Forensic Science International-Genetics最新文献_第8页

Non-destructive identification of forensically relevant body fluid stains using a portable electronic nose: A pilot study 使用便携式电子鼻对法医相关体液污渍进行无损鉴定：一项试点研究

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-21 DOI: 10.1016/j.fsigen.2025.103347

Daijing Yu , Niu Gao , Tian Wang , Liwei Zhang , Jun Zhang , Jiangwei Yan

Identification of bodily fluid stains is crucial for forensic investigations. While current molecular detection methods provide high accuracy, their destructive sampling nature imposes significant limitations on trace samples by compromising sample integrity and subsequent short tandem repeat (STR) profiling. To date, no non-destructive method for fluid identification has been reported. This study introduces a novel portable electronic nose (e-nose) technology that facilitates non-invasive differentiation through the detection of volatile organic compounds (VOCs) in bodily fluids. Requiring only 3–4 min per test while preserving DNA integrity, this approach effectively distinguishes morphologically similar fluids such as blood and menstrual blood, offering an innovative solution for the non-destructive analysis of forensic body fluid. In this study, VOCs from 200 body fluid samples—including blood, saliva, semen, vaginal secretions (VS), and menstrual blood (MB)—were analyzed using electronic nose technology. Samples were collected via sterile swabs (n = 100) and toilet paper (n = 75). Radar plots indicated that sensor S7 (W1W) exhibited peak responses across both carriers. Linear discriminant analysis of the 175 samples revealed distinct clustering patterns (ANOSIM R = 0.088, p < 0.001). The random forest-based predictive models demonstrated that the general model—non-carrier-specific with n = 175—achieved accuracies of 100.00 % for VS, 90.91 % for blood, 81.82 % for semen, 72.73 % for MB, and 63.64 % for saliva; resulting in an overall accuracy of 81.82 %. Notably, when specifically utilizing toilet paper as a carrier, accuracy improved to 92.00 %, with saliva, MB, and VS achieving perfect scores at 100.00 %, while blood and semen reached accuracies of 80.00 %. An external validation set comprising n = 25 was employed to assess accuracy using sterile swab carriers; results indicated an overall accuracy rate of 84.00 %. Specifically: both blood and saliva achieved perfect scores at 100.00 %, whereas semen and MB recorded accuracies of 80.00 %. Conversely, the lowest accuracy was observed for VS at 60.00 %. This study marks the first use of a portable e-nose to differentiate common body fluids, offering a promising, low-cost, non-destructive and user-friendly approach for forensic analysis.

鉴定体液污渍对法医调查至关重要。虽然目前的分子检测方法提供了很高的准确性，但它们的破坏性采样性质通过损害样品完整性和随后的短串联重复（STR）分析，对痕量样品施加了显著的限制。迄今为止，尚无无损流体识别方法的报道。本研究介绍了一种新型便携式电子鼻（e-nose）技术，该技术通过检测体液中的挥发性有机化合物（VOCs）来促进非侵入性区分。每次检测只需要3-4 分钟，同时保持DNA的完整性，这种方法有效地区分了形态相似的液体，如血液和经血，为法医体液的非破坏性分析提供了创新的解决方案。在这项研究中，使用电子鼻技术分析了来自200个体液样本的挥发性有机化合物，包括血液，唾液，精液，阴道分泌物（VS）和经血（MB）。通过无菌拭子（n = 100）和卫生纸（n = 75）采集样本。雷达图显示，传感器S7 （W1W）在两个载波上都表现出峰值响应。175个样本的线性判别分析显示出不同的聚类模式（ANOSIM R = 0.088, p <； 0.001）。基于随机森林的预测模型表明，一般模型（n = 175）对VS、血液、精液、MB和唾液的准确率分别为100.00 %、90.91 %、81.82 %、72.73 %和63.64 %；总体精度为81.82 %。值得注意的是，当专门使用卫生纸作为载体时，准确性提高到92.00 %，唾液，MB和VS达到100.00 %的完美分数，而血液和精液的准确性达到80.00 %。采用外部验证集n = 25来评估无菌拭子载体的准确性；结果表明，总体准确率为84.00 %。具体来说：血液和唾液都达到了100% %的完美分数，而精液和MB记录的准确性为80.00 %。相反，VS的最低准确率为60.00 %。这项研究标志着首次使用便携式电子鼻来区分常见的体液，为法医分析提供了一种有前途的、低成本的、非破坏性的和用户友好的方法。

