体液法医学鉴定miRNA检测方法的建立与应用

目的建立针对体液特异性miRNA的SYBR Green检测方法,用于体液鉴定,探究法医实践中体液鉴定新方法。方法根据文献选出6条miRNAs为靶点,以合成的标准miRNA作为模板进行体系构建,再对实际样本进行验证,检测6条miRNAs在外周血、月经血、唾液、精液中的相对表达量。结果 miRNAs205在不同体液间表达差异不明显;miRNAs451、miRNAs144可明显的区分血与非血;miRNAs214可鉴定月经血;miRNAs888、miRNAs891在精液中高表达。结论建立的针对miRNAs的SYBR Green检测方法合理有效,可用于法医实践中体液鉴定。     

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids, ,

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body‐fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR‐Duet^? kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand‐alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification.     

Frequencies of morphological characteristics in two contemporary forensic collections: implications for identification

Positive identification relies on comparison of antemortem and postmortem data. Some identifications are based on morphological features such as fracture, pathological condition, and surgical hardware, despite little literature indicating the frequencies of such traits. This study examines whether such features are sufficiently rare as to be deemed individualizing. Data were collected on two modern North American skeletal collections (N=482 individuals). Presence/absence of features was scored by skeletal element and side. Results indicate that frequencies vary by geographic region (higher frequency of fractures and pathological conditions in New Mexico while individuals in Tennessee were more likely to have surgical interventions), many features such as fractures are remarkably common and that even suites of traits may not be individualizing. Caution is warranted when using written data rather than radiographic comparisons as the primary source of identification. The implications of these findings to missing person databases are also discussed.     

个体识别SNPs位点组合筛选与法医学应用价值初探

目的筛选用于包括中国主要民族在内的多个群体个体识别的SNPs位点组合体系。方法以Kidd实验室筛选的86个SNPs位点、欧洲SNPforID组织构建的52-plex SNPs复合检测体系为基础,收集和整理这些位点在HapMap数据库中11个人群的分型数据,计算各位点杂合度和Fst值,筛选杂合度〉0.4,Fst值〈0.06,并在研究人群中处于Hardy-Weinberg和连锁平衡的位点组合。针对这些位点,采用MassARRAY分子阵列技术对自行收集的8个人群（尼日利亚人、坦桑尼亚查加人、印度人、丹麦人、俄罗斯汉特人、中国汉族、藏族、维吾尔族）308份样本进行分型,统计群体遗传学参数。结果按本文标准共筛选出66个SNPs位点,均符合Hardy-Weinberg平衡,之间互不连锁,平均杂合度和Fst值分别为0.475、0.014。在本文收集的8个人群中的随机匹配概率在1.45E-24~4.72E-27之间,累积非父排除率为0.999 995 608~0.999 997 876之间。结论本文筛选的SNPs组合系统具有较强的个体识别能力,可用于本文调查的HapMap数据库中11个人群和本文收集的8个人群的个体识别鉴定。     

Identification of Saliva Using MicroRNA Biomarkers for Forensic Purpose

In the forensic science community, microRNA (miRNA) profiling has started to be explored as an alternative tool for body fluid identification. Several origins of body fluid can be distinguished by measuring differential expression patterns of particular miRNAs. However, most of reported saliva miRNAs are nonoverlapping and debatable. The aim of this study was to develop a strategy of identifying saliva using miRNA biomarkers for forensic purpose. Eight miRNA candidates were selected to examine expression abundance in forensically relevant body fluids using hydrolysis probes quantitative real‐time PCR (TaqMan qPCR). Results revealed that none of them was truly saliva specific, and only miR‐200c‐3p, miR‐203a, and miR‐205‐5p were higher or more moderate expression in saliva. A stepwise strategy that combines each of three miRNAs with different body fluid‐specific miRNAs was developed, and three miRNA combinations could effectively differentiate saliva from other body fluids.     

