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

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

My research focuses on the molecular quantification of viral and bacterial pathogens in complex systems such as stormwater, wastewater, beach waters, drinking water, and shellfish.

How severely has your region been impacted by the pandemic?

The area around the Institute of Marine Sciences, which is at the coast, hasn’t been as severely hit as some of the more urban areas of North Carolina and the United States. We do have very limited ICU and emergency room capacity here though because it is a small town. As of October, those were 50% full, which is concerning.

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

We were on the pulse of the outbreak as it was evolving and were already talking about wastewater work as early as mid-February, but we never realized or thought that we would be working on the topic this close to home. Initial conversations were held among myself, my laboratory manager Denene Blackwood, and John Griffith at Southern California Coastal Water Research Project. I now have multiple projects funded.

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

The goal was to use our extensive knowledge around sample processing in difficult matrices, our knowledge of viral pathogens in wastewater, and droplet digital PCR to come up with protocols, and to share our expertise with other laboratories across the state who were not as well equipped.

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

We’ve been using both the CFX Connect Real-Time PCR Detection System and the QX200 Droplet Digital PCR System. We have actually created quite the business and equipment sale run for Bio-Rad during this process because of their excellent ddPCR technology!

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

We have been using droplet digital PCR technology for over seven years to quantify other viral RNA pathogens such as norovirus in complex water samples. We were having fantastic success with it, so we knew from the start that it was going to meet our needs for sensitivity and reproducibility that were higher than qPCR. In recent studies that’s exactly what we have found.

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

We have multiple projects, spanning rural systems (4,000–12,000 people), semi-urban systems (50,000–80,000 people), and urban (>80,000 people).

What were the key outcomes/findings from your project?

Although our project is not complete yet, here are a few salient points:

• Rural systems seem to have lower levels of community prevalence and SARS-CoV-2, which correlates with rural areas having reduced human interactions.
• We’ve been able to quantify the virus across a wide range of systems and begin to account for additional flows to systems caused by precipitation through the process of inflow and infiltration to wastewater infrastructure.
• We inadvertently studied wastewater effluent for a few months because a sampling treatment plant was sampling the wrong location. It was fortuitous because we demonstrated our approaches work well on effluent and that viral RNA was quantifiable in the effluent.
• We’ve built a pathogen tracking program across five university teams in North Carolina and have incorporated spatial modeling, disease mapping, epidemiological assessments, methodological optimizations, and dormitory sampling.
• We’ve established the capacity for advanced molecular quantification using Bio-Rad technology. We hope the state will move forward with developing a more proactive surveillance system.

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

We are asking ourselves that question — Are there epidemiological components to how people interact in rural systems that would help us further control COVID-19 spread in metropolitan areas that we are not currently taking advantage of?

We want to bring down the cost of doing these types of analyses, and we are working towards that. We have also developed new technologies that can be used for other applications, such as for antibiotic resistance genes, fecal human marker genes, and multiple viral pathogens in a single sample.

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