单细胞检验技术用于混合上皮细胞的分离和检验

目的建立单细胞显微捕获联合低体积扩增技术,用于混合上皮细胞检材分离检验。方法取5名男性口腔上皮细胞拭子浸泡液30μL,分别滴加到5份含同一女性皮肤表皮细胞拭子上,制成5份混合上皮细胞样本为实验组,同时制备同样的5份样本为对照组。实验组样本采用显微捕获单个口腔上皮细胞,并使用低体积扩增技术进行扩增;对照组用M48纯化试剂盒提取DNA,Identifiler试剂盒复合扩增,扩增体系为10μL。所有产物均用ABI 3130遗传分析仪进行STR分型。结果 5份实验组样本均得到男性STR分型结果,5份对照组样本则均仅得到混合分型结果。将该方法应用于1例强奸杀人案例检验,取得了满意效果。结论单细胞显微捕获联合低体积扩增技术可用于混合上皮细胞样本的分离检验。     

龙胆紫染色法在显微捕获单细胞分离技术中的应用

目的建立用于显微捕获单细胞技术的龙胆紫染色法并评价其应用效果。方法制作20枚口腔拭子脱落细包悬液,配制0．05g／mL龙胆紫染液。取100μL口腔细胞悬液加入0．15、0．25、0．5、1．0、2．0μL染色液,考察最佳染色浓度;争别染色5、10、30、60min,考察最佳染色时间;用最佳条件染色后抓捕3个细胞,采用联合LV-PCR技术扩增并进行DNA分驯佥测,进行重复试验20次,并对同一检材未经染色的抓捕细胞进行检测,用于比对STR分型成功率。将龙胆紫染色法用于案例检材的口腔上皮细胞分离检验。结果1001μL细胞悬液加入0．5止0．05g／mL龙胆紫染液,染色5min,胞核呈紫工色,与胞浆对比明显;染色时间延长不影响染色效果及细胞分离检验结果。优化后的龙胆紫染色法对低体积扩增无归显抑制（P〉0．05）。应用此染色法于案例检材,胞核标识清晰,STR分型结果达同一认定标准。结论龙胆紫染色法刚于细胞核的发现,有助于提高显微捕获单细胞技术的检测效能。     

脱落上皮细胞类生物检材的自动化提取

目的建立一种自动化提取脱落上皮细胞类生物检材DNA的方法。方法附着于不同载体上的脱落上皮细胞类生物检材共278份,应用Eppendorf epMotion 5075LH工作站结合DNA IQTM系统提取模板DNA,并用Identifiler试剂盒进行STR检验。结果在278份被检的生物检材,其中126份检材获得13个基因座以上的STR分型,不同类型的检材其检出率不相同,最高达73.44%,最低为10.89%。结论脱落上皮细胞类检材应用自动化工作站提取DNA模板可在法医日常检案中广泛应用。     

微量细胞DNA分型的法医学应用研究进展

微量细胞DNA分型已成为目前法医学研究的热点之一。如果能够从犯罪现场遗留的微量疑难生物检材中提取到细胞,并获得DNA分型,这无疑将会大大拓宽检材的范围。同时可以为刑事案件的侦查提供线索、为法庭量刑提供有力的证据。本文从微量细胞的捕获、DNA模板的制备、扩增和检测及应用前景几个方面综述了微量细胞DNA分型在法医学中的研究进展。     

微量细胞DNA分型的法医学应用研究进展

微量细胞DNA分型已成为目前法医学研究的热点之一。如果能够从犯罪现场遗留的微量疑难生物检材中提取到细胞，并获得DNA分型，这无疑将会大大拓宽检材的范围。同时可以为刑事案件的侦查提供线索、为法庭量刑提供有力的证据。本文从微量细胞的捕获、DNA模板的制备、扩增和检测及应用前景几个方面综述了微量细胞DNA分型在法医学中的研究进展。     

