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

Abstract

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

Figure 1

Workflow of the LAMPore assay for detection of SARS-CoV-2 in wastewater. (1) Wastewater sample collection and RNA extraction. (2) Sequential barcoding is conducted on 96 samples by using RT-LAMP forward inner primer (FIP) (8 barcodes in rows) and Rapid Barcoding Kit for nanopore sequencing (12 barcodes in columns) pooled together for analysis. (3) Library loading for nanopore sequencing and identification of SARS-CoV-2 reads by sequence analysis.

Figure 2

(A) Overall results of SARS-CoV-2 positive reads that correspond to 96 RT-LAMP products. (B and C) F1 score curve and receiver operating characteristic (ROC) curve with AUC value for the LAMPore assay. (D) IGV read alignment against SARS-CoV-2 reference genome for LAMPore assay on wastewater samples collected from pre- and postvariant emergence periods. Black lines indicate mismatched positions against the reference. Histogram on top of each compilation of aligned reads indicates normalized coverage of the reads with positions. Red (T), green (A), blue (C), and brown (G) colored bars indicate the base substitutions against the reference.