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引用次数: 0

Epigenetic differentiation of monozygotic twins in forensic investigations using nanopore sequencing 单卵双胞胎在法医调查中的表观遗传分化使用纳米孔测序

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-19 DOI: 10.1016/j.fsigen.2025.103344

Kuo Zeng , Kai-bo Yang , Jiang Du , Allah Rakha , Zhenze Liu , Dan-yang Wang , Yun-zhou Chen , Si-wen Wang , Mao-ling Sun , Hongbo Wang , Yi-long Wang , Atif Adnan , Jun Yao

Monozygotic twins (MZTs) pose a significant challenge in forensic genetics due to their identical nuclear DNA, rendering conventional markers like STRs and SNPs ineffective. This limitation highlights the need for novel approaches to distinguish MZTs in criminal investigations, disaster victim identification, and paternity testing. Current methods lack the resolution to differentiate MZTs, creating a gap in human identification. Epigenetic markers, such as DNA methylation, offer a solution by reflecting environmental and stochastic differences post-twinning. However, traditional sequencing technologies often fail to meet forensic requirements for resolution, speed, or reliability. To address this, we employed Oxford Nanopore sequencing to analyze genome-wide DNA methylation patterns in six MZT pairs, identifying robust epigenetic biomarkers for forensic discrimination. Our approach leverages long-read sequencing and single-base resolution to overcome conventional limitations. The study aimed to identify differentially methylated loci (DMLs) as stable, heritable biomarkers for distinguishing MZTs, even in degraded or trace samples, while demonstrating the forensic utility of nanopore sequencing. We identified 3820 shared DMLs enriched in metabolic and neural pathways, localized to promoter (1.84 %) and intergenic (88.03 %) regions. Nanopore sequencing achieved > 99.5 % alignment efficiency and > 13 kb N50 read lengths, enabling rapid, PCR-free analysis. This study demonstrates that nanopore-based methylation profiling effectively distinguishes MZTs by capturing environmentally influenced epigenetic differences, providing actionable biomarkers for forensic discrimination. By bridging epigenetics and forensics, our findings advance precision in human identification, offering transformative tools for criminal casework, disaster victim identification, and paternity testing, addressing a longstanding limitation in resolving cases involving genetically indistinguishable individuals.

由于同卵双胞胎具有相同的核DNA，使得传统的标记如STRs和SNPs无效，因此对法医遗传学提出了重大挑战。这一限制突出了在刑事调查、灾难受害者鉴定和亲子鉴定中区分mzt的新方法的必要性。目前的方法缺乏区分mzt的分辨率，造成了人类识别的空白。表观遗传标记，如DNA甲基化，通过反映双胞胎后的环境和随机差异提供了一个解决方案。然而，传统的测序技术往往不能满足法医对分辨率、速度或可靠性的要求。为了解决这个问题，我们使用牛津纳米孔测序分析了六个MZT对的全基因组DNA甲基化模式，确定了强大的表观遗传生物标志物，用于法医鉴定。我们的方法利用长读测序和单碱基分辨率来克服传统的限制。该研究旨在确定差异甲基化位点（dml）作为区分MZTs的稳定、可遗传的生物标志物，即使在降解或痕量样品中也是如此，同时证明纳米孔测序的法医实用性。我们发现3820个共享的dml富集于代谢和神经通路，定位于启动子（1.84 %）和基因间（88.03 %）区域。纳米孔测序实现了>； 99.5 %的比对效率和>； 13 kb N50读取长度，实现了快速，无pcr分析。这项研究表明，基于纳米孔的甲基化分析通过捕获环境影响的表观遗传差异，有效地区分MZTs，为法医鉴定提供可操作的生物标志物。通过连接表观遗传学和法医学，我们的发现提高了人类鉴定的准确性，为刑事案件工作、灾难受害者鉴定和亲子鉴定提供了变革性的工具，解决了在解决涉及基因难以区分的个体的案件方面的长期限制。

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引用次数: 0

Making AI accessible for forensic DNA profile analysis 使人工智能可用于法医DNA分析

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-19 DOI: 10.1016/j.fsigen.2025.103345

Abel K.J.G. de Wit , Claire D. Wagenaar , Nathalie A.C. Janssen , Brechtje Hoegen , Judith van de Wetering , Huub Hoofs , Simone Ariëns , Corina C.G. Benschop , Rolf J.F. Ypma