国产磁珠结合自动化工作站批量提取生物检材DNA的应用

目的建立国产磁珠结合自动化工作站批量提取案件中生物检材DNA的方法。方法采用国产磁珠结合Bio-Robert Universal System自动化工作站对案件中常见的生物样本进行DNA提取,检测Identifiler系统16个STR基因座,在ABI3130XL遗传分析仪上进行STR分型。其中210份样品同时在ABI7500型荧光定量PCR仪上进行定量。结果9100份10类生物检材应用国产磁珠结合自动化工作站,大部分可提取到足够的DNA进行STR检验。STR检验成功率最高的为口腔拭子、肌肉,达100%,接触细胞检材的成功率较低,为50.0%。结论国产磁珠结合自动化工作站可用于案件中常见的大部分生物样本的DNA提取。     

混合生物检材的个人识别及研究进展

混合生物检材一般指两个或多个生物学成分混杂在一起的一类检材,包括不同个体来源的精液、阴道分泌液、血液、尿液、汗液等形成的混合斑迹和含有少量胎儿细胞的母体外周血等。这类检材的个体识别是法医学实际检案中经常遇到。由于不同个体的遗传物质混合存在,往往给检验工作带来很大的麻烦。多年来,学者们就这一问题进行了广泛的探讨,积累了不少成功的经验。与分析单纯生物检材一样,分析混合生物检材所使用的遗传标记一般也为血型、多态蛋白等表型标记及DNA多态标记,但在检验方法及分析策略上则有所不同。本文就各类遗传标记在混合检材中…     

定量PCR技术在法医学中应用的研究

目的研究荧光定量PCR技术在法医学中的应用。方法应用Taqman技术对法医各种生物检材进行DNA定量。结果该定量PCR技术对各种法医生物检材进行了准确定量,并判断检材中是否存在抑制物,从而指导了后续STR的检验。结论定量PCR技术是法医DNA检验中一项不可缺少的辅助技术。     

生物检材采集与保存套管的应用

目的探讨生物检材采集与保存套管在法医学中的应用价值。方法在不同温、湿度环境下，观察悬空放置在生物检材采集与保存套管、有孔与外界相通的套管及密闭管内的湿润棉签的干燥时间30次；分别用生物检材采集与保存套管和医用棉签采集纸袋保存口腔细胞、血液、皮肤脱落细胞样本各20例，磁珠法提取DNA并进行DNA定量；用生物检材采集与保存套管采集口腔脱落细胞和血样进行DNA直接扩增。结果在温度4～30℃、相对湿度21％～90％的环境下，湿润棉签在套管内的平均干燥时间为7．89h，有孔管内的为23．30h，密闭管内观察15天仍不干燥，出现霉斑。生物检材用套管采集保存比医用棉签采集纸袋保存方式获得的DNA量显著提高，平均高达0．968倍；用套管采集口腔细胞和血样进行直接扩增，操作简单方便，成功率高。结论生物检材采集与保存套管具有快速干燥、对检材无损耗和浓缩等优点，可提高检材DNA的提取效率，且适合直接扩增。     

改良EVO150-8方法在DNA自动化检案中的应用

目的探讨改良EVO150-8方法在批量生物检材DNA检验中的应用价值,建立一种自动化、简单、快速的DNA提取方法。方法采用改良EVO150-8自动化核酸提取纯化仪器与DNA IQ磁珠法纯化试剂盒,对各现场提取的880份血迹、烟蒂、口香糖、精斑（混合斑）、组织、骨骼、脱落细胞等常见生物检材进行DNA提取与纯化,采用Identifiler试剂盒进行扩增检验,用3130XL电泳,GeneMapper ID V3.2分析软件进行分析比对。结果在880份生物检材中,有836份检材成功获得STR分型;检验92份检材仅需时128min。结论改良EVO150-8适合批量生物检材的自动化提取。     

激光显微捕获口腔上皮细胞的DNA分型

目的探索激光显微技术(lasercapturemicrodissectionsystem,LCM)捕获口腔上皮细胞,并进行STR-DNA分型检测的方法。方法用VERITAS显微切割仪红外低能激光显微捕获一定数量口腔上皮细胞,进行ProfilerPlus试剂盒STR复合扩增,检测DNA基因型。结果7～8个口腔上皮细胞能成功获得STR-DNA分型。3～4个口腔上皮细胞不能成功获得STR-DNA分型。结论激光显微捕获作为一种分离单个细胞的新技术,对于微量口腔上皮细胞的STR-DNA分型是可行的。     

激光捕获显微切割技术用于分离混合斑中精子细胞

目的 评估激光捕获显微切割(laser capture microdissection.LCM)技术在分离混合斑中少量精子细胞的应用价值.方法 配制不同比例的精液-阴道上皮细胞混合液.分别用差异裂解法和LCM法分离精子细胞,用磁珠法提取精子细胞DNA,并用IdentifilerTM试剂盒进行STR基因型检测.结果 LC...     

