Fig. 2: Development of RPA-dipstick assay with carbon black nanoparticles (RPA-dipstick-CBNP) for rapid SARS-CoV-2 fragments detection in wastewater.
From: Towards ultra-sensitive and rapid near-source wastewater-based epidemiology
a Schematic of RPA-dipstick assay for wastewater SARS-CoV-2 surveillance. RNA extracted from wastewater was amplified to generate E gene amplicons, which can bind to a complementary test line on the dipstick strip. The use of carbon black nanoparticles as sensing probe allows test results to be visualized by naked eye or through camera capture. b Examples of test strips showing a typical positive and negative result. c Analysis of the RPA-dipstick-CBNP assays show LoDs of 71 gc/reaction (95% CI: 34–134) for the E gene assay (green) and 127 gc/reaction (95% CI: 64-249) for RdRp gene assay (blue). The E-gene test was chosen for the further development. The dots show means and error bars show the s.d. of repeat measurements (n = 3 technical replicates and n = 3 measurement replicates for each sample). A stretched exponential regressions are shown by solid lines, and shaded areas show the 95% confidence intervals of the fits. d The sensitivity of the assay was 80% (95% Cl: 65-91) and the specificity was 100% (95% Cl: 84-100) (n = 62). e The semiquantitative nature of the assay has been demonstrated by plotting wastewater sample RT-qPCR cycle threshold (Ct) against the RPA-dipstick assay (with carbon black or gold nanoparticles probe) test line intensities, showing dose-dependent results. RT-qPCR–positive wastewater samples (with CT range from 25 to 40) and dipstick tested positive samples are plotted in green dots (CNP) or yellow dots (GNP), and RT-qPCR–negative samples (Ct > 40) and dipstick tested negative samples are plotted in pink dots (CNP) and red dots (GNP). PCR undetermined samples are plotted in grey dots. Significant differences were determined using one-way analysis of variance (ANOVA) with Tukey’s post hoc test. P = 0.2007 between CNP and GNP groups. The assay is semi-quantitative due to saturation in the regime where there are already many amplicons per nanoparticle (so there is a marginal increase in nanoparticle-test line binding rate with increasing amplicon concentration). Source data are provided as a Source Data file.
