Improving Consistency in Wastewater Testing

Wastewater based epidemiology, or WBE, is a method of testing wastewater for the genetic material of pathogens which can be used as a biomarker for disease spread. Wastewater testing has already been proven to be effective in monitoring for rare cases of disease.(1) However, results from current WBE studies are difficult to compare at a larger scale due to variations in sampling, processing and measurement techniques. (2)

Consistency is a challenge for wastewater testing

Wastewater Sample Processing
Overview of wastewater sample processing and testing for SARS-CoV-2. Figure from CDC COVID-19 Wastewater Surveillance Testing Methods.

A series of preparation steps are required to prepare raw sewage for molecular quantification. Typically, a sample is first collected and  concentrated, then the nucleic acids are purified, and measured (Figure 1).

Depending on the method of sample collection (e.g. grab versus composite), the exact workflow and steps used can vary significantly between sites and groups. Since there are many methods of concentration, extraction, and measurement, each step adds new variables which complicate the ability to compare results broadly. 

Controls are useful tools for data normalization

Controls can provide tangible metrics for normalization of downstream results and help limit the impact of sample preparation variability. Process controls, such as Bovine Coronavirus (BCoV) or Human Coronavirus 229E, are viruses similar to SARS-CoV-2 that can be inactivated and added to wastewater samples before any processing takes place. These controls can then be measured in a similar manner as SARS-CoV-2 targets. This type of control provides insights into the recovery efficiency of upstream processing steps.  

In wastewater epidemiology studies, the amount of feces (fecal load) per sample is important to consider when evaluating the abundance of a particular target of interest. Quantifying a target that is commonly shed in feces (Ex: Pepper Mild Mottle virus (PMMoV) or crAssphage) across all samples tested can help to normalize data even when the fecal load varies between those samples. The ratio of pathogenic targets to the total amount of a normal fecal control target can be used to compare datasets from samples with variability in upstream collection methods. For example, these ratios can be used to compare samples collected from sewersheds with contributions from different population sizes or can be used to compare samples using a variety of upstream concentration methods.

Quantification with RT-qPCR adds more variables

After processing wastewater samples, the SARS-CoV-2 targets must be measured. To date, these measurements are performed using many different assays on many different instruments. (3)  Furthermore, RT-qPCR – the most commonly used method – reports a threshold cycle and 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.

With digital PCR, absolute quantification is possible in wastewater testing

When comparing quantitative wastewater epidemiology data, it is essential that your measurement technique introduces as few variables as possible. Digital PCR (dPCR) divides the PCR reaction mixture into a very large number of separate small volume reactions, such that zero or one target molecule is present in any individual reaction. At the end of cycling, each partition is interrogated for the presence of a signal. Then, positive partitions can be counted, providing an absolute quantification of the targets present in a given sample – with no need or reliance on a standard curve. 

Without standard curves, the measured quantities of both the process and internal controls can be used to more accurately compare wastewater datasets – even when upstream preparation methods vary. The Absolute Q utilizes a micro-molded plastic plate for digital PCR using microfluidic array partitioning (MAP) technology to create 20,000 precise partitions. Using this platform, more than 95% of the loaded PCR mix is analyzed across 20,480 fixed partitions per reaction. This exceptionally high total analyzed sample volume paired with a consistent partitioning strategy helps make rare target detection more consistent and accurate. 

Read more about our unique microfluidic array partitioning technology in our blogpost

Interested in learning more about wastewater testing with digital PCR? Speak with an application scientist by sending us an email at


  1. Asghar H, et al., Environmental surveillance for polioviruses in the global polio eradication initiative. J Infect Dis. 2014;210:S294–S303.
  2. Michael-Kordatou IM, et al., Sewage-analysis as a tool for the COVID-19 pandemic response and management: the urgent need for optimised protocols for SARS-CoV-2 detection and quantification. J Environ Chem Eng. 2020;8:104306.
  3. Hamouda M, el al., Wastewater surveillance for SARS-CoV-2: Lessons learnt from recent studies to define future applications. Sci Total Environ. 2020;Nov 7:143493.

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