Deep learning has the potential to be a powerful tool for automating allele calling in forensic DNA analysis. Studies to date have relied on bespoke model architecture and painstaking manual annotations to train models, which makes it challenging for other researchers to work with these techniques. In this study, we explore the possibility of training a well-performing model using data gathered as part of casework, and employing a widely adopted architecture: the U-Net. In this approach, annotations are created from alleles called during casework. The model, dubbed ‘DNANet’, then classifies each scan point in the electropherogram (EPG) as part of an allele or non-allele, building on the task of segmentation in computer vision. We evaluate performance on unseen case data and on independent mixture research data, taking analyst annotations as ground-truth. We further compare DNANet’s performance with analyst performance on the research data, taking actual donor alleles as ground-truth. DNANet reached an F1 score of 0.971 on analyst annotated alleles on case data not seen during training, and 0.982 on the research data. On actual donor alleles, DNANet reached an F1 score of 0.962, equal to the F1 score computed from analyst annotations. Our results show that DNANet’s performance is comparable to human annotations following standard procedures. This illustrates the potential for obtaining good results with standard data and architecture. Future work may focus on what aspects of data, annotations or model architecture are key in shaping performance. We make our code, model weights and research data publicly available to aid the community. Lastly, we call for an effort to establish a standardized benchmark to aid in quantitative comparisons between allele calling systems.

深度学习有可能成为法医DNA分析中自动调用等位基因的强大工具。迄今为止的研究都依赖于定制的模型架构和艰苦的手工注释来训练模型，这使得其他研究人员使用这些技术具有挑战性。在这项研究中，我们探索了使用作为案例工作的一部分收集的数据来训练一个性能良好的模型的可能性，并采用了广泛采用的架构：U-Net。在这种方法中，注释是根据案例工作期间调用的等位基因创建的。该模型被称为“DNANet”，然后在计算机视觉分割任务的基础上，将电泳图（EPG）中的每个扫描点分类为等位基因或非等位基因的一部分。我们在未见的案例数据和独立的混合研究数据上评估性能，将分析师的注释作为基本事实。我们进一步将DNANet的性能与分析师在研究数据上的性能进行比较，以实际供体等位基因为基础。DNANet对训练中未见的病例数据的分析师注释等位基因的F1得分为0.971，对研究数据的F1得分为0.982。在实际供体等位基因上，DNANet的F1得分为0.962，与分析者注释计算的F1得分相等。我们的结果表明，DNANet的性能与遵循标准过程的人类注释相当。这说明了使用标准数据和体系结构获得良好结果的可能性。未来的工作可能会集中在数据、注释或模型架构的哪些方面是塑造性能的关键。我们将代码、模型权重和研究数据公开，以帮助社区。最后，我们呼吁努力建立一个标准化的基准，以帮助在等位基因呼叫系统之间的定量比较。

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引用次数: 0

Effective use of the degradation index from human DNA quantification kits to improve STR and Y-STR profiling 有效利用人类DNA定量试剂盒的降解指数来改善STR和Y-STR分析

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-19 DOI: 10.1016/j.fsigen.2025.103342

Seiki Nakao, Misa Kitagawa, Koichi Suzuki, Takako Sato

Short tandem repeat (STR) genotyping is primarily used for human identification in various forensic biological samples. However, samples collected from crime scenes or mass disasters are often exposed to environmental factors that cause considerable DNA degradation. As a result, degraded DNA yields significantly less polymorphic information than non-degraded DNA due to a reduction in the effective copy number of STR loci available for amplification. To obtain reliable STR results, it is crucial to consider the degree of DNA degradation alongside accurate DNA quantification. In this study, we investigated the impact of DNA degradation on allele detection in STR and Y-chromosome STR (Y-STR) analyses to improve the estimation of degraded DNA quantity for PCR amplification and enhance allele detection rates. Specifically, we analyzed the relationship between the degradation index (DI) provided by the Quantifiler HP DNA Quantification Kit and allele detection rates in STR and Y-STR analyses using various amounts of artificially fragmented or UV-irradiated DNA. Our results demonstrate that the DI serves as a valuable indicator of DNA degradation, aiding in the estimation of the appropriate amount of degraded DNA for PCR amplification. Furthermore, STR and Y-STR profiles and allele detection rates vary depending on the degradation pattern, such as fragmentation or UV irradiation, even when the DI remains the same. Our findings underscore the importance of incorporating DI into forensic workflows to maximize allele recovery from a limited amount of degraded DNA, ultimately enhancing forensic and disaster victim identification.

