Field-deployable assay based on CRISPR-Cas13a coupled with RT-RPA in one tube for the detection of SARS-CoV-2 in wastewater

CRISPR-based nucleic acid detection is easy to implement, field deployable, and always coupled with isothermal amplification to improve the sensitivity. However, the conventional detection requires two separate steps, which can cause long-lasting amplicon aerosol contaminants, hence leading to false-positive results. To address this problem, we proposed a one-tube assay based on CRISPR-Cas13a coupled with reverse transcription-recombinase polymerase amplification to avoid aerosol pollution. The one-tube assay could be completed within 40 min with a sensitivity of up to 180 copies of RNA per reaction, and exhibited no cross reactivity with two related coronaviruses. Our technology showed reproducibility with relative standard deviation of 4.6% responding to 1 fM nucleic acid for three times. It could be used to detect SARS-CoV-2 nucleic acids in raw wastewater with a limit of detection of 103 copies/mL. We also validated the practicability of this technique for viral detection in environmental water samples by detecting SARS-CoV-2 in wastewater, which were not detectable by RT-qPCR technology, showing resistance of this technology to wastewater matrix. It is anticipated that the robustness and high sensitivity will significantly promote the development of a point-of-care method in environmental virus monitoring.