Evaluation of RT-qPCR Primer-Probe Sets to Inform Public Health Interventions Based on COVID-19 Sewage Tests

Sewage
surveillance is increasingly employed as a supplementary
tool for COVID-19 control. Experiences learnt from large-scale trials
could guide better interpretation of the sewage data for public health
interventions. Here, we compared the performance of seven commonly
used primer-probe sets in RT-qPCR and evaluated the usefulness in
the sewage surveillance program in Hong Kong. All selected primer-probe
sets reliably detected SARS-CoV-2 in pure water at 7 copies per μL.
Sewage matrix did not influence RT-qPCR determination of SARS-CoV-2
concentrated from a small-volume sewage (30 mL) but introduced inhibitory
impacts on a large-volume sewage (920 mL) with a ΔCt of 0.2–10.8.
Diagnostic performance evaluation in finding COVID-19 cases showed
that N1 was the best single primer-probe set, while the ORF1ab set
is not recommended. Sewage surveillance using the N1 set for over
3200 samples effectively caught the outbreak trend and, importantly,
had a 56% sensitivity and a 96% specificity in uncovering the signal
sources from new cases and/or convalescent patients in the community.
Our study paves the way for selecting detection primer-probe sets
in wider applications in responding to the COVID-19 pandemic.