短串联重复序列（STR）基因分型主要用于各种法医生物样本的人类鉴定。然而，从犯罪现场或大规模灾难中收集的样本经常暴露在导致相当大的DNA降解的环境因素中。因此，由于可用于扩增的STR位点的有效拷贝数减少，降解的DNA产生的多态性信息明显少于未降解的DNA。为了获得可靠的STR结果，在精确的DNA定量的同时考虑DNA降解的程度是至关重要的。在本研究中，我们研究了DNA降解对STR和y染色体STR （Y-STR）分析中等位基因检测的影响，以改进PCR扩增对降解DNA数量的估计，提高等位基因的检出率。具体来说，我们分析了Quantifiler HP DNA定量试剂盒提供的降解指数（DI）与使用不同数量的人工片段化或紫外线照射的DNA进行STR和Y-STR分析时等位基因检出率之间的关系。我们的研究结果表明，DI是DNA降解的一个有价值的指标，有助于估计用于PCR扩增的降解DNA的适当数量。此外，即使在DI保持不变的情况下，STR和Y-STR谱和等位基因检出率也会根据降解模式（如破碎或紫外线照射）而变化。我们的研究结果强调了将DI纳入法医工作流程的重要性，以最大限度地从有限的降解DNA中恢复等位基因，最终增强法医和灾难受害者的识别。

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引用次数: 0

An Integrated proteomic workflow for body fluid classification and single amino acid variant identification: Advancing towards body fluid source attribution 体液分类和单氨基酸变异鉴定的集成蛋白质组学工作流程：体液来源归属研究进展

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-19 DOI: 10.1016/j.fsigen.2025.103343

Thomas P. Shehata , Shirin Alex , Stijn N.C. van Lierop , Maarten J. Blom , Jantine van de Wetering -Tieleman , Nadine Prust , Jeroen Demmers , Marcel de Puit

A particularly challenging subject in the investigation of forensic human biological traces is analyzing samples containing mixtures of body fluids from multiple donors. Ideally, researchers want to identify each type of body fluid present. However, traditional methods, like mRNA and DNA profiling, often struggle with sensitivity, specificity, and efficiency, especially in complex mixtures. This proof-of-concept study has two primary aims: first, to classify body fluids within a mixture using discriminatory protein markers, and second, to evaluate the feasibility of using single amino acid variants (SAAVs) to trace the source of specific body fluids back to individual donors. To achieve this, we employed proteomic analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-independent acquisition (DIA) mode, developing a reliable approach for accurate body fluid classification. Through comprehensive proteomic profiling, we characterized a diverse array of discriminatory proteins present in peripheral blood, semen, saliva, urine, and vaginal fluid. Using advanced data analysis techniques, including t-distributed stochastic neighbor embedding (t-SNE), we demonstrated that these proteins could reliably distinguish between different body fluids, even in mixed samples. Additionally, our findings reveal that SAAVs within certain proteins, such as those in saliva, hold promise for source attribution in a forensic context. Challenges, including contamination and limited sample sizes, highlighted the need for strict quality controls and further large-scale studies. With these improvements, proteomic analysis could greatly enhance body fluid identification, classification, and source attribution in forensic investigations, improving both accuracy and reliability in forensic science.

在调查法医人类生物痕迹方面，一个特别具有挑战性的课题是分析含有多个供体体液混合物的样本。理想情况下，研究人员希望能识别出存在的每种体液。然而，传统的方法，如mRNA和DNA分析，经常在敏感性，特异性和效率方面挣扎，特别是在复杂的混合物中。这项概念验证研究有两个主要目的：第一，使用歧视性蛋白质标记物对混合物中的体液进行分类，第二，评估使用单氨基酸变体（saav）追踪特定体液来源的可行性，以追溯到个体供体。为了实现这一目标，我们采用数据独立采集（DIA）模式，通过液相色谱-串联质谱（LC-MS/MS）进行蛋白质组学分析，开发了一种可靠的准确体液分类方法。通过全面的蛋白质组学分析，我们表征了外周血、精液、唾液、尿液和阴道液中存在的多种歧视性蛋白质。使用先进的数据分析技术，包括t分布随机邻居嵌入（t-SNE），我们证明这些蛋白质可以可靠地区分不同的体液，即使在混合样本中。此外，我们的研究结果表明，某些蛋白质中的saav，如唾液中的saav，在法医环境中具有来源归属的希望。污染和样本量有限等挑战凸显了严格质量控制和进一步大规模研究的必要性。有了这些改进，蛋白质组学分析可以大大提高法医调查中的体液鉴定、分类和来源归属，提高法医科学的准确性和可靠性。

