Study On Forensic Application Of Species Specificity Detection Of V3 Region Of Soil Bacteria's 16S RRNA Genes

The research on soil sample in various forms have been applied in forensic investigations on thousands of years.In the field of forensic science, soil as suspicious evidence at the scene had been distincted based on the main features of geology. However, most of the soil samples is invovled as trace evidence, which limit the extensive application of soil research in forensic biology.The rapid development of the molecular biology technology have enriched the knowledge of the soil microbes and expanded people's understanding of the soil microbes, and the research on soil microbial DNA diversity is growing increasingly.Microbes is a group of low-organisms ,which is in tiny shape and simple structure.Microbes in soils are much more in the numbers and types than other environmental microbes.Because of the nutritional and ecological point of the competition, mutual influence and constraints , microorganisms in the soil form a dynamic balance of the soil microbial communities.Soil microbial substances in soil not only is the driving force of material circulation, but also the metabolism is the nutritional content,whose activities can directly affect the soil physical and chemical properties.The types and quantities of soil samples in different locations show a marked diversity because of the variation of the soil composition and the surrounding environment between the microbial groups. So the trace of soil samples for microbiological testing has an important role in forensic application.In ecological and environmental sciences research, particularly on soil microorganisms, the soil microbial diversity is increasingly focused. A large number of experimental studies show that macromolecules such as protein, RNA and DNA can be seen as a molecular evolutionary clock.In this large number of biological macromolecules, the most appropriate to reveal all kinds of biological relationship is rRNA, especially the 16S rRNA.Ribosomal 16S RNA (16S rRNA) gene is a bacterial rRNA corresponding to coding DNA sequence on chromosome, with collection of conservative and variability, and is target genes which is the most extensive application in molecular microbiology detection. At present, there is no the relevant research on soil bacteria 16 SrRNA in domestic field of forensic medicine. We used the denaturing gradient gel electrophoresis (DGGE) technology to analyse soil microbial groups genomic DNA and compare the 16S rRNA genetic information between the different soil microorganisms to understand the diversity of microorganisms. we can judge the identity of soil samples and provide a new way to analys the trace evidence of soil samples.Specific research ideas are as follows: CTAB, lysozyme and proteinase K crack cells, with PEG8000 precipitation and purification of DNA , extract directly DNA of the soil microbes,amplify 16S rDNA V3 area with specific primers F357GC and R₅₁₈ and get a different number of electrophoresis band by the isolation of DGGE. Through the location and comparison of electrophoresis band, we can judge the identity of soil samples and provide a new way to analys the trace evidence of soil samples.MATERIAL AND METHODS1. Select randomly sample points (A, B) respectively from the surface, 10cm, 20cm, 30cm depth and four different amounts of soil samples (300mg, 200mg, 100mg, 80mg, 60mg).2.Extract DNA by CTAB-lysozyme-proteinase K-freeze-thaw method, purify and precipitate DNA by PEG8000.3.Amplify soil bacteria 16S rDNA V3 area by specific primers F357GC and R₅₁₈.4.Detect DNA quality by ultraviolet spectrophotometer and agarose gel electrophoresis.5.The amplified products were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) and denaturing gradient gel electrophoresis (DGGE).RESULT1.Soil samples is detected by ultraviolet spectrophotometer ,and OD_260/280 were distributed in between 1.5 to 1.68. 2.The 230 bp amplified fragment is detected by agarose gel electrophoresis.3.The identity of soil samples can be determined by the PCR-DGGE techonology.CONCLUSIONPCR-DGGE technology is employed to separate the different amplified DNA fragments of bacteria 16S rDNA V3 region.. It is an effective technology to detect microbial diversity , and gradually applied to the field of forensic trace evidence of soil testing.