A triplex RPA-LFA assay for the simultaneous detection of forensically important necrophagous insects in complex DNA...

Accurate species identification of necrophagous insects is crucial in forensic investigations. To overcome the limitations of complex morphological identification-especially for larval stages-and time-consuming DNA sequencing methods, we developed a triplex recombinase polymerase amplification combined with a lateral flow assay (RPA-LFA). This assay targets three common forensically important insects: Chrysomya megacephala, Lucilia sericata, and Sarcophaga peregrina. We comprehensively evaluated the specificity, sensitivity, and mixed DNA detection capability of the assay. The results demonstrated extremely high specificity; the specific test lines (T-lines), indicated by different positions, accurately differentiated the target species without cross-reactivity with four other common necrophagous insects. Furthermore, the sensitivity analysis revealed low limits of detection, ranging from 62 pg to 250 pg. Additionally, the assay accurately identified the target species within complex mixed DNA samples. In conclusion, the developed triplex RPA-LFA system enables rapid and visual species identification of forensic insects, providing an efficient and reliable novel tool for forensic field investigations.