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引用次数: 0

Development and validation of a capture sequencing panel containing 9000 SNPs for inferring distant relatives in East Asian populations 包含9000个snp的捕获测序面板的开发和验证，用于推断东亚人群的远亲

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-18 DOI: 10.1016/j.fsigen.2025.103341

Kuo Zeng , Wenting Zhao , Zhixiao Fang , Jing Li , Jing Liu , Dong Zhao , Bofeng Zhu , Caixia Li

Inferring distant relatives has long presented a significant challenge in forensic science. Recently, forensic researchers have increasingly focused on single nucleotide polymorphisms (SNPs) as a potent tool for this purpose. In this study, we developed and validated a capture sequencing panel comprising 9000 SNPs specifically aimed at inferring distant relatives within East Asian populations. Initially, we screened the 9000 SNPs from four data sources: the Infinium Global Screening Array, the Infinium Chinese Genotyping Array, the Single-Nucleotide Polymorphism database, and the 1000 Genomes Project. Subsequently, we established a likelihood ratio (LR)-based algorithm utilizing pedigree genotyping data from Han Chinese populations. Next, we constructed a sequencing method for the 9000 SNPs employing hybridization capture sequencing technology. Finally, the 9000 SNP panel was evaluated following the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), including studies on repeatability, concordance, sensitivity, species specificity, PCR inhibition, DNA degradation, DNA mixture and casework-type samples. The results demonstrated that the 9000 SNPs exhibited considerable genetic polymorphism within East Asian populations, with an average minor allele frequency of 0.4521. The panel of 9000 SNPs was demonstrated to reliably identify relatives up to the 5th degree and certain 6th degree using the GSA SNP array for pedigree genotyping. Furthermore, the 9000 SNP panel yielded robust and reliable genotyping results for trace DNA (1.953 ng), degraded DNA (50 bp), and mixed DNA (19:1 ratio), showing specific species specificity and resistance to PCR inhibition. In conclusion, this study highlights the significant potential of the 9000 SNP panel for inferring distant relatives in East Asian populations, providing a valuable tool for forensic applications. Further validation in a larger sample size is needed to confirm our observations.

长期以来，推断远亲一直是法医学的一个重大挑战。最近，法医研究人员越来越关注单核苷酸多态性（snp）作为这一目的的有力工具。在这项研究中，我们开发并验证了一个包含9000个snp的捕获测序面板，专门用于推断东亚人群中的远亲。最初，我们从四个数据源中筛选了9000个snp: Infinium全球筛选阵列、Infinium中国基因分型阵列、单核苷酸多态性数据库和1000基因组计划。随后，我们利用汉族人群的家系基因分型数据建立了基于似然比（LR）的算法。接下来，我们利用杂交捕获测序技术构建了9000个snp的测序方法。最后，根据DNA分析方法科学工作组（SWGDAM）的验证指南对9000个SNP面板进行评估，包括重复性、一致性、敏感性、物种特异性、PCR抑制、DNA降解、DNA混合和案例类型样本的研究。结果表明，9000个单核苷酸多态性在东亚人群中表现出明显的遗传多态性，平均小等位基因频率为0.4521。使用GSA SNP阵列进行家谱基因分型，9000个SNP被证明可以可靠地识别到5度和某些6度的亲属。此外，9000 SNP小组对痕量DNA(1.953 ng)，降解DNA（50 bp）和混合DNA（19:1比例）的基因分型结果稳健可靠，显示出特定的物种特异性和对PCR抑制的抗性。总之，本研究强调了9000个SNP面板在推断东亚人群远亲方面的巨大潜力，为法医应用提供了有价值的工具。需要在更大的样本量中进一步验证来证实我们的观察结果。

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引用次数: 0

Potential application of hair shaft for human identification by mRNA polymorphism 毛干mRNA多态性在人体鉴定中的潜在应用

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-15 DOI: 10.1016/j.fsigen.2025.103340

Jinding Liu , Bing Du , Yuxin Zhang , Hailing Yang , Jiangwei Yan , Gengqian Zhang

