IdentiFLY: The Development and Validation of a 15-Plex SNP Assay for Forensic Identification of UK Blowfly Species (Calliphoridae)

Members of the blowfly family (Calliphoridae) are usually the first insect species to arrive at a corpse, using the body as an oviposition site, and, as such, they are the most important group of insects used to estimate the post-mortem interval (PMI). PMI estimations are based on species-specific developmental timings; therefore, accurate species identification is crucial. Current identification methods are based on morphological characteristics, which are time-consuming and difficult to perform on damaged, immature specimens and closely related species. Advances have led to specimens being identified via a host of molecular techniques, mainly DNA sequencing. Although molecular identification is becoming increasingly more common, there is currently a lack of genetic data regarding UK Calliphoridae species. This study aimed to address this issue. We present the development and validation of an identification assay capable of differentiating six UK species (Calliphora vicina, Calliphora vomitoria, Lucilia sericata, Lucilia illustris, Lucilia caesar, and Protophormia terranovae). The sequencing of six genes, including both nuclear (28S rRNA and Elongation factor 1 alpha) and mitochondrial markers (Cytochrome oxidase I and II, Cytochrome b and 16S rRNA) identified 298 species-specific single nucleotide polymorphisms (SNPs). Fifteen SNPs from six genes were chosen for inclusion in a SNaPshot™ multiplex assay. The developed assay is capable of differentiating the species based on between 4 and 12 SNPs. Validation following guidelines by the International Society of Forensic Genetics (ISFG) demonstrated the assay to be accurate, reproducible, sensitive, and specific.