Environmental DNA ‐based RPA ‐ CRISPR /Cas12a assay for on‐site detection of chironomid larvae in aquatic environments

BACKGROUND Freshwater chironomid midges pose a significant threat to drinking water quality and freshwater ecosystems in South Korea by disrupting ecological balance and causing biofouling in drinking water treatment systems. To enable rapid detection and provide early warnings of chironomid presence, we developed a highly sensitive and accurate diagnostic assay based on environmental DNA (eDNA) analysis. This method integrates recombinase polymerase amplification (RPA), CRISPR/Cas12a‐mediated cleavage, and visualization through either fluorescence‐based detection or a lateral flow assay (LFA). RESULTS The RPA‐CRISPR/Cas12a assays demonstrated remarkable specificity for chironomid eDNA, as confirmed through field surveys and microscopic observations. Although both fluorescence‐based and LFA‐based RPA‐CRISPR/Cas12a assays exhibited comparable detection limits of 10 copies/μL, the LFA method proved more convenient for on‐site applications due to its ease of deployment in the field. CONCLUSION Our eDNA‐based RPA‐CRISPR/Cas12a assay addresses the limitations of traditional biomonitoring by requiring only a small sampling volume, thereby minimizing habitat disturbance, while offering much higher sensitivity. This assay represents a valuable tool for on‐site detection of invasive species and for monitoring environmental and public health threats (e.g., chironomids).