Identity of the numerous bloodstains at the murder scene: molecular identification of fly artifacts and fly species by CO1 analysis

Crime scenes may contain insect artifacts as well as samples of human origin. While the presence of insects can be important evidence in forensic medicine and forensic entomology, the insect artifacts sometimes interfere with the interpretation of bloodstain pattern analysis (BPA) which can be critical for accurate crime reconstruction. Fly artifacts are especially complicated to distinguish from true bloodstains. Indeed, we encountered a murder scene with numerous bloodstains inconsistent with the cause of death and had trouble interpreting them. The morphological method has been developed to distinguish them, but this method has to rely on the analyst’s experience and opinion. This study aims not only to distinguish fly artifacts from true bloodstains but also to identify fly species by detecting fly DNA in small amounts of bloodstains at the scenes. Melt curve analysis of real-time PCR (qPCR) targeting cytochrome c oxidase subunit 1 (CO1) of mitochondrial DNA (mtDNA) was able to detect fly DNA in bloodstains from a murder scene. The fly DNA was sequenced from the qPCR product, and the fly species were identified by BLAST search. Fluorescence-labeled specific primers for four species of necrophagous flies were designed based on the sequences of the CO1 region, and differences in the length of the amplification products were used to identify fly species from trace amounts of fly DNA in the artifacts.