检材采集与保存方式对DNA提取效率的影响

目的 研究检材采集与保存方式对DNA提取效率的影响。方法 使用生物检材采集与保存套管棉签、尖头棉签和普通医用棉签采集血样,分别放置于生物检材采集与保存套管或纸质物证袋中保存1周。剪取全部血样于96孔板中,用磁珠法结合自动化工作站提取DNA,以ABI7500型荧光定量PCR仪定量。结果 生物检材采集与保存套管采集保存的血样所提取的DNA浓度平均为(2.54±0.63)ng/μL,而尖头棉签、普通医用棉签采集并分别用纸袋保存的血样提取的DNA浓度平均为(2.06±0.44)ng/μL和(0.93±0.59)ng/μL。结论 生物检材采集与保存套管较之于尖头棉签或普通医用棉签采集、纸袋保存方式,其获得的DNA浓度显著提高,DNA提取率高。     

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     

生物检材采集与保存套管结合直接扩增检案1例

1案例资料 某日，13岁的女中学生阮某报案称，其在回家上楼途中被张某强行拉到楼梯间僻静处猥亵。根据受害人的供述，法医将生物检材采集与保存套管的棉签头部用附送的滴瓶内的Low TE液1滴湿润后，在受害人的左乳头周围轻柔擦拭采集了左乳头拭子中，用同样的方法采集了右乳头拭子。     

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.     

免疫磁珠法分离混合样本中血液成分的探究

目的 基于免疫磁珠法分离脱落上皮细胞中的白细胞，消除或减弱混合检材中血液来源STR分型对结果分析的干扰。方法 分别取两名不同个体的血液和口腔脱落上皮细胞，按不同比例制备成混合样本作为实验组，并同时制备细胞量相等的对照组。应用免疫磁珠法分离实验组样本中白细胞，并与对照组以相同条件进行DNA提取、扩增、分型，对比两组STR分型结果。结果 当血液量较少时，对照组为混合STR分型，经免疫磁珠法分离混合检材中白细胞后，实验组为单一来源STR分型；当血液量较多时，此时对照组为混合STR分型，但口腔脱落上皮细胞来源的STR分型谱带峰高较低甚至丢失，经免疫磁珠法后，实验组仍为STR混合分型，但口腔脱落上皮细胞来源的STR分型谱带成为主峰；当混合样本中口腔脱落上皮细胞微量，血液占比极大时，此时对照组为血液来源的单一STR分型结果，经免疫磁珠法后，实验组为STR混合分型，包含口腔脱落上皮细胞来源的所有等位基因分型。结论 该方法可用于分离脱落上皮细胞中血液成分，降低血液来源的DNA比例，能够改善此类混合检材中目的细胞的STR分型结果，为刑事案件中含有血迹浸染的混合生物检材的检验提供了一种新思路。     

衣物载体上人体脱落细胞的负压吸附采集法

目的建立脱落细胞负压吸附方法,用于DNA检验中衣物等载体上人体脱落细胞的采集。方法检材包括由志愿者佩带1、5、10、20m in纱线手套,穿着5、10、20m in的内衣以及外套、鞋、帽子、头套、袜子、水杯、矿泉水瓶等。在吸尘器的进风口处连接特制的吸筒,一端覆盖具有拦截和静电吸附细胞能力的特制吸附膜,选择适当负压对各检材相应部位进行吸扫。收集吸附膜上的脱落细胞,采用Chelex-100法提取DNA,AmpFLSTR Identifiler复合扩增试剂盒扩增检测。结果上述检材用本文负压吸附法采集人体脱落细胞,经检验均获得清晰、完整的STR分型图谱,并与检材提供者的基因型一致。结论本文建立的脱落细胞负压吸附技术可有效吸附载体上的脱落细胞,适用于DNA检案实践。     

改良直接扩增法应用于生物检材DNA检验

Identifiler（R） Plus试剂盒性能卓越,PCR反应液经过优化,抗抑制剂能力强,适用于案件生物检材DNA检验[1].本文利用纳米银溶液和生物物证提取棉签、植绒拭子对案件常见的血痕类、唾液斑和肋软骨等进行检材转移,采用Identifiler（R）Plus扩增系统配以Prep-n-Go Buffer进行直接扩增检验,以探讨直接扩增技术对此类检材的有效性和可靠性.     

自动化高通量提取脱落细胞检材DNA

正脱落细胞是目前法医DNA检验中的重要检材之一,此类物证具有体积小、较为隐蔽、不易察觉、不易毁灭等特点。通过提取脱落细胞中的DNA,可以直接获取犯罪现场人员信息,为案件侦破提供重要技术支持。但在现场检材中通常仅能采集到微量的脱落细胞,且常常带有扩增抑制物,手工进行DNA提取操作复杂,提取效果因人而异,给DNA检验带来了诸多困难。为有效提高脱落细胞的获取量,必然要增加现场采样的数量,这又大大增加了后续DNA提取的工作     

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。     

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。     

Optimization of the collection and analysis of touch DNA traces

The aim of this work was to optimize a strategy for detection, collection, and analysis of touch DNA traces. 4N6FLOWSwabs™ and cotton swabs were compared by collecting touch DNA traces from glass slides and gun shell casings. Shed cells were visualized using Diamond™ nucleic acid dye and a digital fluorescent microscope. Different collection and extraction methods were tested. Collected samples were amplified with the AmpFlSTR® NGMSElect™ kit and the Precision ID mtDNA Whole Genome Panel.     

