Using high-resolution melting to identify Calliphoridae (blowflies) species from Brazil

Abstract

Forensic entomology is the study of insects and other arthropods used in the solution of crimes. Most of entomological evidences strongly depend on accurate species identification. Therefore, new methods are being developed due to difficulties in morphological identification, including molecular methods such as High-Resolution Melting. In this study, we reported a new HRM primer set to identify forensically important Calliphoridae (blowflies) from Brazil. For such purpose, Calliphoridae species of forensic importance in Brazil were listed and confirmed by specialists. Mitochondrial COI sequences of those species were downloaded from databases and aligned, and polymorphic variations were selected for distinction between species. Based on it, HRM primers were designed. Forty-three fly samples representing six species were tested in the HRM assay. All samples had the COI gene sequenced to validate the result. Identifying and differentiating the six species proposed using a combination of two amplicons was possible. The protocol was effective even for old insect specimens, collected and preserved dried for more than ten years, unlike the DNA sequencing technique that failed for those samples. The HRM technique proved to be an alternative tool to DNA sequencing, with advantage of amplifying degraded samples and being fast and cheaper than the sequencing technique.

Keywords: BOLD; DNA Barcoding; Diptera; Forensic entomology; Melting curves; Species identification.

Figure 1. High-resolution melting analysis using COI primers (82 bp amplicon) for identification of Calliphoridae (blowflies) species.

(A) Normalized melting curves. (B) Difference plot curves using C. megacephala as reference genotype. Orange: L. eximia; Blue: L. cuprina; Black: C. megacephala and C. albiceps; Red: Co. macellaria; Green: C. putoria. (i) C. putoria; (ii) Co. macellaria; (iii) C. megacephala and C. albiceps; (iv) L. cuprina; (v) L. eximia.

Figure 2. High-resolution melting analysis using COI primers (124 bp amplicon) for forensic species.

(A) Normalized melting curves. (B) Difference plot curves using C. albiceps as reference genotype. Yellow: C. megacephala; Red: Co. macellaria; Brown: C. albiceps; Black: L. cuprina and L. eximia; Green: C. putoria. (i) C. putoria; (ii) L. cuprina and L. eximia; (iii) C. albiceps; (iv) Co. macellaria; (v) C. megacephala.

Figure 3. Flowchart showing species identification of blowflies using each of the HRM primers proposed, (A) 82 bp amplicon and (B) 124 bp amplicon.