激光捕获显微分离技术分离多人混合精斑

目的利用激光捕获显微分离技术(LCM)分离多供体混合精斑。方法将3名自愿者供给的精液混合后,制备成混合精斑,并涂于覆膜玻片上。用PALM激光捕获显微分离系统切割并捕获样本中的单个精子,利用低体积扩增技术(LV-PCR)扩增并进行检测,并对分型结果进行统计。结果联用LCM和低体积扩增技术捕获样本中单个精子细胞进行扩增,可获得每位自愿者的Y-STR分型,分型成功率为73.15%。结论激光捕获显微分离联用低体积扩增技术可完成多供体混合精斑的分离检验。     

尼龙膜套管分离技术对混合斑中精子细胞DNA的提取

目的建立新型的混合斑中精子细胞分离的方法,并评价其应用价值。方法收集性侵案件中的40份混合斑检材,分别采用常规差异裂解法和尼龙膜套管分离技术进行精子细胞分离,使用硅膜试剂盒(Forensic DNA Extraction Kit for Soft Tissues)提取精子细胞DNA,Amp FlSTR~ Identifiler~ Plus PCR扩增试剂盒进行PCR扩增,3500x L基因分析仪进行电泳检测,并对两种分离方法的结果进行对比。结果 40份混合斑检材中,采用尼龙膜套管分离技术的检材仅3份有女性成分微弱残留,余均获得了完整的单一男性分型。而采用常规差异裂解法分离的检材中,有25份完全未检出男性精子细胞STR分型,15份检出男性精子细胞STR分型(7份不完整男性精子细胞STR分型,6份有女性成分残留,2份单一的男性精子细胞STR分型)。结论本研究建立的尼龙膜套管分离技术适用于混合斑中精子细胞的分离,特别是对含有大量女性细胞而精子细胞较少的检材提取效果明显,整个提取实验成本低廉、快速简便。     

显微操作法提取混合斑中精子细胞方法的探讨

目的尝试建立一种提取混合斑中精子细胞的检测方法。方法在人为控制条件下,制备精液—阴道液混合斑,分别使用显微操作法与差异裂解法分离精子细胞,提取DNA,进行STR基因型检测。结果采用显微操作法检测成功率11/12,差异裂解法成功率1/12,两者有显著性差异。结论显微操作法可有效获取精子细胞,排除女性物质和其它杂质的干扰,STR分型成功率优于差异裂解法。     

精子细胞定向捕获与分离技术初步研究

目的探索并研究精子细胞定向捕获与分离技术,初步建立精子细胞特异性分离与DNA提取的方法与试剂体系。方法通过特异性定向捕获复合体（精子特异性抗体一磁性纳米微球）的制备,在一定的试剂体系环境下,实现精子细胞的定向捕获与分离。结果能够实现精子细胞的定向富集与分离,通过后续的提取过程,获得了高质量的DNA,并获得了相应的完整STR分型结果。     

FISH联用激光捕获显微分离技术对混合斑进行分离检验

目的建立荧光原位杂交(FISH)联用激光捕获显微分离技术(LCM)精确分离男女混合斑中精子的检验方法。方法收集健康志愿者精子和女性阴道上皮细胞制备模拟混合斑,经过预处理后用Vysis CEPX SpectrumOrangeTMY SpectrumGreenTM试剂盒进行荧光原位杂交,并用PALM激光捕获显微分离系统分离男、女性细胞,使用Identifiler试剂盒结合低体积扩增技术分别对男女成分进行STR扩增。结果荧光原位杂交后,可清晰分辨混合斑中的男女细胞。捕获20个精子细胞可以得到完整的STR分型,检出率为80%。随着精子数目增多,检出率提高而等位基因丢失率降低。30个精子检出率最高,为95%。结论激光捕获显微分离联用FISH技术可用于混合斑中男女细胞DNA的分离检验。     