Simplified field preservation of tissues for subsequent DNA analyses

Successful DNA-based identification of mass disaster victims depends on acquiring tissues that are not highly degraded. In this study, multiple protocols for field preservation of tissues for later DNA analysis were tested. Skin and muscle samples were collected from decaying pig carcasses. Tissues were preserved using cold storage, desiccation, or room temperature storage in preservative solutions for up to 6 months. DNA quality was assessed through amplification of successively larger segments of nuclear DNA. Solution-based storage, including a DMSO/NaCl/EDTA mixture, alcohols, and RNAlater preserved DNA of the highest quality, refrigeration was intermediate, and desiccation was least effective. Tissue type and extent of decomposition significantly affected stored DNA quality. Overall, the results indicate that any tissue preservation attempt is far superior to delaying or forgoing preservation efforts, and that simple, inexpensive methods can be highly effective in preserving DNA, thus should be initiated as quickly as possible.     

Tissue storage solution for preservation and transfer of forensic specimen in high ambient-temperature

Storage of tissue samples in high ambient-temperature can affect the quality of forensic evidence. Experiments were conducted to investigate the potential use of 3 tissue storage solutions for the preservation and transfer of forensic specimen in high ambient temperature conditions, i.e., DMSO, Longmire’s buffer, and trehalose solution. Results showed that DNA in tissue was best preserved in DMSO buffer. Samples preserved in Longmire’s buffer gave DNA analysis results for temperatures up to 60 °C, however, amplification between replications were not reproducible. For those tissue samples preserved in trehalose solution, DNA markers larger than 300 bp were absent, and irreproducible amplification results were detected at a higher level when the storage temperature increased, and storage time was over 2 weeks. Tissue storage condition at high temperature over 1 week is not recommended. Experimental results here provided an alternative collection and preservation method for tissue samples at ambient temperature (without cold-storage) for subsequent DNA analysis. These can potentially be implemented in forensic biological evidence collection, preservation and transfer in hot climates.     

不同保存时间和不同精子数量精斑样本DNA分型比较

目的比较不同保存时间和不同精子数量精斑样本DNA分型的效果。方法制备精斑样本,保存10d的样本采用激光显微捕获30、20、15、10、5、1个精子,用于不同数量精子分型比较;保存10d、214d、375d的样本分别捕获30、20、10个精子,用于不同保存时间分型比较。比较各组检出率、等位基因丢失率和非特异性扩增率,采用χ2检验进行差异比较。结果①不同精子数量分型：捕获10个精子即可得到完整的DNA分型,且随着精子数增多,检出率逐渐提高而等位基因丢失率逐渐降低,30个精子等位基因丢失率为0%,1个精子则可达58.89%;②不同保存时间分型：总趋势是保存时间越短,捕获精子越多检出率越高,10个精子与20、30个精子组比较,均有显著性差异（P〈0.05）;等位基因丢失率及非特异性扩增率则随保存时间的延长而增加,相同保存时间的不同精子数量组之间和相同的精子数量的不同保存时间组之间比较,差异均具有统计学意义（P〈0.05）。结论激光显微捕获精子数目和检材保存时间对DNA分型结果有直接影响。     

The use of DNA stabilizing solution to enable room temperature storage and transportation of buccal and trace sample swabs

It is proposed that a DNA stabilizing solution (DNA Genotek Inc.) designed to preserve DNA in saliva samples at room temperature can be extrapolated to the storage of swab heads. The aim of this study was to evaluate the effectiveness of the solution for the preservation of reference swabs (buccal) and trace samples (facial swabs). To this end, the solution was used during a twin-site DNA transfer project assessing background levels of carer DNA present in children. Tubes containing 400 μl of solution were used to store and transport swab heads. At the laboratory, samples were extracted using the QIAamp DNA Mini Kit (Qiagen), quantified using the Quantifiler Duo Kit and profiled using the AmpF?STR^® SGM Plus^® PCR Amplification Kit (both Applied Biosystems). Twenty-eight PCR cycles were applied to all samples. Thirty-four cycles or a longer electrophoresis injection time was applied to trace samples where necessary. All Reference swabs produced high quantities of DNA and full DNA profiles after 28 cycles. Profile morphology indicated good quality DNA with no degradation. Of the trace samples, sufficient profiles were achieved to study the transfer of carer DNA making the solution fit for continued use in this project. DNA stabilizing solution enables the storage and transportation of swabs without freezing. This is convenient, reduces transportation costs and enables instant analysis of samples upon arrival at the laboratory. This is a useful alternative for a multi-site research project as well as a reliable storage tool for use in remote areas.     

