbial detection more difficult (11, 22, 23). To evaluate the presence of PCR inhibitors and overall assay performance in wastewater samples, GAPDH/EPC was used for SARS-CoV-2 analysis, while mtDNA served as a performance indicator for Adenovirus detection. Both targets are consistently present in wastewater, as they originate from stool and other human biological fluids.

The ddPCR technology represents a highly sensitive approach for detecting pathogens such as SARS-CoV-2, as documented in our previous studies (12, 24). In this study, we demonstrated ddPCR’s ability to deal with PCR inhibition in wastewater analysis, which often compromises the performance of routinely used qPCR. Using the optimized extraction protocol and ddPCR assays, we successfully detected both SARS-CoV-2 and adenoviruses in wastewater samples. The presence of positive signals for the controls (EPC for RNA detection and mtDNA for DNA detection) indicated the robustness and reliability of the method. On the contrary, qPCR and RT-qPCR showed limitations in the detection of limited amounts of viral nucleic acids, despite the successful amplification of controls (CT value of 32.64 for EPC and 26.49 for mtDNA). These findings are consistent with our previous study on SARS-CoV-2, which demonstrated that the sensitivity of samples with low detection limits can be significantly enhanced by selecting an appropriate combination of enrichment, isolation, and detection methods (12, 24). In another study, Wang et al. showed high sensitivity of ddPCR for SARS-CoV-2 variant detection in clinical and wastewater samples (25). In Adenovirus detection, ddPCR was successfully applied to monitor viral load in children following hematopoietic stem cell transplantation, whereas qPCR showed lower sensitivity and reproducibility than ddPCR (26). In another epidemiological study, Veneri et al. used digital PCR (dPCR) combined with molecular typing to investigate the prevalence and diversity of human adenovirus in wastewater (27).

Importantly, our results highlight the broad applicability of ddPCR technology in monitoring pathogens regardless of nucleic acid type. It should be noted, however, that the WBE approach is strictly limited to pathogens shed in body fluids and stools (28). Despite this limitation, this approach allows for a wide range of applications in monitoring not only local outbreaks, such as hepatitis C, tuberculosis, or influenza, but also global pandemics, as demonstrated during the SARS-CoV-2 pandemic.