一种收集衣服上脱落细胞的新方法

目的建立一种生物脱落细胞的微量提取新方法。方法利用一套自制的“生物细胞提取仪”无损提取衣物等载体上的人体脱落细胞,采用Chelex-100法提取DNA,用不同的试剂盒进行STR复合扩增检验。结果10例检材都得到16个基因座成功分型。结论用该方法提取微量细胞DNA,可获得满意的DNA分型。     

Chelex-100法提取脱落细胞检材DNA的实时定量研究

目的研究脱落细胞检材DNA检验的简便有效的提取方法。方法对76份包括帽子、眼镜、牙刷、剃须刀、梳子、口香糖、羊水在内的7种脱落细胞检材采用Chelex-100法提取DNA,在ABI7500型荧光定量PCR仪上进行定量,同时用Identifiler复合扩增系统扩增,在ABI3130遗传分析仪上进行STR分型。结果从帽子（头套）中获得的脱落细胞DNA平均含量为8.31ng,眼镜擦拭物上获得的脱落细胞DNA平均含量为6.20ng,牙刷上获得的脱落细胞DNA平均含量为49.40ng,剃须刀擦拭物上获得的脱落细胞DNA平均含量为6.92ng、梳子擦拭物上获得的的脱落细胞DNA平均含量为10.68ng,口香糖的脱落细胞DNA平均含量为16.30ng,羊水的脱落细胞DNA平均含量为320ng。以上76份检材性别及10个以上STR位点分型成功率为75.8%。结论从帽子、眼镜、牙刷、剃须刀、梳子、口香糖、羊水等检材提取的脱落细胞可用Chelex-100法提取DNA作STR分型。     

两种纯化试剂盒对人体脱落细胞检材STR检出率比较

正随着DNA检验技术的普及和发展,办案人员应用DNA技术的意识不断增强,采集人体脱落细胞进行DNA分析应用逐渐广泛。采用常规提取方法难以获得高质量的DNA模板,影响后续的STR分型。本研究分别采用QIA DNA Investigator试剂盒(德国QIAGEN公司)、PrepFiler~?Express BTA Forensic DNA Extraction试剂盒(美国AB公司,下文简称为PrepFiler~?Express BTA试剂盒)对129份不同载体上人体脱落细胞检材进行检验,评估两种方法的     

大体积液体检材过滤装置研发及提取技术

目的研究一种新型的"液体检材过滤提取仪",用于过滤、富集大体积液体检材中的脱落细胞。方法在液体检材过滤提取仪中分别安装8种不同孔径的滤膜(0.45μm、10μm、20μm、30μm、60μm、80μm、120μm的尼龙膜、无纺布),依次过滤、富集64份200 mL洗手水样本;采用M48磁珠法提取滤膜上富集的细胞DNA;再分别对提取到的DNA用7500荧光定量PCR仪进行定量;根据定量结果进行DNA-STR检验。结果 10μm和20μm孔径的尼龙网格膜成为该过滤提取仪首选,无纺布次之。结论 "液体检材过滤提取仪"可用于法庭科学实际案例中大体积液体检材中脱落细胞的过滤、富集。     

擦拭直扩法和粘取磁珠法检测衣物脱落细胞DNA的比较

<正>法医检案中经常遇到对衣物接触DNA检验的要求,目前多为先用真空吸附或胶带粘取的方法富集衣物上的脱落细胞,然后用Chelex-100法、磁珠法或硅珠法提取DNA进行STR分型检测[1-5]。上述方法操作均较繁琐,且容易污染。本研究用生物检材采集与保存套管的尖头棉签擦拭穿过的衣物上的脱落细胞,然后剪取少量棉签头部,采用直接扩增法及用脱落细胞粘取器粘取后磁珠提取法获得脱落细胞DNA,并     

