The Availability Of COI Gene For The Identification Of Forensically Important Flies(Diptera:Mascidae) In China

BackgroundForensic entomology is a new cross-edge discipline of forensic medicine and arthropod science combined in a wide range of areas and affect each other, involved in criminal, civil and administrative cases in the range of applications, provide technical assistance for forensic inspection case. The main research methods of the Forensic entomology are inspecting, testing, classifying, clearing insect species, which are collected from within the body, the bodies of surface or near the body. And also determine a particular insect growth and development of state, after the combining with the Ecological community succession of the insects on the corpse and the living habits to help forensic completion of the postmortem interval. The premise of the insect community succession law in the application on the body to solve related forensic issues is to identify the types of insects.After the death of the body, there will be a lot of sarcosaphagous flies arrive and lay eggs, therefore, the types of the determination corpse sarcosaphagous flies and their pupae made childbearing is important entomological data to estimate time of death. However, so many insect species, only specially trained professionals in order to grasp the morphology of certain types of group identification points. Morphological characterization of flies cannot get a good popularization, also a great obstacle to the practical inspection of forensic workers case. Developed in recent years, molecular biology techniques will be good to solve this problem. This study through the use of mitochondrial cytochrome oxidase subunit I (CO Ⅰ) from the genetic level to design primers to explore the identification of insect species, and making the insect species identification is more accurate and scientific.Objective: The purposes of our study are to obtain the accurate species identification of sarcophagous flies, and then we base on the a272base pair region of Cytochrome oxidase subunit I (COI) in Mitochondrial DNA (mtDNA) to take Sequence analysis and phylogenetic tree construction. Accumulate data for the establishment of regional sarcosaphagous insect gene bank. For the method may precisely estimate postmortem interval (PMI) or the time of death in practical.Methods:Samples Collection:Specimens of Muscidae were collected from Hunan and neighboring provinces in China from2009to2011, and some samples were pre-collected by our research department from2007-2008. And all samples were trapped using animal (rabbit and rat) corpse-baited traps either by the authors or by members of interests group of forensic students of the Central South University, and stored at room temperature. Invitation of the Hunan Agricultural University entomology experts selected a sample of the morphological identification. The use of Cetylteriethylammnonium bromide (CTAB) extraction of DNA samples, and detecting by0.8%agarose gel electrophoresis. he extracted DNA were amplified by PCR amplification protocol, and tested by vertical non-denaturing polyacrylamide gel electrophoresis and Silver staining technology, finally the PCR products isolated by QiaQuick PCR Purification Kit. Purified product was sent to the Genomics Institute (Wuhan) sequence measured;MEGA4package for sequence analysis and phylogenetic tree was constructed.Result:A portion of272bp fragment of the mitochondrial COI gene was amplified and sequenced. We assessed the COI sequence as a potential marker for the identification of Muscidae family flies from China. The intra versus interspecific variation also marked the clear threshold levels for species identification based on this COI region. All the collected specimens from the Muscidae family are divided into4genus and5species:(1)Muscina: Muscina stabulans (Fall,1823)(9specimens);(2)Musca:Musca domestica(Linnaeus,1758)(7specimens);(3)Ophyra: Hydrotaea spinigera (Stein)(5specimens); Hydrotaea chalcogaster(Wiedemann)(6specimens);(4) Stomoxys Geoffroy: Stomoxys calcitrans (Linnaeus)(4specimens).Conclusion:DNA-based method can provide a quick and reliable species determination and is relatively insensitive to the preservation methods. The272bp region of the COI sequence in our study revealed the potential of mtDNA for using as a discriminatory tool in forensic investigation and also showed that mitochondrial nucleotide sequences can offer a means of identifying forensically important Chinese insects. Therefore DNA-based method can be used as a supplemental means of morphological method.