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

Chamteut Oh, Guanhua Xun, Stephan Thomas Lane, Vassily Andrew Petrov, Huimin Zhao, Thanh H. Nguyen

Research output: Contribution to journalArticlepeer-review

Abstract

The Argonaute protein from the archaeon Pyrococcus furiosus (PfAgo) is a DNA-guided nuclease that targets DNA with any sequence. We designed a virus detection assay in which the PfAgo enzyme cleaves the reporter probe, thus generating fluorescent signals when amplicons from a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay contain target sequences. We confirmed that the RT-LAMP-PfAgo assay for the SARS-CoV-2 Delta variant produced significantly higher fluorescent signals (p < 0.001) when a single nucleotide polymorphism (SNP), exclusive to the Delta variant, was present, compared to the samples without the SNP. Additionally, the duplex assay for Pepper mild mottle virus (PMMOV) and SARS-CoV-2 detection produced specific fluorescent signals (FAM or ROX) only when the corresponding sequences were present. Furthermore, the RT-LAMP-PfAgo assay does not require dilution to reduce the impact of environmental inhibitors. The limit of detection of the PMMOV assay, determined with 30 wastewater samples, was 28 gc/μL, with a 95 % confidence interval of [11,103]. Finally, using a point-of-use device, the RT-LAMP-PfAgo assay successfully detected PMMOV in wastewater samples. Based on our findings, we conclude that the RT-LAMP-PfAgo assay can be used as a portable, SNP-specific duplex assay, which will significantly improve virus surveillance in wastewater.

Original languageEnglish (US)
Article number168701
JournalScience of the Total Environment
Volume912
DOIs
StatePublished - Feb 20 2024

Keywords

  • Diagnostic assay
  • PfAgo
  • Portable
  • Single nucleotide polymorphism detection
  • Virus
  • Wastewater

ASJC Scopus subject areas

  • Pollution
  • Waste Management and Disposal
  • Environmental Engineering
  • Environmental Chemistry

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