Wastewater-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.

In non-open environments, pathogenic microorganisms are more likely to invade the human respiratory tract due to their limited diffusion in the environment, which has received little attention. In this study, we explored the distribution characteristics of particulate matter (PM) in non-open environments, and included sewage treatment plants and farms, which are occupational exposure risks, and G-series high-speed trains and waiting rooms, which are crowded. The results showed orders of magnitude differences in PM and microbial concentrations and the DNA/PM values of adsorption in the different non-open spaces. The concentration of PM with a size in the 4.7–10.0 μm range was higher than those of PM in the 1.1–4.7 μm and 0.43–1.1 μm ranges in all three types of places, accounting for 74.64%, 46.59%, and 51.49%, respectively. The DNA/PM value for the 1.1–4.7 μm range was higher than those for PM in the other two ranges in all three types of places at 0.175, 3.78 × 10−3, and 9.98 ng/μg, respectively. Although the relative abundances of Class II potentially pathogenic bacteria with sizes ranging from 1.1 to 4.7 μm were higher in all three types of places, the total abundance and the relative abundance of identified pathogenic microorganisms with sizes ranging from 4.7 to 10.0 μm were higher in all three types of places. Here, in non-open spaces, the pathogen exposure risk associated with PM10, particularly the coarse fraction of PM10, deserves special attention. Infectious diseases caused by aerosol transmission of pathogens in non-open environments should receive more attention and require further investigation in the future.