SNP Typing Using Multiplex Real-Time PCR Assay for Species Identification of Forensically Important Blowflies and Fleshflies Collected in South Korea (Diptera: Calliphoridae and Sarcophagidae)

Medicolegal entomology—a subfield of forensic entomology—is mainly used in medicolegal investigations to estimate the postmortem interval (PMI). The minimum PMI of a corpse invaded by necrophagous immature insects can be estimated because the PMI is near to or earlier than the oviposition time of the larvae that hatched and fed on the corpse. As the growth speeds of larvae differ depending on temperature and species, species-specific growth data are used to estimate the minimum PMI. While morphological identification of adult necrophagous flies can be done by a well-trained entomologist, identification of larvae is relatively difficult. Larvae can only be identified up to the family level and developmental stage by observing the posterior spiracles. For these reasons, the molecular biology method of DNA barcoding has been developed. DNA barcoding that targets the mitochondrial cytochrome c oxidase subunit I (COI) gene is commonly used. COI sequences are currently acquired using polymerase chain reaction (PCR) and Sanger sequencing, which are too time-consuming and complex for practical use in medicolegal investigations. To compensate for these limitations and facilitate the use of entomology for medicolegal investigation, we designed a multiplex real-time PCR system to identify nineteen forensically important species of Calliphoridae and Sarcophagidae flies collected in South Korea. In contrast to the Sanger nucleotide sequencing process, this technology only requires a one-step real-time PCR with melt curve analysis of amplicons generated by primers targeting species-specific single nucleotide polymorphisms (SNPs). Multiplex real-time PCR was performed for twelve species of Calliphoridae (four reactions) and for seven species of Sarcophagidae (three reactions). This assay is expected to make it easier and faster for investigating authorities to identify major species of necrophagous flies at beginning of investigation and to increase the utilization of entomological evidence in forensic investigations