染色混合精斑涂片DNA检验方法的研究

建立了染色混合精斑涂片DNA的提取方法,通过两步扩增的技术与垂直聚丙烯酰胺凝胶电泳及银染色法,成功检测了3个STR位点.结果显示,所有染色涂片DNA分型均与对照血样一致,苏木素-伊红(HE)染色比酸性复红-美蓝(Baecchi)染色对DNA的降解作用要小;涂片灵敏度检验可达到少于10个精子,且室温保存一年以内的涂片其灵敏度光显著差异.该方法简单、快速、可靠,为染色混合精斑涂片的PCR分析提供了新方法.     

Demonstration of a gastric bioptic specimen mix-up by laser capture microdissection (LCM) and DNA fingerprinting

We demonstrate here the successful use of laser capture microdissection (LCM) and DNA fingerprinting in the identification of a case of gastric bioptic specimen mix-up. A 70-year-old man, suffering from chronic atrophic gastritis, underwent to a gastric biopsy and received a diagnosis of gastric cancer. In the absence of any clinical evidence of gastric cancer, a specimen mix-up was suspected. LCM was used to retrieve gastric cells from the histologic slide, classified as gastric carcinoma, and suspected to be mislabelled. DNA was extracted from microdissected cells, and a total of 16 different genetic loci were analyzed, using an identity test. Comparison of the results with those obtained using DNA extracted from a control slide, and from patient's saliva, demonstrated a distinct DNA fingerprint pattern in all genetic markers examined, clearly indicating the occurrence of a specimen mix-up. The combined use of LCM and DNA fingerprinting represents the most accurate and sophisticated method available for the identification of specimen mix-up, especially when only the tissue on the suspected slide is available.     

激光显微捕获技术用于尿液脱落细胞DNA检测

目的采用激光显微捕获技术（LCM）捕获尿液脱落细胞,并进行STR分型。方法收集10份健康成人尿液样本,根据储存时间分组,其中新鲜尿液组（≤24h）分别采用Chelex-100及LCM联合DNA IQTM提取法提取DNA,储存尿液组（〉24h）再分为4℃组和室温组,分别在4～30d内不同时间点采用LCM联合DNA IQTM提取法提取DNA;各组提取的模板DNA进行扩增及SRT分型检验。结果新鲜尿液组采用LCM联合DNA IQTM提取法提取DNA,所有样本均可检出全部基因座（16个）,采用Chelex-100法则在部分基因座上出现等位基因丢失、非特异性扩增、峰值低等现象;4℃储存10d和室温储存4d以内的尿液经检验可明确判读12个以上基因座,4℃20～30d及室温7d,可检出7个以上基因座。结论 LCM技术可用于尿液检材的DNA分型检验,且检材应尽可能4℃保存并尽快检验。     

Locard's Principle of Exchange,Dental Examination and Fragments of Skin

The transfer of materials between victim and perpetrator was first reported by Locard in the nineteenth century. While in recent years DNA testing has been very successful in matching biological material from crime scenes to perpetrators, the following cases demonstrate that other more time‐honored methods remain useful. Two cases of lethal assault are reported where the victims had bitten their assailants resulting in fragments of the perpetrators’ skin being wedged between their teeth which were discovered during post mortem oral examinations. As the fragments were able to be matched to injuries in the perpetrators, identification was established prior to confirmatory DNA testing. In case 1 a criminal conviction for manslaughter resulted, and in case 2 the identity of the assailant was confirmed. Examination of a properly exposed and illuminated oral cavity may provide useful evidence in assault cases. These cases represent an unusual dental variant of Locard's principle.     

