Highly Multiplexed Reverse-Transcription Loop-Mediated Isothermal Amplification and Nanopore Sequencing (LAMPore) for Wastewater-Based Surveillance

• DOI: 10.1021/acsestwater.3c00690
• Publication Date: 2024-02-27T02:02:05Z
• Authors: Seju Kang; Petra Choi; Ayella Maile-Mosk...; Connor L. Brown; Raul A. Gonzalez; Amy Pruden; Peter J. Vikesland
• Keywords: Amplicon; Amplicon sequencing; Nanopore; Primer (cosmetics)

Wastewater-based surveillance (WBS) has gained attention as a strategy to monitor and provide an early warning for disease outbreaks. Here, we applied isothermal gene amplification technique, reverse-transcription loop-mediated (RT-LAMP), coupled with nanopore sequencing (LAMPore) means detect SARS-CoV-2. Specifically, combined barcoding using both RT-LAMP primer the rapid kit achieve highly multiplexed detection of SARS-CoV-2 in wastewater. targeting N region was conducted on 96 reactions including wastewater RNA extracts positive no-target controls. The resulting amplicons were pooled subjected sequencing, followed by demultiplexing based barcodes that differentiate source each amplicon derived from products. criteria developed establish whether detected LAMPore assay indicated high consistency polymerase chain reaction-based gene, sensitivity 89% specificity 83%. We further profiled sequence variations amplicons, revealing number mutations sample collected after viral variants had emerged. results demonstrate potential facilitate WBS emergence