In silico PCR-RFLP identification of forensically important Calliphoridae.

purposeThis study aimed to evaluate a cost-effective and rapid molecular approach for identifying forensically important Calliphoridae using an in silico PCR-RFLP assay designed with degenerate primers.

methodsCOX1 gene sequences from Calliphoridae species were retrieved from the NCBI database. Multiple sequence alignment with MAFFT identified conserved and variable regions for degenerate primer design in Primer3Plus. In silico PCR was simulated with Serial Cloner, and primer specificity was assessed using Primer-BLAST. Restriction enzyme screening was performed with NEBcutter and RFLP-inator to evaluate single and double digestions, and to generate species-specific fragment patterns.

resultsAnalysis of 47 COX1 sequences identified a highly variable 178-bp fragment suitable for species-level differentiation. The degenerate primers amplified all target species without cross-reactivity with human DNA. Single digestion with AluI differentiated four of six species, while double digestion with AluI/BsaJI resolved seven of nine. Additional enzymes (MseI, BsmAI) further discriminated challenging species pairs, enabling differentiation of 17 out of 18 species.

conclusionThe in silico PCR-RFLP assay presented here provides a promising molecular tool for identifying forensically relevant Calliphoridae species, with potential to improve postmortem interval (PMI) estimation. Experimental validation is required to confirm its applicability in forensic casework.