| Background:Forensic entomology is a new study of applying knowledge about the insects in legal issues and homicide investigations. It is aimed to estimate the postmortem interval (PMI). The important insect evidence contains Diptera flies and Coleoptera beetles. Among of these, sarcosaphagous flies are mostly applied in study and practice, as their special growth habit of feeding on the body to corruption, they often provide helpful clues in case of homicide.Different flies arrived in a corpse are usually in different stages of the body after death, showing a strong succession of phenomena. Blow flies are usually the first fly to arrive in a corpse. Especially Luciliinae, Calliphorinae have strongly sensitivity to the corpus, the body, and lay eggs on it within a few hours after death. Unsurprisingly, the blowfly larvae collected from death scenes and also determined as the best entomological indicator for PMI estimation.In the early and mid period after death, the sarcosaphagous insects mainly in a non-mature stage, the insect eggs and pupas early larvae are very similarly, so it is very difficult to identify by using traditional morphological method. In order to reslove the problem, and with a rapid development in molecular biology techniques in recent years, we intend to use the molecular biology techniques to resolve the problem. A primer of 16S rDNA designed by ourselves is applied to provide fundamental data to species identification.Objective:According relevant reports, a primer of short segment 16S Ribosomal DNA (16S rDNA) gene sequences was designed by ourseflves. The purposes of our work are to identify species of Calliphoridae based on a 289 base pair region of the gene for 16S Ribosomal DNA in mitochondrial DNA (mtDNA) and to explore common molecular markers for sarcosaphagous flies. The method can provide technical supports for PMI.Methods:Samples were collected in several regions. Samples of blowflies were collected from animal carcass which were set outdoors, then the flies mitochondrial DNA (mtDNA) were extracted by the improved small insects DNA homogenate method. Then, the entire extracted DNA were amplified by Eppendorf 5331 and tested by vertical non-denaturing 7% polyacrylamide gelectrophoresis and 0.8% agarose gel electrophoresis. PCR products were purified by using the nucleic acid purification kit and then sequenced. Sequence analysis were carried out by software and phylogenetic trees.construction was also made by MEGA 4.Result:All the 58 specimens in family Calliphoridae were sequenced after the amplified 16S Ribosomal DNA. Phylogenetic analyses using sequence data from the 16 S ribosomal DNA region illustrate relationships among all the 58 specimens, including four genus (Lucilia, Chrysomya, Achoetandrus, Calliphora) in total 10 species (Lucilia sericate (Meigen), Lucilia cuprina (Wiedemann), Lucilia caesar (Linnaeus), Lucilia illustris (Meigen), Lucilia porphyrina (Walker), Lucilia bazini (Seguy), Chrysomya megacephala (Fabricius), Chrysomya ruffifacies (Macquart), Calliphora vicina (Robineau-Desvoidy), Calliphora vomitoria (Linnaeus)).Conclusion:1.16 SrDNA sequences is an effective instrumental for identification of species within family Calliphoridae, especially genus Lucilia. It enables the reliable identification of species used for providing the estimation of postmortem interval.2. There are some limitations in the short 16 SrDNA segments, because of all the Chrysomya ruffifacies were formed a cluster, separating from the group of Chrysomya megacephala. So the method of molecular marker technology to identify species is a necessary complement to traditional morphological method.3. The geographical variation is not obviously in the study for it is maybe the quantity of samples in every place was too few. So it should rise the numbers of the same species to find the variation between locations in future study.
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