Informativeness of NGS Analysis for Vaginal Fluid Identification

The identification of vaginal fluids in forensic examinations plays an important role in crime scene reconstruction. Molecular detection of vaginal bacterial communities can lead to the correct discrimination of body fluids. These kinds of studies can be performed through multiplex real‐time PCR using primers for a specific selection of bacteria. The availability of next‐generation sequencing (NGS) protocols provided for the extension of the analysis to evaluate the prokaryotes present in specimens. In this study, DNA was extracted from 18 samples (vaginal, oral, fecal, yoghurt) and analyzed by real‐time PCR and NGS. The comparison between the two approaches has demonstrated that the information developed through NGS can augment the more conventional real‐time PCR detection of a few key bacterial species to provide a more probative result and the correct identification of vaginal fluid from samples that are more forensically challenged.     

Messenger RNA Profiling for Forensic Body Fluid Identification： Research and Applications

Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link betw een sample donors and actual criminal acts. How ev-er, the conventional body fluid identification methods are prone to various limitations, such as time con-sumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Re-cently, the analysis of cell-specific messenger RNA expression （mRNA profiling） has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group （EDNAP ） in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profil-ing, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibilitywith current DNA extraction and analysis procedure. In the current review ,we provided an overview of the present know ledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possi-ble practical application to forensic casew ork.     

Raman spectroscopy offers great potential for the nondestructive confirmatory identification of body fluids

Raman spectroscopy was used to compare body fluids commonly found at crime scenes in a nondestructive manner. The dry traces of semen, vaginal fluid, sweat, saliva, and blood were analyzed using confocal Raman microscopy with a 785-nm excitation. The results show that the five fluids can be differentiated from one another by visual comparison of their Raman spectra, and that the laser radiation does not damage the sample. The Raman signature of each body fluid is specific and correlates with the known composition of the fluid. Dry traces of human and canine semen exhibited distinctly different Raman signatures. Overall, this preliminary study demonstrates the great potential of Raman spectroscopy for nondestructive, confirmatory identification of body fluids for forensic purposes.     

Determination of an effective housekeeping gene for the quantification of mRNA for forensic applications

The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the α, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood.     

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.     

Cytochemical detection of ABH antigens in human body fluids

The use of the peroxidase-anti-peroxidase (PAP) technique has been described previously for the detection of cellular antigens and in particular ABO antigens from tissue samples (Pedal and Hülle 1984; Pedal and Baedeker 1985; Pedal et al. 1985). In this survey, the PAP method has been employed to study the detection of ABO antigens in cells from body fluids of particular interest to forensic science, namely buccal cells and vaginal cells. Also tested, but in a limited number, were mixtures of body fluids and semen samples. No false reactions were obtained from buccal cells, all samples corresponding to the ABO blood type of the donor. Preliminary results from vaginal cells, vaginal/buccal cell mixtures, and semen were encouraging but must be treated with caution due to the limited number tested. Vaginal smears contaminated with semen showed varying degrees of nonspecificity.     

RNA cell typing and DNA profiling of mixed samples: Can cell types and donors be associated?

Forensic samples regularly involve mixtures, which are readily recognised in forensic analyses. Combined DNA and mRNA profiling is an upcoming forensic practice to examine donors and cell types from the exact same sample. From DNA profiles individual genotypes may be deconvoluted, but to date no studies have established whether the cell types identified in corresponding RNA profiles can be associated with individual donors. Although RNA expression levels hold many variables from which an association may not be expected, proof of concept is important to forensic experts who may be cross examined about this possible correlation in court settings. Clearly, the gender-specificity of certain body fluids (semen, vaginal mucosa, menstrual secretion) can be instructive. However, when donors of the same gender or gender-neutral cell types are involved, alternatives are needed. Here we analyse basic two-component mixtures (two cell types provided by different donors) composed of six different cell types, and assess whether the heights of DNA and RNA peaks may guide association of donor and cell type. Divergent results were obtained; for some mixtures RNA peak heights followed the DNA results, but for others the major DNA component did not present higher RNA peaks. Also, variation in mixture ratios was observed for RNA profiling replicates and when different donor couples gave the same two body fluids. As sample degradation may affect the two nucleic acids and/or distinct cell types differently (and thus influence donor and cell type association), mixtures were subjected to elevated temperature or UV-light. Variation in DNA and RNA stability was observed both between and within cell types and depended on the method inducing degradation. Taken together, we discourage to associate cell types and donors from peak heights when performing RNA and DNA profiling.     

