Identification of forensically important blowfly species (Diptera: Calliphoridae) by high-resolution melting PCR analysis

We describe here the successful coupling of real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis to rapidly identify 15 forensically important species of blowfly from the family Calliphoridae (Diptera), which occur in Poland. Two short regions (119 and 70 base pairs, respectively) of cytochrome oxidase gene subunit I with sufficient sequence diversity were selected. In the case of lacking taxa (e.g., reference species) these amplicons can be synthesized using sequences deposited in gene banks. The technique utilizes low template DNA concentration and is highly reproducible. The melting profile was not altered up to a 10,000-fold difference in DNA template concentration (ranging from 5 pg to 50 ng). The several HRM runs performed on different specimens from Poland belonging to the same species and on different days resulted in only minor variations in the amplification curves and in melting temperatures of the peaks. Intraspecific variation in a larger scale was tested using synthesized oligonucleotides from cosmopolitan Lucilia illustris originating from Poland, France, Great Britain, India, and USA. As HRM PCR analysis is sensitive to even single base changes, all geographic variants of this species were identified. This technique is also cost-effective and simple, and it may even be used by non-geneticists. A working protocol was ultimately constructed for the purpose of rapid and accurate species identification in most countries in Europe regardless of which stage or which part of a blowfly was collected.