Use of a commercially available hydrogel as a novel DNA surface sampling tool

A common requirement in the military, law enforcement, and forensic mission space is the need to collect trace samples from surfaces using a method that not only readily captures the sample but also retains its integrity for downstream identification and characterization. Additionally, collecting samples from three-dimensional objects (e.g., shell casings) is a challenge for which there is currently no validated standardized approach. Recently, hydrogels have been shown to have the potential for surface collection of trace bacterial spores, amino acids, and DNA. To test whether these hydrogels can serve as a viable collection medium for sampling DNA from surfaces, we carried out a series of preliminary tests examining collection efficiency and suitability of hydrogel material to recover samples of diluted, dried human DNA on a smooth polycarbonate surface. The recovery of surface DNA using a commercially available hydrogel was examined, and the efficiency compared to samples collected using a standard foam collection swab. DNA collected using the hydrogel and swab methods was then examined using quantitative polymerase chain reaction (qPCR) and short tandem repeat (STR) analysis to determine whether the collection material was compatible with these downstream processes. The hydrogel material used for this study collected the experimental DNA with comparable efficiency to standard collection swabs. In addition, qPCR and STR analyses demonstrated compatibility with the hydrogel collection and extraction process. These data suggest that hydrogels have the potential to be used as sample collection materials and deserve further characterization to elucidate their utility in collection from irregularly shaped, three-dimensional surfaces/materials.     

24个基因座复合扩增系统的应用

目的建立24个基因座的复合扩增系统,并对其性能指标进行评价。方法选择24个基因座（包含D8S1179、D5S818、D2S1338、D18S51、D6S1043、D2S441、D3S1358、vWA、D19S433、D16S539、CSF1PO、Penta D、D22S1045、D13S317、D1S1656、D7S820、TPOX、Penta E、D10S1248、TH01、D12S391、D21S11、FGA等23个STR基因座及1个Amelogenin基因座）;合成引物,上游端标记荧光染料;收集DNA数据库建库血痕及口腔拭子样本及相关11种动物样本,采用本文方法进行复合扩增;并对方法的准确性、灵敏度、稳定性、种属特异性、检材适用性、混合样本的检验等系统性能指标进行验证。结果本文复合扩增系统对最长保存9年的各种血痕检材完整分型成功率为100%,且均衡性良好,无非特异性扩增,口腔拭子的成功率为97.8%,灵敏度达125pg,对含有抑制剂样本,在血红素浓度≤600μmol/L,腐殖酸浓度≤50ng/μL时检出效果稳定,种属特异性好。结论本文24个基因座复合扩增系统在DNA数据库建设中具有较好的应用价值。     

压力循环技术用于人指甲DNA提取及方法学评价

目的将压力循环技术（PCT）用于指甲DNA提取,并对方法学进行评价。方法收集10份人指甲样本,剪碎约为1mm×1mm大小,采用10%漂白粉水,10%SDS,10%漂白粉水,无菌水清洗样本。10份样本各分成两组,1组用压力循环技术处理,另1组不作处理,提取DNA经复合扩增并进行STR分型检测,用于评价压力循环技术的作用。取5份指甲样本用血浸泡,5份用去离子水浸泡,之后采用上述清洗方法各清洗1-3次,收集各次清洗用的无菌水提取DNA,经STR分型检测,用于评价清洗对去除外源性DNA的效果。结果 10份经压力循环技术处理的样本中有7例比相应未经处理样本DNA提取量更高,但两组进行统计学处理,差异不具有统计学意义（P〉0.05）;两组样本中提取DNA含量在0.026 ng以上的样本均得到完整的STR分型,与相应口腔拭子样本对照准确无误。血污染和非血污染样本清洗二次以上,均可避免外源性DNA的污染。结论使用压力循环技术并配合本文清洗方法,可有效提高人指甲DNA的提取效率,并避免外源性生物DNA的干扰,保证DNA分型结果的准确。     

联苯胺预试验处理血痕后样本DNA定量的研究

目的探讨联苯胺血痕预试验处理后样本DNA含量的变化及对STR分型检测的影响。方法选取10名无关个体EDTA抗凝血液制成滤纸血痕,保存干燥时间分0．5h、1h、3h、6h、12h、24h六个实验组,并采用磁珠提取法、QIAcubeDNA提取纯化法、chelex-100提取法提取样本DNA,应用RT-PCR定量技术检测样本DNA含量,同时应用PCR-STR技术和Idfiler-plus试剂盒检测相应样本STR分型。结果联苯胺血痕预试验后处理样本,随保存时间的延长,其样本DNA含量显示逐渐降低的趋势。回归线性对数分析显示,磁珠提取法：Y=-0．40871n（x）＋0．7044R0—0．7633;QIAcubeDNA提取纯化法：Y=-0．23931n（x）＋0．4764R0—0．8715;chelex-100提取法：Y=-0．11781n（x）＋0．2302R2=0．9571。不同DNA提取方法对同一保存时间的联苯胺血痕预试验试剂处理样本DNA含量间差异有极显著性,P〈O．01。结论联苯胺血痕预试验后对后续STR分型影响较大,联苯胺血痕预实验后的血痕不能继续进行STR分型检测。