Recently, RNA has shown great potential for use in forensic genetics. Our previous work indicated that hair shafts possess detectable RNA levels. Fallen hair samples at crime scenes are common, but human identification is difficult because of the degeneration of traceable nuclear DNA. We aimed to establish a new mRNA polymorphism assay for the identification of hair shafts in humans. In this study, we utilized polymorphic mRNAs to obtain a human identification profile that is more detailed than that of any previously reported method. Ten to fifteen pieces of 5-cm hair shafts were used to extract total RNA from 40 individuals. RNA was transcribed into cDNA and typed on a BGISEQ T7 platform using a massively parallel sequencing assay encompassing 404 coding region and untranslated region (UTR) single nucleotide polymorphisms (SNPs) from 78 genes. The multiplex assay was evaluated for sensitivity, species specificity, capability for aged hair shafts, consistency of the typing results for hair borne from different body parts, and genomic DNA (gDNA)/mRNA of the same individual. We also obtained the genetic parameters for human identification in a Chinese population. Genes that did not meet this threshold were excluded from the analysis. Ultimately, 71 genes containing 284 SNPs, amplified with 228 amplicons were retained. Polymorphisms were observed in 210 amplicons. The random match probability (RMP) values ranged from 1.24 × 10⁻⁴⁴ to 1.14 × 10⁻⁷² (median = 4.36 × 10⁻⁵⁰). When one piece of 5-cm hair shaft was used, 70.31–72.05 % of the amplicons could be detected, and 46.72–62.01 % of the amplicons showed the same genotype as 15 pieces of hair shafts. A total of 44–82 amplicons were detected in hair shafts from four common animals (cats, dogs, rabbits, and rats). However, the genotyping of most SNP/microhaplotype (MH) markers was inconsistent with the database records. This study provides a new strategy for human identification of hair shafts.

近年来，RNA在法医遗传学中显示出巨大的应用潜力。我们之前的工作表明，毛干具有可检测的RNA水平。犯罪现场掉落的头发样本很常见，但由于可追溯的核DNA退化，人类鉴定很困难。我们的目的是建立一种新的mRNA多态性测定方法来鉴定人类毛干。在这项研究中，我们利用多态mrna获得了比以前报道的任何方法更详细的人类鉴定概况。用10到15根5厘米长的毛干提取40个人的总RNA。将RNA转录成cDNA，并在BGISEQ T7平台上进行大规模平行测序，包括来自78个基因的404个编码区和非翻译区（UTR）单核苷酸多态性（snp）。评估多重检测的敏感性、物种特异性、对衰老毛干的检测能力、不同身体部位毛发分型结果的一致性以及同一个体的基因组DNA /mRNA。我们还获得了在中国人群中进行人类鉴定的遗传参数。不符合这一阈值的基因被排除在分析之外。最终，保留了71个基因，包含284个snp，扩增了228个扩增子。210个扩增子存在多态性。随机匹配概率（RMP）取值范围为1.24 × 10−44 ~ 1.14 × 10−72，中位数为4.36 × 10−50。以1根5 cm毛干为样本，扩增子的检出率为70.31 ~ 72.05 %，与15根毛干基因型一致的扩增子为46.72 ~ 62.01 %。在四种常见动物（猫、狗、兔和大鼠）的毛干中共检测到44-82个扩增子。然而，大多数SNP/微单倍型（MH）标记的基因分型与数据库记录不一致。本研究为人类毛干鉴定提供了一种新的策略。

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引用次数: 0

Quantitative differential analysis of tsRNAs for forensic body fluid identification: RT-qPCR-based discrimination derived from epithelial cell fluids screening 法医体液鉴定tsRNAs的定量差异分析：基于上皮细胞液筛选的rt - qpcr鉴别

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-12 DOI: 10.1016/j.fsigen.2025.103338

Zhilong Li , Bin Zhou , Min Su , Hongwu Ren , Jiaru Li , Li Wang , Lin Zhang

Identification of body fluid types at crime scenes is a critical step in forensic science, providing essential context for criminal investigations and the interpretation of evidence. Epigenetic markers, particularly small non-coding RNAs (sncRNAs), have attracted increasing attention in forensic body fluid identification, with various small RNA species demonstrating potential as biomarkers for distinguishing different body fluid types. A novel class of sncRNAs, tRNA-derived small RNAs (tsRNAs), has been detected in various biological samples, yet their potential application in forensic body fluid identification remains unexplored. In this study, we identified differentially expressed tsRNAs in saliva and vaginal secretions, two epithelial cell-derived body fluids. Using stem-loop reverse transcription followed by SYBR Green quantitative polymerase chain reaction (RT-qPCR), we measured tsRNA abundance with U6-snRNA as the reference control. After implementing quality control measures to ensure detection accuracy and adjusting Cq values, we identified 10 tsRNAs that were differentially expressed between saliva and vaginal secretions. To further investigate the potential of tsRNAs in forensic body fluid identification, we extended our analysis to include semen, peripheral blood, and menstrual blood. Our results revealed 18 tsRNAs with significant differential expression patterns across these five body fluids. By integrating quantitative expression data with a logistic regression machine learning model, a panel of 9 tsRNAs achieved 98 % accuracy in leave-one-out cross-validation. While our findings demonstrate the promising potential of tsRNAs as biomarkers for forensic body fluid identification, additional body fluid-specific tsRNAs would likely enhance prediction accuracy while potentially reducing the number of required markers. This preliminary validation establishes tsRNAs as valuable candidates for forensic body fluid identification applications.

