SARS-CoV-2 Detection in Wastewater using Droplet Digital PCR by Walter Betancourt, Ph.D.

Before the COVID-19 pandemic, what was your primary role or field of research?

My work is focused on environmental virology — virus reduction through wastewater treatment, water processing for potable and non-potable reuse applications, and examining the presence of viruses in surface waters and environmental surfaces as part of the exposure risk assessment framework to determine the probability of infections in humans.

How severely has your region been impacted by the pandemic?

The infection rates seem to be declining when you look at the Arizona Department of Health website. However, the concentration of SARS-CoV-2 in wastewater has remained the same — around 106 GC/L.

How did this wastewater project come about? Who seeded the idea and when?

The University of Arizona put together a task force that is made up of epidemiologists, administrators, faculty members, and even students. Their goal was to “Test, Trace, and Treat” the coronavirus — those three T’s are the key. Because our center was already focused on wastewater epidemiology, we realized that since the virus is excreted in feces, we can therefore track the virus in sewage. From there, we can then take action based on the concentration. So, we offered our expertise — biology, epidemiology, and engineering — to do surveillance of the virus.

What was the primary goal? What did you hope to determine or achieve?

The major concern was ensuring a safe environment for the students, staff, and faculty during the pandemic. We developed a program for the fall semester that was focused on testing individual dormitories for SARS-CoV-2.

What Bio-Rad ddPCR platforms did you use to perform your research?

We are using the QX200 Bio-Rad ddPCR System, and all reagents including enzymes (master mix) and primers/probes are based on the SARS-CoV-2 ddPCR assay for the 2019-nCoV CDC Triplex assay, also from Bio-Rad.

Why did you choose to use ddPCR technology to measure SARS-CoV-2?

We have seen that droplet digital PCR technology is far more sensitive for detection of the virus. We compared several samples and found that ddPCR assays could detect virus even when RT-qPCR gave a negative result. So, from then on we started applying droplet digital PCR technology. RT-qPCR allows you to detect the virus when it's present in high concentrations, but when the concentration is not yet at those levels ddPCR testing offers an advantage. Wastewater also has a lot of inhibitors, but we can be confident in our detection with droplet digital PCR technology — we know each test will be a true positive or negative. Another advantage of the droplet digital PCR instruments is the high throughput. Since we can use the machine in a 96-well plate format, we can test for different reactions for the same target. It’s also obviously more accurate because you are expressing an absolute quantification of virus in your sample, which is correlated with the incidence of the disease in the population. So that's important. Recently, we have also started testing for specific SARS-CoV-2 variants, and ddPCR technology lets us do that.

What was the scope of the project — for example, how many people were in the wastewater catchment area?

The facility where we sample from since March 2020 serves a population of ~500,000. However, we were able to distinguish between individual university dorms with our sampling strategy.

What were the key outcomes/findings from your project?

We were able to define different levels of concern based on concentrations of SARS-CoV-2 in the wastewater, which was critical for taking action. Even the first day the university reopened, and students returned to campus, we tested one of the dormitories and found the virus in one of the samples at a level of 2x105 GC/L. This elevated the level of concern for that particular dorm, so the university individually tested all three hundred students in that dormitory. Three of the students tested positive — even though they were asymptomatic at the time — and so we isolated them. This is an example of how the wastewater testing strategy is successful, because we are able to minimize exposure of other students to infected individuals and prevent outbreaks on campus.

How do you think wastewater testing using ddPCR might help stop the spread of SARS-CoV-2 in different communities?

The key takeaway is that wastewater testing using ddPCR allows researchers to gain a true reflection of what is happening in a community with regards to the spread of a virus like SARS-CoV-2. If we look at plots that show the viral genome copies per liter in wastewater compared to the incidence of disease within that community, we see a very strong relationship between those two variables. Wastewater-based epidemiology obviously has to be done in conjunction with clinical testing, but it is an important part of pandemic strategy. There have been many groups in different countries doing this, but for us, having preexisting expertise in wastewater testing and knowing methods for concentrating viruses in wastewater was key. These skills allowed us to establish a robust water-based epidemiology program that can actually provide specific measurements for a successful surveillance program that can help minimize the spread of the disease in the population.

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