微量口腔脱落细胞检材的DNA检验

目的寻求提高微量口腔脱落细胞检材的DNA检验成功率的简便有效的提取方法。方法对不同载体上的100份微量口腔脱落细胞检材采用小体积Chelex-100法提取DNA,在ABI7500型荧光定量PCR仪上进行定量,同时用IdentifilerTM复合扩增系统扩增,在ABI3130遗传分析仪上进行STR分型。结果从25根饮料吸管上提取的DNA量在0.72～116.7.8ng,16个水杯杯缘提取的DNA量在2.15-142.5ng,31个饮料瓶(罐)口提取的DNA量在1～34.65ng,10根筷子上提取的DNA量在3.35～26.6ng,12个果核中提取的DNA量在0.294～21.4ng,6份吃剩的骨头中提取的DNA量在0.88～5.88ng。100份检材性别及9个以上STR位点分型成功率平均为59.38%。除了使用者的个人原因外,检材的提取送检方式、检材的质地、饮料的性质对提取的DNA量有显著影响,是否加蛋白酶K对提取的DNA量无显著影响。结论采用小体积Chelex-100法可对60%左右的微量口腔脱落细胞检材提取DNA进行STR分型。     

3种方法联合运用提取人体脱落上皮细胞DNA

目的寻求提高法医物证检验中附有人体脱落细胞载体检材的DNA检出率的方法。方法根据案件检材的具体条件．选择3种提取方法即Chelex-100法、Chelex-100联合有机法和Chelex-100联合磁珠法。结果采用了分步提取DNA法,可充分、有效地提取微量DNA,提高了人体脱落上皮细胞有效DNA的检出率。结论根据第一步提取方法的检测结果,来调整下一步的提取策略,如是否加以纯化浓缩,可增加人体脱落上皮细胞有效DNA的检出率。     

微量体表脱落上皮细胞的DNA检验

目的建立微量体表脱落上皮细胞的DNA检验方法. 方法采用Chelex-100法提取DNA,以Microcon-100纯化柱纯化浓缩DNA,用Profiler Plus 试剂盒PCR扩增后用310基因分析仪检测. 结果 10种常见粘附有体表脱落上皮细胞的样本100个,其中7种70个样本成功检测到10个STR位点的分型,检出率为100%,其余3 种样本检出率分别为50%、20%、10%,将该方法应用于2例实际检案,取得满意效果. 结论所建立方法稳定可靠,易于操作,适用于多种检材,为微量体表脱落上皮细胞的DNA检验提供了确实可行的检验方法.     

磁珠直接吸附法对体态检材游离DNA的提取

目的采用磁珠直接吸附法对人体尿液、唾液、血液3种体态生物检材中的游离DNA进行提取检验,为法医物证中游离DNA的研究及检验工作提供参考。方法对3种生物检材采取离心吸取上清液的方法分离游离DNA,然后采用磁珠直接吸附法进行提取纯化,Identifiler-Plus试剂盒进行复合扩增后常规STR检测。结果在3种检材中均检出了游离DNA,其中血液中游离DNA检出率为100%,唾液为90%,尿液为70%。结论人体体态生物检材中存在游离DNA,同时磁珠直接吸附法可高效、快捷的提取生物检材中的游离DNA。     

指纹脱落细胞的STR检验

正现场遗留的帽子、口罩、衣服、手套等生物学检材,一般含有较多脱落细胞,DNA检验比较容易,然而对于指、掌纹短时间接触部位,由于所含脱落细胞少,且部分细胞核DNA容易降解,STR分型成功率较低。本研究对指纹脱落细胞分别应用Chelex-100、QIAamp DNA Investigator Kit、Mag Attract M48 DNA Manual Kit磁珠及直接扩增法进行STR检验,并比较分析。     

签字笔上附着的脱落上皮细胞STR分型

目的 探讨遗留在签字笔上微量脱落细胞DNA分型的可行性以及保存时间对分型的影响.方法 17名志愿者每人使用7支签字笔,每支笔每天使用20 min,为期1个月,分别保存1、3、5、7、14、21和28 d,运用硅珠法提取签字笔上微量脱落细胞中的DNA,应用荧光标记PCR-STR技术进行DNA分型,同时采集上述17名志愿者口腔拭子作为对照,分析签字笔作为检材进行DNA分型的可行性以及保存时间对DNA分型的影响. 结果以基因座检出个数为指标,签字笔脱落细胞和口腔拭子的DNA分型结果随保存时间变化而产生的差异具有统计学意义(P＜0.01).签字笔保存1、3、5、7、14、21和28 d后进行DNA分型检出的基因座个数与对应的口腔拭子DNA分型检出的基因座个数相比差异均有统计学意义(P＜0.01).签字笔使用后保存1 d进行DNA分型.可明确判读12个以上基因座的占41.2%. 结论签字笔上附着的微量手指脱落细胞可作为一种法庭生物检材进行DNA分型,但其保存时间会影响DNA分型.     

