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Two of rove beetles (Staphylinidae) species: Creophilus maxillosus (Linnaeus, 1758), Ontholestes murinus (Linnaeus, 1758). Four carrion beetles (Silphidae) species: Thanatophilus sinuatus (Fabricius, 1775), Thanatophilus rugosus (Linnaeus, 1758), Oiceotoma thoracicum (Linnaeus, 1758), Necrodes littoralis (Linnaeus, 1758). There were analyzed specimens belonging to 12 species from 4 families. Amplicon 197 bp lenght spans from 257 - to 454 bp (numbering according to Thanatophilus sinuatus HQ165288.1).

ResultsД«ased on multiple sequence alignment of COI sequences of Coleoptera species important for forensic entomology were designed pair of primer for HRM-PCR: forvard - silphfv GCHACWYTWCATGGAACWCAA and reverse silphrv TASAATTGCAAAHACSGCHCC. The resulting normalized curves were subsequently used to construct difference graphs, depicting the variation in fluorescence of a sample to a selected reference. Therefore, the raw data were normalized in two regions (region 1: 71вЂ“72вЂЇвЊ region 2: 88вЂ“89вЂЇвЊ) to allow the comparison of all melting curves from the same starting and ending signal level.

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The resulting fluorescence data were first visualized by difference plotting using the software Rotor-Gene 1.7.27. Melting curve data were generated by monitoring the changes in fluorescence during the increase of temperature from 60 to 95вЂЇвЊ at 0.1вЂЇвЊ per second. Amplification was performed under following thermal program: an initial denaturation step of 95вЂЇвЊ for 3вЂЇmin followed by 40 cycles at 95вЂЇвЊ for 30вЂЇs, 61вЂЇвЊ for 30вЂЇs, and 72вЂЇвЊ for 30вЂЇs. Each PCR reaction mix was prepared using 10вЂЇОјl Real Time 2вЂЇГ—вЂЇPCR Master Mix EvaGreen (A&A Biotechnology, Gdynia, Poland), 2вЂЇОјl of 5вЂЇОјM each primer, 2вЂЇОјl DNA extract, and 4вЂЇОјl H 2O to total 20вЂЇОјl volume. Real time PCR reactions and the subsequent HRM analysis were performed in a Rotor-Gene 6500 thermal cycler (Corbett Research, Mortlake, NSW, Australia). In recent years, the developments in molecular biology opened the door for a variety of DNA sequence-based alternative approaches for determining organisms.

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Moreover, the quality and level of taxonomic resolution achieved depends upon the individual knowledge and experience of the respective investigator, while professional taxonomists simultaneously are getting rare.

The morphological identification of Coleoptera species from varying life stages to species level can be challenging, time- consuming and costly. Identification of Coleoptera species is however more difficult that Calliphoridae. Especially interesting is Coleofauna present at all stages of cadaver decomposition. Further stages of cadaver decomposition are associated with other entomofauna.

Application of Calihoridae for post mortem interval estimation is well suited for initial period of cadaver decomposition but limited to of their larval stage. Precise estimation time of death is one of key task of forensic entomology.

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