Wastewater Monitoring with Digital PCR

Overview

As the COVID-19 pandemic continues to have a lasting global impact, effective methods for monitoring communities for early signs of disease spread are critical. Screening wastewater, or sewage, for the presence of SARS-CoV-2 viral RNA can be an effective monitoring method. Wastewater monitoring enables broad collection of surveillance data – even in areas that have limited access to healthcare or testing facilities. Because natural sewage is highly heterogeneous, a method capable of identifying very rare target RNA from a mixture of non-target nucleic acid molecules is required. While quantitative PCR (qPCR) is the current standard for COVID-19 clinical testing, the resulting data can be highly variable due to inadequate sample dilution or chemical contamination. 1,2 The Absolute Q Digital PCR (dPCR) platform is better suited for detecting SARS-CoV-2 targets from wastewater samples because it overcomes the problem of variability, reduces the impact of many PCR inhibitors, and eliminates the need for standard curves.  

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Testing Wastewater Samples for SARS-CoV-2 Using the Absolute Q

This study explores using the Absolute Q for screening of wastewater RNA samples with questionable or near limit qPCR results.

Fight Outbreaks with Early Warning

Viral RNA has been detected in environmental samples before symptoms appear.3 The Absolute Q facilitates accurate and sensitive detection of these rare molecules. The video below demonstrates reliable quantification of viral load without a standard curve for several sample types – including those collected from wastewater. In these studies, rare SARS-CoV-2 targets that were originally missed by qPCR were detected on the Absolute Q.

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References

  1. Sean C. Taylor et al. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports. 2017 May 25;7(1):2409.
  2. Dong, Lianhua, et al. “Highly Accurate and Sensitive Diagnostic Detection of SARS-CoV-2 by Digital PCR.” MedRxiv, Cold Spring Harbor Laboratory Press, 1 Jan. 2020.
  3. Peccia, Jordan, et al. “SARS-CoV-2 RNA Concentrations in Primary Municipal Sewage Sludge as a Leading Indicator of COVID-19 Outbreak Dynamics.” MedRxiv, Cold Spring Harbor Laboratory Press, 1 Jan. 2020, doi.org/10.1101/2020.05.19.20105999.

Application Highlights

Digital PCR Turnaround Time

1st demonstration of a true 1-step RT-PCR on a digital PCR platform in under 80 minutes

Digital PCR Workflow

Low limit of detection for viral load quantification demonstrated down to 5 target copies per reaction among high human genomic DNA background

Digital PCR Multiplexing

Potential to enable more efficient quantification of viral targets with up to 4-color multiplexing

The COVID-19 pandemic has drawn heightened concern, with over eleven million positive SARS-CoV-2 cases confirmed worldwide by July 2020.¹ RT-qPCR currently serves as the clinical standard for the diagnosis of COVID-19. However in a recent study, it was demonstrated that digital PCR (dPCR) provided better sensitivity for identifying patients who ultimately were diagnosed with COVID-19.²

Reverse transcription digital PCR (RT-dPCR) is a valuable technique which enables improved consistency and lower limits of detection compared to qPCR. Quantification of extremely rare viral RNA target material is also possible without the need for a comparative standard curve.

Dynamic range of SARS-CoV-2 quantification on Absolute Q using |Q| SARS-CoV-2 Triplex Probe Assay

Results of dynamic range testing for SARS-CoV-2 RNA targets among 50 nanograms (~15,000 copies) of human genomic DNA. Individual points are plotted.

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Combinat’s Absolute Q platform is for research use only and has not been validated for use in diagnostic procedures.

Additional Resources

SARS-CoV-2 Reference Material Quantification on the Absolute Q dPCR Platform

SARS-CoV-2 Reference Material Quantification on the Absolute Q dPCR Platform

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1-Step RT-dPCR in under 80 Minutes on the Combinati Absolute Q

1-Step RT-dPCR in under 80 Minutes on the Combinati Absolute Q

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References

  1. (WHO), World Health Organization. “Coronavirus Disease (COVID-19) – Situation Report 169.” Coronavirus Disease (COVID-2019) Situation Reports, 7 July 2020, 10:00 CEST, www.who.int/docs/default-source/coronaviruse/situation-reports/20200707-covid-19-sitrep-169.pdf?sfvrsn=c6c69c88_2.
  2. Dong L, Zhou J, Niu C, et al. Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR. medRxiv. March 2020:2020.03.14.20036129. doi:10.1101/2020.03.14.20036129