Cao, HaoruiMao, KangRan, FangXu, PengqiZhao, YirongZhang, XiangyanZhou, HourongYang, ZhugenZhang, HuaJiang, Guibin2022-10-042022-10-042022-08-30Cao H, Mao K, Ran F, et al., (2022) Paper device combining CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification for SARS-CoV-2 detection in wastewater. Environmental Science and Technology, Volume 56, Issue 18, September 2022, pp. 13245-132530013-936Xhttps://doi.org/10.1021/acs.est.2c04727https://dspace.lib.cranfield.ac.uk/handle/1826/18514Wastewater-based surveillance of the COVID-19 pandemic holds great promise; however, a point-of-use detection method for SARS-CoV-2 in wastewater is lacking. Here, a portable paper device based on CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with excellent sensitivity and specificity was developed for SARS-CoV-2 detection in wastewater. Three primer sets of RT-LAMP and guide RNAs (gRNAs) that could lead Cas12a to recognize target genes via base pairing were used to perform the high-fidelity RT-LAMP to detect the N, E, and S genes of SARS-CoV-2. Due to the trans-cleavage activity of CRISPR/Cas12a after high-fidelity amplicon recognition, carboxyfluorescein-ssDNA-Black Hole Quencher-1 and carboxyfluorescein-ssDNA-biotin probes were adopted to realize different visualization pathways via a fluorescence or lateral flow analysis, respectively. The reactions were integrated into a paper device for simultaneously detecting the N, E, and S genes with limits of detection (LODs) of 25, 310, and 10 copies/mL, respectively. The device achieved a semiquantitative analysis from 0 to 310 copies/mL due to the different LODs of the three genes. Blind experiments demonstrated that the device was suitable for wastewater analysis with 97.7% sensitivity and 82% semiquantitative accuracy. This is the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater via different LODs, demonstrating a promising point-of-use method for wastewater-based surveillance.enAttribution-NonCommercial 4.0 Internationalhttp://creativecommons.org/licenses/by-nc/4.0/SARS-CoV-2CRISPR/Cas12aRT-LAMPpaper devicewastewaterArticle