利用SSOs演替进行PMI推断的法医学研究

The estimation of postmortem interval (PMI) of putrescent corpses has long been a hot spot and difficulty in Forensic practice. It is well known that specific spoilage organisms(SSOs) are closely related to the progress of postmortem putrefaction. Preliminary experiments of our research group, coupled with previous research reports have supported that new insight and initial method for the precise deduction of PMI would prospectively be granted first of all by the distinctive distribution of SSOs of various parts of rat body; and then by the fact that succession pattern has been shown in type and quantity of SSOs. This project，in a further approach，takes miniature pigs as the model and sets comparison group contacted by insects. After that, by applying the standard bacteria-ring method, it collects the samples of SSOs in different parts of miniature pigs including mouth cavity, nasal cavity, peri-ocular parts, crissum, genital organ, abdomen and back. Subsequently, combined with the process of mark amplification of 16S rDNA, ROCHE-454 high-throughput sequence measuring, and QIIME program analysis, this project defines the classification and abundance of bacteria by dint of traditional CFU counting and ECO-BIOLOGO method; and it also, by light of R software, makes the statistical analysis and establishes multiple linear regression equation. The project strives to thoroughly investigate the distribution，succession patterns of classification and quantity of spoilage organisms in pig cadavers of different seasons and different PMI，and thus, to explore the pattern of PMI deduction via SSOs. It is also worth mentioning that, in order to guarantee the verification, we attempt to select samples among practice cases of the 100 healthy adult cadavers with positive case details and definite PMI.

腐败尸体死亡时间（PMI）推断是法医工作难题和研究热点。特定腐败细菌（SSOs）与尸体腐败进程关系密切。已有研究提示及本课题组采用大鼠尸体预实验验证，尸体上不同部位SSOs的分布具有特异性，在种类、数量等方面均呈现出一定的演替现象，有望为PMI的精确推断提供新的思路和方法。本课题进一步的研究以猪为实验模型，以尸体所处不同条件为对照，用标准取菌环法采集猪尸体口腔、鼻腔、眼周、肛周、生殖器、腹部、背部的SSOs，运用CFU计数法和ECO-BIOLOGO法，结合16S rDNA分子标记扩增、ROCHE-454高通量测序及QIIME程序分析，明确细菌种类及数量，用R软件进行统计分析，建立多元线性回归方程。全面获取不同季节不同PMI猪尸体上SSOs的种类、分布及数量演替，探索利用SSOs进行PMI推断的规律。在实际案例中选择案情及PMI明确的约100例健康成人尸体上相应部位的SSOs进行验证。

腐败尸体死亡时间（PMI）推断是法医工作难题和研究热点。特定腐败细菌（SSOs）与尸体腐败进程关系密切，广泛存在于自然界中，有推断PMI的潜能。本课题以SD大鼠、猪为实验模型，以尸体所处不同条件为对照，使用无菌棉签采集动物尸体口腔、鼻腔、眼周、肛周、生殖器、胸部、腹部、背部的SSOs，运用CFU计数法和ECO-BIOLOGO法，结合16S rDNA分子标记扩增、Miseq高通量测序及QIIME程序分析，选取有明显变化趋势的，用realtime-PCR TaqMan荧光探检测，建立非线性回归方程。在实际案例中选择案情及PMI明确的约30例健康成人尸体上相应部位的SSOs进行验证。尸体不同部位细菌群落演替随时间进展发生的变化有相应的规律性，变形菌门丰度增高，厚壁菌门及拟杆菌门丰度逐渐下降。腐败晚期尸体不同部位的细菌群落逐渐趋于相似，微生物多样性与死亡间隔时间负相关。尸体不同部位细菌群落构成不同，眼周细菌多样性较口腔及阴道低，直肠细菌多样性最高。口腔的链球菌属对早期PMI具有指示作用，Ignatzschineria菌属、大肠杆菌和奇异变形杆菌对晚期PMI具有指示作用。直肠的普氏菌属对早期PMI具有指示作用，变形菌属对晚期PMI具有指示作用。环境中的生物及非生物因素会影响尸体腐败：嗜尸性昆虫活动加快尸体腐败，也可以使腐败微生物演替进程加快，但对微生物群落物种分类学构成无显著影响；温度与尸体腐败速度正相关。人和大鼠的尸体之间的细菌演替规律有着很多的相似性，进一步证实了所得结论具有一定的普适性。