荧光原位杂交技术在法庭科学DNA检验中的应用

目的运用荧光原位杂交技术结合激光显微切割技术,分离法医物证男女混合样本中的男性细胞和女性细胞,并进行DNA分型。方法通过双色荧光原位杂交,Y染色体标记上绿色信号,X染色体标记上红色信号,在荧光显微镜下识别男性细胞和女性细胞,并通过激光显微切割技术分别获得男性细胞和女性细胞进行DNA分型。结果运用荧光原位杂交技术,能够分别标记法医物证男女混合样本中的男性细胞和女性细胞,并通过激光显微切割技术获得各自的DNA分型。结论荧光原位杂交技术结合激光显微切割技术,可应用于法医物证男女混合样本的检验,提高个体识别能力。     

The Use of Laser Microdissection in Forensic Sexual Assault Casework: Pros and Cons Compared to Standard Methods

Sexual assault samples are among the most frequently analyzed in a forensic laboratory. These account for almost half of all samples processed routinely, and a large portion of these cases remain unsolved. These samples often pose problems to traditional analytic methods of identification because they consist most frequently of cell mixtures from at least two contributors: the victim (usually female) and the perpetrator (usually male). In this study, we propose the use of current preliminary testing for sperm detection in order to determine the chances of success when faced with samples which can be good candidates to undergo analysis with the laser microdissection technology. Also, we used laser microdissection technology to capture fluorescently stained cells of interest differentiated by gender. Collected materials were then used for DNA genotyping with commercially available amplification kits such as Minifiler, Identifiler Plus, NGM, and Y‐Filer. Both the methodology and the quality of the results were evaluated to assess the pros and cons of laser microdissection compared with standard methods. Overall, the combination of fluorescent staining combined with the Minifiler amplification kit provided the best results for autosomal markers, whereas the Y‐Filer kit returned the expected results regardless of the used method.     

Fixation of cells for analysis by laser microdissection--comparative studies in forensic trace material

This paper is focused on the preparation of samples for laser microdissection (LM) in forensic casework. In forensic genetics, it is essential to preserve and separate cellular traces during sample preparation, as they are usually gathered in very small amounts and are often contaminated with undesired cells. This is made possible by laser microdissection, a technique developed to cut cells or tissue of a certain type from a microscopical specimen by UV laser and catapult them directly into a PCR reactor. This method minimizes the risk of getting inconclusive, mixed DNA profiles due to contamination by foreign DNA and also supplies information about the cellular origin of a DNA profile. A method for optimized fixation and staining of spermatozoa for laser microdissection was established. Four different fixation methods combined with two staining methods were tested on two different microscope slides. Moreover, the effect of a blocker pen to contain the specimen on the slide was investigated.     

Standard 28-cycle amplification of X/Y-FISH labelled epithelial cells for DNA profiling

The examination of sexual assault evidence frequently involves the analysis of samples that comprise mixtures of male and female cells. Separating male and female cells benefits analysis as the results are more likely to be simplified into profiles from single contributors. Some separation methods have focussed on separation of sperm from epithelial cells, but samples without sperm also require separation (vasectomised males, licked skin, etc.). X/Y chromosome FISH labelling when combined with laser micro-dissection (LMD) is a reliable method to separate male and female epithelial cells, but has mostly been combined with increased cycle PCR to create DNA profiles, limiting its use in many forensic laboratories. This study aimed to determine the limits of cell numbers collected by LMD for standard 28-cycle DNA profiling, and to test the effects, if any, on stochastic variation normally caused by sampling effects. Male and female epithelial cells were stained using the Vysis CEP X/Y DNA Probe kits, and collected using a Leica LMD6000. DNA was extracted and amplified by the ESR in-house one-tube method, using standard 28-cycle PCR with the AmpFISTR Identifiler™ (Applied Biosystems) multiplex kit. Full IdentifilerTM DNA profiles were produced using standard 28-cycle PCR, and partial profiles suitable for submission were produced from even relatively low numbers of cells collected. Profiling results were compared with low-copy number PCR on low numbers of cells stained and collected in the same manner, and the observed effects on heterozygote balance are discussed.