Molecular Identification of Sarcosaphagous Flies in Luoyang Based on 28S rRNA Gene Sequence / 法医学杂志

Objective To detect 715 bp sequence of 28S rRNA in sarcosaphagous flies, and to identify their common species for solving the problem of morphological identification, as well as providing technical support for postmortem interval (PMI) estimation. Methods Twenty-nine common sarcosaphagous flies were collected in Luoyang and classified by morphological characteristics. The DNA was extracted from the fly's legs by Chelex-100 method and then the fragments of 28S rRNA were amplified and sequenced. The results were compared with twenty-eight corresponding fly species of GenBank and EMBL databases. All the sequences were analyzed by MEGA7.0 software, and sequence alignment was performed by the searching in BLAST. The nucleotide composition was analysed, and the intraspecific and interspecific ge-netic distance and phylogenetic tree were established. Results Twenty-nine sarcosaphagous flies were classified into 6 species of 5 genera, 3 families by morphological characteristics. In the obtained 715 bp sequence of 28S rRNA , the comparison result of online BLAST showed that the similarity was 100％. Five species were well clustered by a phylogenetic tree. Between different groups, the interspecific and intraspecific differences ranged from 0.007 to 0.045 and 0 to 0.001, respectively. Conclusion The 28S rRNA target gene sequences shows a good identification capability, which can be a new genetic marker for the identification of sarcosaphagous flies.

Identification of sarcosaphagous flies by amplification and sequence analysis of COⅠ gene fragment from DNA extracts / 中国法医学杂志

Objective To identify the species of sarcosaphagous flies by amplifying cytochrome oxidase subunitI (CO I) gene fragment,combined with morphological characteristics.Methods The DNA of sarcosaphagous flies was extacted by modified Tris balanced phenol-Tris saturated phenol protocol,the amplificationand sequencing of CO Ⅰ fragment were conducted,and the results were then compared with the database for analysis.Results The modified DNA extration method by Tris balanced phenol-Tris saturated phenol could obtain effective DNA of sarcosaphagous flies,and be applied for CO Ⅰ fragment amplification,and thus for identification of the species of sarcosaphagous flies.Conclusion The DNA extracted from sarcosaphagous flies by modified Tris balanced phenol-Tris saturated phenol protocol could be used astemplate for the amplification of CO Ⅰ fragment,and the system could identify the species of sareosaphagous flies after sequencing and alignment with the sequences of database.Compared with traditional identification methods of using morphological characteristics,the current system is more accurate and could be more widely applied.

Application of mtDNA COI genes in common sarcosaphagous flies species identification / 中国法医学杂志

Objective To observe the application of mtDNA COI genes in common sarcosaphagous flies species identification. Methods 30 sarcosaphagous fly samples were indentified by morphological method which collected in different regions belonging to 2 families, 4 genera and 6 species. MtDNA was extracted for the PCR amplification reaction in COI gene. The PCR products were purified through agar gel electrophoresis and sequenced. Sequences of 498 bp in COI gene were disposed by multiple-alignment software of DNAMAN. Sequences divergence of 498 bp between and within species of COI gene were processed by software of MEGA. Results It was showed that there is a certain sequence differences between the 30 samples from 6 species. The intraspecific and interspecific divergence of sequence variation ranged from 0.1% to 1.6% and 2.2% to 11.2% respectively. All the species can be identified successfully by this method. Conclusion Species identification of sarcosaphagous flies can be conducted by sequence analysis and phylogenetic tree of COI gene. This method can be effectively used in fast and accurate identification in forensic entomology.