鉴定犯罪现场的体液类型是法医学的一个关键步骤，为刑事调查和解释证据提供必要的背景。表观遗传标记，特别是小非编码RNA (sncRNAs)，在法医体液鉴定中引起了越来越多的关注，各种小RNA物种显示出作为区分不同体液类型的生物标记物的潜力。一类新的sncRNAs， trna衍生的小rna (tsRNAs)，已经在各种生物样品中检测到，但它们在法医体液鉴定中的潜在应用仍未探索。在这项研究中，我们在唾液和阴道分泌物（两种上皮细胞来源的体液）中发现了差异表达的tsRNAs。采用茎环反转录和SYBR Green定量聚合酶链反应（RT-qPCR），以U6-snRNA为对照，测定tsRNA丰度。在实施质量控制措施以确保检测准确性和调整Cq值后，我们鉴定出10个在唾液和阴道分泌物中差异表达的tsRNAs。为了进一步研究tsrna在法医体液鉴定中的潜力，我们将分析范围扩大到精液、外周血和经血。我们的研究结果揭示了18种tsRNAs在这五种体液中具有显著差异的表达模式。通过将定量表达数据与逻辑回归机器学习模型相结合，9个tsrna的面板在留一交叉验证中达到了98% %的准确率。虽然我们的研究结果表明tsrna作为法医体液鉴定的生物标记物具有很大的潜力，但额外的体液特异性tsrna可能会提高预测的准确性，同时可能减少所需标记物的数量。这一初步验证确立了tsrna作为法医体液鉴定应用的有价值候选者。

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引用次数: 0

A septennium review of wildlife forensic DNA analysis in South Africa 南非野生动物法医DNA分析的九十年回顾

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-11 DOI: 10.1016/j.fsigen.2025.103339

Marli de Bruyn , Desiré Lee Dalton , Cindy K. Harper , Mamadi Theresa Sethusa

The application of scientific research tools and technologies in wildlife forensic analysis is fundamental to support law enforcement in the regulation and enforcement of illegal criminal activities. Validated genetic technologies and techniques have proven to be critical in securing successful prosecutions specifically through the examination of DNA from physical exhibit material. In South Africa, DNA techniques and tools have been implemented to identify and characterise biological evidence of wildlife, in answering questions that arise during crime investigation and prosecution. Here, we describe, and review wildlife forensic cases analysed in South Africa (by South African National Biodiversity Institute (SANBI) and the Veterinary Genetic Laboratory (VGL)) over a seven-year period (August 2017 to July 2024). In total, 3 763 wildlife forensic cases were analysed. The taxonomic representation was skewed towards mammals encompassing 94.1 % of all cases due to large amount of wildlife cases involving black and white rhinoceros, African elephant, lion and antelope. These cases were predominantly from the north-eastern parts of the country including Limpopo, Mpumalanga and KwaZulu-Natal provinces which have previously been classified as a ‘hotspot’ for poaching. The type of analysis requested varied between the different taxonomic groups with 90 % of mammal cases submitted for DNA comparison, while bird, reptile, fish and invertebrate cases were mainly submitted for species identification (>87 %). This paper further reviews the successes and challenges encountered from a South African perspective and provides future recommendations.

在野生动物法医分析中应用科学研究工具和技术对于支持执法部门监管和执法非法犯罪活动至关重要。经过验证的遗传技术和技术已被证明是确保成功起诉的关键，特别是通过检查物证材料中的DNA。在南非，已经实施了DNA技术和工具来识别和描述野生动物的生物证据，以回答犯罪调查和起诉期间出现的问题。在这里，我们描述并回顾了南非（由南非国家生物多样性研究所（SANBI）和兽医遗传实验室（VGL））在七年期间（2017年8月至2024年7月）分析的野生动物法医案例。共分析了3 763宗野生动物法证个案。由于大量的野生动物病例涉及黑、白犀牛、非洲象、狮子和羚羊，因此分类代表性偏向于哺乳动物，占所有病例的94.1% %。这些案件主要发生在该国东北部，包括林波波省、姆普马兰加省和夸祖鲁-纳塔尔省，这些省以前被列为偷猎的“热点”。不同分类类群所要求的分析类型不同，哺乳动物的90 %用于DNA比较，而鸟类、爬行动物、鱼类和无脊椎动物的DNA鉴定主要用于物种鉴定（>87 %）。本文从南非的角度进一步回顾了成功和遇到的挑战，并提出了未来的建议。