Applicability of two commercially available kits for forensic identification of saliva stains

The RSID-saliva test and the SALIgAE-saliva test are two recently developed forensic saliva detection kits. In this study, we compared the sensitivity and the specificity of the two test kits with the Phadebas amylase test by analyzing amylases from various sources including human, animals, plants, and micro-organism. The data demonstrate that the RSID-saliva test and the SALIgAE-saliva test offer higher sensitivity and specificity for the detection of saliva than the Phadebas amylase test. The detection limits of the RSID-saliva test, the SALIgAE-saliva test, and the Phadebas amylase test equate to 10, 4, and 1000 nL, respectively for human saliva. The RSID-saliva test and the SALIgAE-saliva test were further evaluated by analyzing semen, vaginal secretion, breast milk, blood, urine, sweat, and feces. The results of the two tests are in good agreement. The two tests reacted with urine, breast milk, and feces, but not with semen, vaginal secretion, blood, and sweat.     

The use of orthopedic surgical devices for forensic identification

Surgically implanted devices have become increasingly common in modern skeletal material. Therefore, having the knowledge of the variety of implanted orthopedic devices, their manufacturer, and where to find and how to use identifying numbers in such implants can assist in the identification process when traditional methods are not applicable. Orthopedic device manufacturers are required by the Safe Medical Devices Act of 1990 and the FDA Modernization Act of 1997 to track permanently implanted devices. Manufacturer information on orthopedic devices associates the orthopedic surgeon who implanted the device with the patient. By providing a current list of the most common orthopedic device manufacturers in the U.S.A. and the associated contact information, investigators will have updated tools for the individuation process. Despite numerous complicating factors regarding how device data are tracked, the information presented here can assist forensic professionals with obtaining presumptive and/or positive identifications.     

microRNAs及其法医学应用前景

microRNAs(miRNAs)是一类内源性的非编码小分子RNA,平均长度约22nt,广泛存在于各种真核细胞中,并参与调节细胞生长发育、分化、凋亡、衰老、疾病及肿瘤的发生等众多重要生命活动。基于其生物学功能,miRNAs可能在法医学体液来源鉴定、个体年龄推断、同卵双生子甄别等方面具有广阔的应用前景。     

Routine forensic use of the mitochondrial 12S ribosomal RNA gene for species identification

Since July 2004, Mitotyping Technologies has been amplifying and sequencing a approximately 150 base pair fragment of mitochondrial DNA (mtDNA) that codes for 12S ribosomal RNA, to identify the species origin of nonhuman casework samples. The approximately 100 base pair sequence product is searched at http://www.ncbi.nlm.nih.gov/BLAST and the species match is reported. The use of this assay has halved the number of samples for which no mtDNA results are obtained and is especially useful on hairs and degraded samples. The availability of species determination may aid forensic investigators in opening or closing off lines of inquiry where a highly probative but challenging sample has been collected.     

Issues in the global applications of methodology in forensic anthropology

The project and research reported in this collection of articles follows a long-term historical pattern in forensic anthropology in which new case work and applications reveal methodological issues that need to be addressed. Forensic anthropological analysis in the area of the former Yugoslavia led to questions raised regarding the applicability of methods developed from samples in other regions. The subsequently organized project reveals that such differences exist and new methodology and data are presented to facilitate applications in the Balkan area. The effort illustrates how case applications and court testimony can stimulate research advances. The articles also serve as a model for the improvement of methodology available for global applications.     

Development of a Real‐Time PCR‐Based Method for Analyzing Semen‐Specific Unmethylated DNA Regions and Methylation Status in Aged Body Fluid Stains

The detection of semen in forensic investigation is considered important evidence of sexual assault. In this study, we report the development of a real‐time polymerase chain reaction‐based method for identifying semen that can simply and rapidly analyze the semen‐specific unmethylated region of the DACT1 gene. Using two fluorescent probes designed for the methylated or unmethylated status, this method could perform quantitative analysis of the methylation status in this region. Furthermore, this method was used to analyze various body fluid samples, including 29‐year‐old semen and blood stains. The results showed that this method can detect almost exclusively semen or nonsemen signals even in highly decomposed samples, while a few semen or nonsemen samples showed slight signals of the other fluorescence probe. Although there is still a need for further analysis such as setting thresholds to analyze unknown samples, this method could be a useful supplementary tool for identifying semen, especially in old stains such as those in cold‐case investigations.     

