Portable, single nucleotide polymorphism-specific duplex assay for virus surveillance in wastewater

Area of Science:

• Molecular Biology
• Biotechnology
• Environmental Science

Background:

• The Argonaute protein from Pyrococcus furiosus (PfAgo) is a versatile DNA-guided nuclease.
• Existing virus detection methods can be limited by inhibitors in environmental samples like wastewater.

Purpose of the Study:

• To develop and validate a novel virus detection assay utilizing the PfAgo enzyme combined with reverse transcriptase loop-mediated isothermal amplification (RT-LAMP).
• To assess the assay's specificity for single nucleotide polymorphisms (SNPs) and its utility in detecting viruses in wastewater samples.

Main Methods:

• A dual assay combining RT-LAMP with PfAgo nuclease activity was designed to detect viral amplicons.
• The assay's performance was evaluated for SARS-CoV-2 Delta variant detection using SNP-specific probes.
• The assay's ability to detect Pepper mild mottle virus (PMMOV) in wastewater was determined using a point-of-use device.

Main Results:

• The RT-LAMP-PfAgo assay showed significantly higher fluorescent signals for the SARS-CoV-2 Delta variant when a specific SNP was present (p < 0.001).
• The duplex assay demonstrated specific fluorescent signals for both PMMOV and SARS-CoV-2.
• The assay exhibited robustness against environmental inhibitors without requiring sample dilution, with a limit of detection of 28 gc/μL for PMMOV in wastewater.

Conclusions:

• The RT-LAMP-PfAgo assay is a portable, SNP-specific, and duplex detection method.
• This assay offers improved virus surveillance capabilities, particularly in wastewater monitoring.