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引用次数: 0

Establishment and application of a triplex RTqPCR assay system for the identification of forensic body fluids 法医体液鉴定三重RTqPCR检测系统的建立与应用

IF 3.1 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics

Pub Date : 2025-08-05 DOI: 10.1016/j.fsigen.2025.103337

Shuxiao Hu , Liping Chen , Jiayao Duan , Sheng Hu , Yixia Zhao , Yang Li , Ruiqin Yang , Anquan Ji , Jie Lian , Qifan Sun

MicroRNAs (miRNAs) have emerged as valuable biomarkers for the identification of forensic body fluids due to their stability and tissue specificity. However, the limited quantity of body fluid at crime scenes often hampers the accuracy of miRNA-based detection methods. In this study, we developed a triplex reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay system that enables the simultaneous detection of three miRNAs, improving throughput and efficiency while overcoming challenges in forensic investigations. First, the primers and probes of the miRNAs were redesigned to meet multiple detection requirements on the basis of a previous study in which five types of body fluid-specific miRNAs and internal genes (miR-451a, miR-891a-5p, miR-144–5p, miR-203a-3p, miR-223–3p and miR-320a-3p) were screened in a laboratory. The primer and probe concentrations, premix concentration and annealing temperature were subsequently optimized to establish a triplex RTqPCR assay system. This system enables the simultaneous reverse transcription of six miRNAs from a single sample, followed by two separate triplex amplification reactions to quantitatively analyze all six miRNA markers. The amplification efficiency, primer cross-reactivity, repeatability, triplex detection and single detection results of the system were subsequently analysed, and a prediction model was constructed by combining the sample data with a kernel density estimation (KDE) method. Finally, the ability of the detection method to identify body fluids was further verified with authentic samples. The results demonstrate that the optimized triplex RTqPCR assay system achieves the same detection performance as the single detection system, but is faster and more cost-effective. This technology is especially suitable for the detection of trace body fluid stains left at crime scenes and effectively solves the contradiction between the requirements of repeated RTqPCR detection of traces and multiple sample sizes. In addition, the body fluid identification model, which was established by the data obtained from the triplex RTqPCR system combined with KDE, was successfully applied to predict and identify simulated samples and actual samples. This system provides an effective tool for the identification of suspicious body fluids and lays the foundation for further research on multiplex RTqPCR assay systems and the construction of more accurate data models.

由于其稳定性和组织特异性，MicroRNAs （miRNAs）已成为鉴定法医体液的有价值的生物标志物。然而，犯罪现场的体液数量有限，往往会影响基于mirna的检测方法的准确性。在这项研究中，我们开发了一种三重逆转录-定量聚合酶链反应（RT-qPCR）检测系统，可以同时检测三种mirna，提高了通量和效率，同时克服了法医调查中的挑战。首先，在先前实验室筛选五种体液特异性mirna及其内部基因（miR-451a、miR-891a-5p、miR-144-5p、miR-203a-3p、miR-223-3p和miR-320a-3p）的基础上，重新设计mirna的引物和探针，以满足多种检测需求。随后对引物和探针浓度、预混料浓度和退火温度进行优化，建立了三重RTqPCR检测体系。该系统能够同时对来自单个样品的六个miRNA进行逆转录，然后进行两个单独的三重扩增反应，以定量分析所有六个miRNA标记物。随后对该体系的扩增效率、引物交叉反应性、重复性、三检和单检结果进行分析，并结合样本数据和核密度估计（KDE）方法构建预测模型。最后，用真实样本进一步验证了该检测方法对体液的识别能力。结果表明，优化后的三联RTqPCR检测系统与单联检测系统的检测性能相同，但速度更快，成本更低。该技术特别适用于犯罪现场遗留的微量体液污渍的检测，有效解决了RTqPCR重复检测痕迹的要求与多样本量之间的矛盾。此外，将三重RTqPCR系统获得的数据与KDE相结合建立的体液鉴定模型成功地应用于模拟样品和实际样品的预测和鉴定。该系统为可疑体液的鉴定提供了有效的工具，为进一步研究多重RTqPCR检测系统和构建更准确的数据模型奠定了基础。

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引用次数: 0