RT-qPCR detection of dsRNA pesticides: Optimization, limitations, and formulation effects

Double-stranded RNA (dsRNA)-based pesticides are emerging as highly specific and environmentally sustainable crop protection agents. However, their environmental fate remains poorly characterized due to the lack of standardized analytical methods. Here, we developed a quantitative reverse-transcription PCR (RT-qPCR) workflow for quantifying naked and chitosan-formulated dsRNA in laboratory preparations and agricultural soils. Using a 297-bp dsRNA targeting Leptinotarsa decemlineata actin (dsACTIN), we assessed nuclease treatment, chitosan formulation, and matrix-matched calibration across three soils differing in pH. Nuclease treatment improved assay specificity, achieving a detection limit of 8 × 10^-8 ng/L, whereas chitosan formulation decreased analytical sensitivity. Rapid adsorption of dsRNA to soil particles underscored the importance of recovering both soluble and particle-bound fractions. Matrix-matched calibration enabled quantification to 10^-3 ng dsRNA/g soil. Repeated applications revealed rapid dissipation of naked dsRNA but enhanced persistence of chitosan-formulated dsRNA. Overall, this study provides a framework for environmental monitoring and risk assessment of dsRNA pesticides.