RT qLAMP--Direct Detection of SARS-CoV-2 in Raw Sewage

The project purpose was to examine ability of a loop-mediated isothermal amplification (LAMP) assay to quantify SARS-CoV-2 in raw sewage, directly, using no preliminary sample processing for virus concentration and RNA extraction. An objective was to take advantage of extensive recently published work to facilitate process development, principally primer selection, and to use readily available off the shelf materials with conventional lab procedure and equipment. Well-developed and referenced primers for ORF1a, E, and N-gene targets were selected and applied, using commercially available synthetic RNA standards, and raw sewage from a local wastewater agency serving 650,000. County health department monitoring provided current COVID-19 data. Testing defined performance characteristics for each primer set, with significant differences between them. Specific amplification of SARS-CoV-2 RNA was observed using each of the primer sets, with E-gene and N-gene primers most effective. Positive analysis results from all raw sewage samples corresponded to calculated concentrations of virus in 5-10 μL raw sewage aliquots for 25 μL reactions. Results show that even at low reported case rates e.g. 1-10/100,000, SARS-CoV-2 is present in raw sewage at > 1-5/ μL, permitting direct LAMP-based detection. Use of RT qLAMP will facilitate wastewater-based epidemiology as an important component for COVID-19 control.