Molecular Detection of Diatom DNA in Necrophagous Blow Fly Larvae (Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae)): A PCR-HRM Proof-of-Concept Bridging Forensic Entomology and Diatomology

Conventional diatom-based drowning diagnosis relies on intact visceral organs, yet this approach fails when corpses recovered from aquatic environments undergo advanced putrefaction. To our knowledge, this study presents the first molecular proof-of-concept demonstrating that necrophagous blow fly larvae can serve as a novel biological substrate for diatom detection. Using a controlled freshwater rat model, larvae of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) were reared on lung tissue from drowned (n = 5) and non-drowned control (n = 5) rats. Diatom DNA was extracted via a glass-bead-vortex-modified method and detected by PCR–high-resolution melting (PCR-HRM) using the diatom-specific primers UPA99 and 18S-V7. All drowning water and drowned lung tissues tested positive, whereas all control tissues remained negative, confirming model validity. Crucially, diatom DNA was detected in 7/10 (70%; Wilson 95% CI: 35.4–91.9%) and 4/10 (40%; Wilson 95% CI: 12.2–73.8%) larvae from the drowning group using UPA99 and 18S-V7, respectively, both comparisons achieved statistical significance (p < 0.05), with a large effect size for UPA99 (φ = 0.74). In this controlled freshwater rat model, PCR-HRM detected diatom DNA in larvae, whereas prior morphological approaches failed. The detection of diatom DNA in larvae should be interpreted as supportive, not conclusive, evidence of antemortem drowning in casework.