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Abstract
In medicolegal investigations, correct identification of the necrophagous fly species collected around and on the corpse is an essential step for estimating the postmortem interval (PMI). Therefore, forensic pathologists and entomologists investigating deaths due to violent crimes need a rapid, easy-to-use protocol to identify fly species found on corpses. A rapid and robust DNA-based tool that can distinguish between various immature and mature species from the Calliphoridae, Muscidae, and Sarcophagidae families would be ideal for such investigations. To date, the DNA barcode initiative is the best approach for identifying species-specific nucleotide sequences. We have developed 3 sequence-characterized amplified region (SCAR)-based identification systems derived from the Abdominal-B homeobox sequences of 17 fly species belonging to the Muscidae and Sarcophagidae. The flies used in this study were collected in Korea. These assay systems can classify 17 forensically important fly species into the dipteran family group and reliably distinguish them from inter- and intraspecific fly species through a 2-step multiplex PCR. This novel approach may also be used as an alternative to conventional DNA-based identification methods.
Figures and Tables
Fig. 1
A phylogenetic tree for 31 species of necrophagous flies, based on Abd-B homeobox sequence. Out-group is used that of Drosophila melanogester.
Fig. 2
Electophoregram showing family-specific SCAR marker for necrophagous flies detected by family identification multiplex PCR system (a; Calliphoridae, b; Muscidae, c; Sarcophagiae).
Fig. 3
Electrophoregram showing each species-specific SCAR markers for 10 Muscidae species detected by the multiplex PCR system.
Fig. 4
Electrophoregram showing each species-specific SCAR markers for 7 Sarcophagidae species detected by the multiplex PCR system.
Table 2
Family-Specific Primer Sets for Calliphoridae, Muscidae and Sarcophagidae
CsF; forward common primer to the families of Calliphoridae and Sacrophagidae flies. MsF; family of Muscidae-specific forward primer, SsF; family of Sarcophagidae specific forward primer, CMS-R: reverse common primer for families of Calliphoridae, Muscidae and Sarcophagidae. Y; C or T, R; A or G. *: optimal concnetraiton for PCR reaction
Table 3
Sequences and Concentration of Multiplex PCR Primers for Amplifying Muscidae and Sarcophagidae Species-Specific SCAR Markers
MsF; Muscidae species-specific forward primer. MsR; Muscidae species-specific reverse common primer. SsF; Sarcophagidae species-specific forward primer. SsR; Sarcophagidae species-specific reverse common primer. Underline; non-template sequences, W; A or T, Y; C or T, R; A or G, *; optimal concnetraion for multiplex PCR reaction
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