双差异分离法对混合斑快速检验的初探

目的建立强奸案中混合斑的分离检验新方法,并评估其应用价值。方法采用双差异分离法对人工制备的3个不同浓度的模拟混合斑样本及相应滤膜进行分离提取,对所得DNA进行定量分析和STR分型检验。结果不同浓度的3个样本经孔径为10μm尼龙网格膜两次离心过膜后精子细胞损耗率均小于3%,同时3个样本沉淀均检出完整单一的男性STR分型。结论该双差异分离法所用滤膜对精子细胞DNA的损耗极少并且具有稳定性;该方法用于精阴混合斑的分离检验,具有高分离效率,装置简单,操作便捷,成本低廉等优势,为性侵案中混合斑的分离检验提供了一种新思路。     

微流控芯片技术应用于精阴混合斑分离的研究进展

在性侵案件中，精阴混合斑检材是常见且具有重要证据价值的物证，如何从中分离出嫌疑人精子细胞并检出其STR分型，是破案的关键，更是法医DNA实验室必须解决的问题。目前，国内实验室主流应用的差异裂解法以及基于此原理的改良方法，都存在难以克服的缺点。微流控技术的不断发展为混合斑中精子细胞的分离带来了新思路，基于不同原理并不断演进的微流控芯片被研制出来以尝试解决此难题。本文从研究和应用进展角度对机械操控法、流体动力和捏流分选融合法、声波差异提取法、介电泳法、SLeX糖芯片法等5种用于精阴混合斑分离的不同类型微流控芯片进行了概述与简要比较。随着相关技术的不断发展和完善，推出具有体积小、速度快、效果稳定等优点，可以广泛适用于各级法医DNA实验室进行案件混合斑分离的芯片将指日可待。     

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。     

激光捕获显微分离技术及其法医学研究进展

激光捕获显微分离技术(laser capture m icrod issection,LCM)是一项显微捕获分离单个细胞和多个细胞的自动化新技术,本文综述了其在法医学领域的研究新进展。经过研究和实践,它将在法医学实践中解决混合和微量物证的DNA检验难题中发挥重要作用。     

应用EZ-tape胶带分离提取混合斑精细胞

在强奸案件中的DNA检验往往需要将混合斑中男性精子与女性阴道上皮细胞彻底分离.常规方法是剪取载体及其上面附着的混合斑迹一起进行差异裂解分离出精子细胞与女性阴道上皮细胞.在实践中,由于载体的介入可能会带来不利因素,加大了混合斑分离的难度,往往得不到男性精斑的DNA分型或者得到的是混合DNA分型.本研究根据混合斑的形成特点,利用EZ-tape胶带既不破坏载体又可多次粘取载体表面混合斑迹的特性,巧妙地克服了载体带来的诸多不利因素,提高了男性精子的检出率.     

激光捕获显微分离技术在DNA检验中的应用研究

目的建立一套显微细胞捕获技术用于法医学生物检材DNA检验方法。方法使用VeritasTM LCM激光捕获仪,采用紫外加红外的捕获方式,对框架覆膜玻片上经苏木素染色口腔上皮细胞进行捕获,采用改良硅珠法提取细胞DNA,使用Identifiler TM试剂盒在5μL体系中进行PCR扩增。结果成功获得20个口腔上皮细胞的13个以上完整STR基因座分型谱带。结论本研究建立的方法适合法医学生物检材制成的染色涂片上细胞的DNA检验。     

Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection

Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.     

Droplet-based optical trapping for cell separation in mock forensic samples

Optical tweezers have a wide range of uses for mechanical manipulation of objects in the microscopic range. This includes both living and static cells in a variety of biomedical and research applications. Single-focus optical tweezers, formed by focusing a laser beam through a high numerical aperture immersion objective, create a significant force, which enables controlled transport of a variety of different cell types and morphologies in three dimensions. Optical tweezers have been previously reported to capture and separate spermatozoa from a reconstituted simulated postcoital sample. We report herein the development of a simplified, more efficient cell transfer protocol that can separate and isolate both spermatozoa as well as leukocytes, with similar efficiencies as those previously reported. The new cell transfer method was used to separate sperm cells from a reconstituted mixture of spermatozoa and vaginal epithelial cells, with complete STR profiles developed from 50 cells with little evidence of contribution from the female contributor to the mixture. This modified protocol was then used to separate 21 samples of enriched leukocytes, with trapped cells ranging from 5 to 22 cells. Complete STR profiles were developed from as few as 10 leukocytes. Thus, with minimal sample preparation and a short trapping time, this method has the potential to provide an alternative to traditional differential extraction methods for separation of sperm:nonsperm mixtures while also providing versatility for separation of cells with differing morphologies.     