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification

Molecular‐based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT‐qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA‐specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.     

circRNA应用于体液斑鉴定的技术可行性研究

目的利用circ RNA检测技术,探索circ RNA分子应用于体液斑迹鉴定的可行性。方法制备精液、唾液、阴道分泌物三种体液斑迹样本,以Qiagen RNeasy Micro试剂盒提取总RNA,经RNase R消化后得到circ RNA,进行逆转录PCR扩增,琼脂糖凝胶电泳检测分析。结果制备的体液斑迹样本均可检测到circ RNA分子,提示circ RNA普遍存在于法医常见体液斑迹中,具备一定的应用价值。结论 circ RNA的检测可与现有DNA检测技术兼容,利用其组织特异性,可成为体液斑迹鉴定的新标记,具备重要的研究价值。     

Age-related changes in pulp cavity of incisors as a determinant for forensic age identification

To find a simple and convenient method for age identification upon age-related pulp cavity narrowing, the mesiodistal diameters of the cervical pulp chamber, the middle and terminal parts of the root canal of the pulp cavity of 620 incisors were measured on radiographs taken in situ in 80 Chinese aged from 15 to 80. It was shown that the three mesiodistal diameters significantly decreased in a negative linear relationship with age (-0.4233 ≤ r ≥ -0.8465) in all incisors, but the narrowing velocity of the cervical pulp chamber and the middle part of the root canal in the maxillary incisors (b = -0.02 mm) was faster than that in the mandibular incisors (b = -0.01 mm). Accordingly, a mathematical model describing the ages as a function of any one of the three mesiodistal diameters of the pulp cavity was deduced, which would be useful for age identification in forensic medicine or archaeology.     

mRNA在体液斑鉴定研究中内参基因的选择与应用

利用mRNA分析技术鉴定生物检材中各种体液斑迹的组织属性来源是近年来法医物证学的一个重要进展。终点逆转录PCR和实时荧光定量PCR技术是法医学实验室常用的mRNA检测技术,为保证实验结果的准确性,研究者通常选用组成性表达的管家基因作为内参,对不同样本提取RNA进行质控和标准化,以正确评估目标mRNA在各样本中的表达量。本文聚焦近年来使用mRNA分析技术进行体液斑迹组织属性来源鉴定的研究报道,对这些工作中内参基因的选择与应用效果进行综述。     

利用腭皱特征进行同一认定的指标体系构建

目的根据腭皱的形态图特征,进行口腔腭皱在法医学同一认定的指标体系构建。方法收集100例成年人腭皱模型,依据腭皱的形状、数量、位置分布等特征对腭皱形态图进行全面系统的编码。编码顺序采用英文字母按照先右侧再左侧,先前部再后部的顺序编码,并且右、左侧编码以破折号连接。最后依据编码,统计分析腭皱形态分布特征。结果 100例腭皱形态图中,个体间未见完全一致者,每个个体不论男性与女性均表现有独特的腭皱形态图;且同一个体左右侧单条腭皱的形态及分布亦不同。波浪形腭皱所占比例最大（23.03%）,三分叉形出现比例最小（0.74%）,不同性别的波浪形及曲线形腭皱所占比例均较大,女性波浪形（22.7%）及曲线形（18.28%）;男性波浪形（24.11%）及曲线形（21.43%）。结论口腔腭皱法医学同一认定的指标体系构建,将为法医学的同一认定提供一种新的方法。     

Assessment of STR Typing Success Rate in Soft Tissues from Putrefied Bodies Based on a Quantitative Grading System for Putrefaction

To date, there is no systematic investigation of the association of short tandem repeat (STR) typing success rate in soft tissues with different signs of putrefaction. Herein, putrefaction was rated using a newly developed 19‐parameter system in soft tissues from a collective of 68 decaying bodies, and DNA yield was determined in 408 samples. DNA integrity was rated using a self‐devised pentaplex PCR generating an “integrity score” (S_i). STR typing success rate was then assessed for selected cases. DNA yield and S_i differed significantly between tissues with kidney on average exhibiting the highest S_i values. Statistical analysis revealed that nine parameters were significantly and positively correlated with S_i. The observed values for each of these nine parameters were summed up to generate a putrefaction score (S_p) for each sample. Our results show that STR typing success rate can be predicted based on S_p before expensive multiplex STR profiling is performed.