Routine wastewater monitoring: why is digital PCR the best approach?

As the world adapts to living with COVID-19, the scientific community has expanded from diagnostics development to monitoring efforts, which will help ease the transition to a post-COVID era. It has become clear that routine wastewater monitoring can help determine the appropriate response measures at all levels, including potentially determining the geographic priority of vaccine deployments in order to thwart emerging outbreaks. In addition, compared to mass individual testing, routine wastewater monitoring is a more cost-effective way to monitor transmission dynamics because sewage influent effectively pools samples from an entire community.(1)

Routine wastewater testing requires accuracy, confidence and simplicity

For any application seeking to routinely monitor a specific target, the ideal sample types and testing workflows generally have the following similar characteristics: 

  1. Relatively simple collection logistics
  2. Simple testing workflow
  3. Consistent and easy to interpret test results

Wastewater is easy to collect, process, and analyze

Unlike typical samples collected from patients such as saliva or nasopharyngeal swabs, the collection of wastewater samples can be done with fewer logistical steps and can reach a broader number of individuals. With appropriate controls, variables like nucleic acid processing or population size can be normalized across different wastewater samples. This would enable more accurate longitudinal monitoring and allow results to be compared across sites. For more information on how digital PCR results paired with effective controls can help normalize data collected from routine wastewater testing efforts, read our recent blogpost Improving Consistency in Wastewater Testing.   

Digital PCR is superior to qPCR in terms of consistency and accuracy for routine wastewater surveillance

Although qPCR is the current standard for clinical COVID-19 testing and other monitoring efforts, digital PCR (dPCR) has been demonstrated to be more sensitive and accurate for low concentration or rare target detection. (2) This is an important consideration for longitudinal or routine wastewater surveillance efforts whose goals are to detect changes over time. Digital PCR (dPCR) uses a count of positive signals at the end of the reaction rather than relying on assay kinetics and standard curves to quantify targets.

Fundamentally, absolute quantification of the total number of targets in a given sample is more straightforward than reporting a cycle threshold value (CT). Furthermore, consistency and reproducibility across measurements can be improved since there is no reliance on the consistency of reference materials between experiments, runs, or even labs.

Multiplexing of targets enables for flexible interrogation of more markers in a single reaction

Finally, for any application requiring repeat or routine measurements, maximizing the amount of data generated saves time, cost, and effort. To aid in the global response to routine wastewater surveillance efforts, Combinati developed a 4-target wastewater specific assay for monitoring SARS-CoV-2. Using this complete kit, extracted RNA from wastewater can be simply and efficiently tested for the presence of 2 SARS-CoV-2 related targets as well as a human fecal normalization control (PMMoV) and a spike-in sample processing control (BCoV) in a single digital PCR reaction that takes under 2 hours to complete from start to finish.

Digital PCR historically has been a labor intensive process, requiring multiple instruments and technical training to maximize the quality of data generated. The Combinati Absolute Q® was specifically designed to be as similar to traditional qPCR as possible in terms of workflow to create a hands-off digital PCR experience. Read more about the Absolute Q and its workflow here.

Read more about our SARS-CoV-2 Wastewater Surveillance Kit here or view our resources page to take a look at recent application and technical notes utilizing the assay.

References

  1. Hart OE, et al., Computational analysis of SARS-CoV-2/COVID-19 surveillance by wastewater-based epidemiology locally and globally: feasibility, economy, opportunities and challenges. Sci Total Environ. 2020;730:138875.
  2. Lianhua Dong, et al., Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR.Talanta.Volume 224, 2021, 121726, ISSN 0039-9140, https://doi.org/10.1016/j.talanta.2020.121726.

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