MMP-11多克隆抗体的制备及其法医学意义

目的制备兔抗人基质金属蛋白酶-11(MMP-11)多克隆抗体,建立用MMP-11抗体检测月经血的可行方法,探讨其法医学意义。方法将用基因工程制备的人MMP-11融合蛋白免疫新西兰白兔,饱和硫酸铵法进行抗体纯化。运用蛋白印迹法检测月经血痕、外周血痕、阴道液斑、精液斑、唾液斑和尿液斑,盲测验证该方法的可靠性。结果高效价的兔抗人MMP-11多克隆抗体检测月经血MMP-11蛋白的阳性率为90.48%(93/105),而外周血痕、精液斑、唾液斑、尿液斑和阴道液斑均未检出MMP-11。结论用自制的抗MMP-11多克隆抗体所建立的蛋白印迹法检测月经血中的MMP-11特异性好,灵敏有效,可用于月经血及外周血的鉴别。     

mRNA在法医学中的应用现状与展望

近年来,随着RNA生物学性质和功能研究的日渐深入,RNA在法医学体液鉴别、斑迹形成时间推断、死亡时间推断、死亡原因分析、损伤时间推断等方面的作用受到了广大法医学研究者的重视,逐渐成为目前法医学应用研究的热点之一。本文综合介绍了mRNA在法医学中的应用现状,并对其未来应用前景进行了展望。     

Multiplex high resolution melt (HRM) messenger RNA profiling assays for body fluid identification

Traditional body fluid identification methods use a variety of technologically diverse techniques that do not permit the identification of all body fluids. Definitive identification of the biological material present can be crucial to a fuller understanding of the circumstances pertaining to a crime. Thus definitive molecular based strategies for the conclusive identification of forensically relevant biological fluids need to be developed. Messenger (mRNA) profiling is an example of such a molecular based approach.Current mRNA body fluid identification assays typically involve either capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the time required for analysis. For qRT-PCR assays, only 3 or 4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays and to reduce the time and cost of analysis, we have developed multiplex high resolution melt (HRM) assays that provide an identification of all forensically relevant biological fluids and tissues.     

Immunohistochemical staining as a potential method for the identification of vaginal epithelial cells in forensic casework

There is currently no accurate method to identify vaginal epithelial cells uniquely. This study aimed to use a cell extraction procedure compatible with routine forensic sampling methods, and to investigate the expression of cytokeratin (CK), estrogen receptor-alpha (ERalpha), and phosphodiesterase 5 (PDE5) in order to distinguish between skin, buccal, vaginal, and external penile epithelial cells. Seminal fluid samples were also examined. Epithelial cell samples were fixed in formalin, embedded in agarose, and processed using histological methods. Antigen-antibody reactions were detected using the DAKO Envision+ detection system. CK was present in all cells from all five sources confirming the origin of cells as epithelial. Both ERalpha and PDE5 positively labeled vaginal, buccal, and skin epithelial cells. Although an antigen unique to vaginal epithelial cells was not identified, we have described a cell extraction procedure for use in the immunohistochemical detection of a wide range of antigens, an approach compatible with forensic diagnostics.     

Optimisation of choline testing using Florence Iodine reagent,including comparative sensitivity and specificity with PSA and AP tests

The detection of semen in forensic science is essential in cases of sexual assault but can be problematic in the absence of spermatozoa. Choline is known to occur in high concentrations in seminal fluid and the Florence Iodine test for its detection has been used in forensic science for many years, however very little is documented regarding its sensitivity and specificity in forensic casework. This paper describes the optimisation of the choline Florence Iodine test (FI) and investigates the sensitivity and specificity of the test against different body fluids, food and drink substances, cleaning products and laboratory chemicals. Comparative testing against Acid Phosphatase (AP) and Prostate Specific Antigen (PSA Seratec®) tests is described and shows that the FI test has greater specificity than the PSA test which cross reacts with a number of body fluids.     

RNA在法医学中的研究应用进展

长期以来,RNA由于其自身的不稳定性、易降解的特征未受到法医学者的关注。近年来,随着定量PCR等分子生物学技术的改进与发展,RNA分析逐渐开始在法医学实验室中开展起来。RNA可用于死亡时间、创口形成时间、斑痕形成时间的推测,并且可取代传统的血清酶学检测用于确定体液类型并扩大了检测范围,还可以辅助诊断细胞或器官的功能状态。本文就近年来RNA在法医学的研究应用进展做一概述。