两种方法提取汗潜手印DNA的比较研究

目的比较M48和DNeasy○R plant Mini两种方法提取汗潜手印DNA的优劣。方法用M48和DNeasy○Rplant Mini两种方法分别提取16对汗潜手印DNA,并进行DNA定量,比较定量结果。结果 M48法明显比plant Mini法提取到的DNA量多（配对t检验：α=0.05,t=3.45,γ=15,0.002     

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。     

3种DNA提取法在污染严重混合斑分型中的应用比较

目的比较Chelex-100法、酚/氯仿法和二氧化硅膜法3种DNA提取法在污染严重混合斑分型中的应用效果。方法从日常案例中收集污染严重的混合斑25份,差异消化法分离精子后同时用Chelex-100法、酚/氯仿法和二氧化硅膜技术3种方法提取DNA,采用PCR-STR技术对D19S253、FGA和CSF1PO 3个基因座进行分型,Gel-Pro软件处理电泳图谱,SPSS软件分析比较不同方法之间的差异。结果采用Chelex-100法提取DNA,25份检材分型结果均未成功;采用酚/氯仿法,25份检材中10份分型成功,3份检材FGA和CSF1PO基因座可分型,4份检材CSF1PO基因座可分型;采二氧化硅膜纯化法,25份检材均成功分型;酚/氯仿法和二氧化硅膜法两种方法比较,结果存在显著性差异(P<0.05)。结论二氧化硅膜纯化技术可以有效去除PCR抑制物,提取的DNA扩增效果明显优于Chelex-100法和酚/氯仿法,具有较高的应用价值。     

DNA IQ^TM SYSTEM在疑难指甲DNA检验中的应用

目的探索DNA IQTMSYSTEM在疑难指甲DNA提取中的应用。方法 15份疑难指甲采用Chelex方法检验没有成功获得STR分型图谱,采用DNA IQTMSYSTEM提取法并纯化,采用Identifiler PLUS试剂盒进行复合扩增,产物经ABI3130XL型DNA基因分析仪检测。结果成功获得15例疑难指甲的STR基因座DNA分型。结论 DNA IQTMSYSTEM方法能快速、有效提取疑难指甲DNA进行STR分型。     

A Study of the Background Levels of Male DNA on Underpants Worn by Females

This study is the first to examine the background level of male DNA on underpants worn by females in the absence of sexual contact. Here, we examined 103 samples from the inside front of underpants from 85 female volunteers. Samples were examined for the presence of male DNA using NGM SElect and PowerPlex Y23 kits. Only five samples gave a “complete” Y-STR profile, even though 83.5% of our volunteers cohabited with a male. In all cases where a partner reference sample was available, the Y-STR profile matched the cohabiting partner. We have demonstrated that a Y-STR profile is not expected on the inside front of underpants worn by females after social contact alone. The results of this study are informative for evaluating the significance of a Y-STR profile on underpants in cases of alleged sexual assault.     

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。     

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.     

Using FastID to analyze complex SNP mixtures from indoor dust

Forensically relevant single nucleotide polymorphisms (SNPs) can provide valuable supplemental information to short tandem repeats (STRs) for investigative leads, and genotyping can now be streamlined using massively parallel sequencing (MPS). Dust is an attractive evidence source, as it accumulates on undisturbed surfaces, often is overlooked by perpetrators, and contains sufficient human DNA for analysis. To assess whether SNPs genotyped from indoor dust using MPS could be used to detect known household occupants, 13 households were recruited and provided buccal samples from each occupant and dust from five predefined indoor locations. Thermo Fisher Scientific Precision ID Identity and Ancestry Panels were utilized for SNP genotyping, and sequencing was completed using Illumina^® chemistry. FastID, a software developed to permit mixture analysis and identity searching, was used to assess whether known occupants could be detected from associated household dust samples. A modified “subtraction” method was also used in FastID to estimate the percentage of alleles in each dust sample contributed by known and unknown occupants. On average, 72% of autosomal SNPs were recovered from dust samples. When using FastID, (a) 93% of known occupants were detected in at least one indoor dust sample and could not be excluded as contributors to the mixture, and (b) non-contributor alleles were detected in 54% of dust samples (29 ± 11 alleles per dust sample). Overall, this study highlights the potential of analyzing human DNA present in indoor dust to detect known household occupants, which could be valuable for investigative leads.