改良差异裂解法结合硅珠法提取混合斑精子DNA

目的采用改良差异裂解法结合硅珠法提取混合斑中精子DNA,并评价其应用价值。方法收集52例经常规差异裂解法检验含有女性分型的混合斑检材,采用改良差异裂解法结合硅珠法提取精子细胞DNA,IdentifilerTM试剂盒进行PCR扩增检验。并将常规差异裂解法结果作为对照。结果52例混合斑检材中,采用改良差异裂解法结合硅珠法检出单一男性精子成分有38例,男性分型检出率达到98．08％。结论改良差异裂解法结合硅珠法适合提取混合斑中精子DNA。     

Alternative direct-to-amplification cell lysis techniques for forensically relevant non-sperm cells

While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to the requirement of a differential lysis step before DNA purification, as well as intricate mixture analysis towards the end of the DNA workflow. Recently, an alternative, direct-to-amplification sperm lysis method (using 1 M NaOH) was identified. However, a direct cell lysis method for non-sperm cells has not been identified yet. Thus, the primary objective of this work was to find an alternative method that is quick, inexpensive, and does not require multiple purification steps for the lysis of non-sperm cells in sexual assault samples. In this study, vaginal swab samples were lysed with the control method, prepGEM™, as well as six alternative reagents: alkaline buffer with 25–200 mM NaOH, high-salt stain extraction buffer, modified radioimmunoprecipitation assay (RIPA) buffer, mammalian protein extraction reagent (M-PER™), digitonin buffer, and urea/thiourea buffer. Quantification using Quantifiler® Trio of vaginal and semen lysates revealed that the alkaline (25 mM NaOH) and M-PER™ methods were efficient for the lysis of vaginal epithelial cells without substantial sperm cell lysis. Following quantification, analysis of STR profiles from vaginal lysates revealed that the M-PER™ method showed promising results across all metrics examined, including the percentage of detected STR alleles, mean peak heights, peak height ratio, and interlocus balance. Thus, this method was recommended as an alternative to the traditional differential lysis method for non-sperm cells given its ability to produce amplification-ready lysates without any DNA purification step.     

混合斑中精子细胞分离及其DNA制备方法

目的尝试建立一种检测混合斑中精子细胞的方法。方法使用显微操作法捕获精子细胞,全基因组扩增(多重置换扩增)精子细胞DNA。结果对10管精斑检材的全基因组扩增,获得了高产、保真的产物。使用50μL体系对20个精子细胞直接进行全基因组扩增,省去了对起始模板的纯化过程,DNA扩增倍数达30000倍以上,片段长度大多在15 kb以上,其STRs复合扩增分型结果有可参照性。结论显微操作法可以有效捕获精子细胞,排除干扰,多重置换扩增可以提供足够量的产物用于法医DNA分析,该方法具有可行性。     

PALM结合低体积扩增技术在混合斑检验中的应用

目的PALM系统联合应用低体积扩增技术,建立一种提取混合斑中精子DNA的分型方法。方法利用PALM切割捕获理想检材的精子细胞,将捕获的细胞直接放置到低体积扩增玻片的反应位点上,裂解细胞之后进行PCR扩增。结果20个精子细胞8次分型中,7次分型结果完全正确。10个精子细胞8次分型中,4次分型结果完全正确。1个精子细胞8次分型中,出现位点的基因座均在10个以上。将该方法应用于两例实际案例的混合斑检材,取得了满意效果。结论联合运用PALM系统和低体积扩增技术对混合斑检材中的精子细胞检验具有重要意义。