Novel Multiplexed Amplicon-Based Sequencing to Quantify SARS-CoV-2 RNA from Wastewater

Area of Science:

• Environmental microbiology
• Molecular epidemiology
• Public health surveillance

Background:

• Wastewater-based epidemiology (WBE) is crucial for global COVID-19 pandemic response.
• Accurate, sensitive, and high-throughput detection of SARS-CoV-2 in wastewater is essential for effective WBE.
• Existing methods like RT-qPCR have limitations in sensitivity and phylogenetic analysis.

Purpose of the Study:

• To introduce the novel ATOPlex platform for sensitive SARS-CoV-2 detection in wastewater.
• To evaluate the ATOPlex platform's performance compared to RT-qPCR.
• To assess the utility of ATOPlex for phylogenetic analysis of SARS-CoV-2 in wastewater.

Main Methods:

• Development and application of the ATOPlex platform, a multiplexed amplicon-based sequencing approach.
• Comparison of SARS-CoV-2 RNA quantification limits between ATOPlex and RT-qPCR.
• Analysis of viral RNA distribution in solid and liquid wastewater fractions.

Main Results:

• The ATOPlex platform quantifies SARS-CoV-2 RNA at concentrations 10 times lower than RT-qPCR detection limits.
• Robust phylogenetic placement is achievable at viral concentrations 4 times lower than RT-qPCR detection limits.
• A significant portion of viral RNA is found in the solid fraction of wastewater.

Conclusions:

• The ATOPlex platform offers a highly sensitive and phylogenetically informative workflow for WBE.
• This advanced analytical method significantly enhances the capabilities of wastewater-based surveillance for infectious diseases.
• Effective viral RNA extraction from both solid and liquid wastewater fractions is critical for comprehensive WBE.