唾液斑DNA检验在性侵害案件中的应用

自从STR-PCR技术应用于生物物证的检验,微量生物学检材的DNA检测已成为可能。血液、毛发、骨骼、组织、人体分泌物(精斑、唾液、汗液)等物证检材均可通过DNA检验技术进行基因型分析。注意物证存在的多样性,广泛提取各种DNA物证成为在侦察破案中充分发挥DNA检验技术作用的关键。     

直接扩增法在血痕、肋软骨和唾液检材中的应用

目的采用Identifiler Direct PCR试剂盒直接扩增法进行棉签擦拭血痕、肋软骨和烟蒂唾液斑DNA分型检验,并评价其应用价值。方法收集棉签擦拭血痕、烟蒂各20份,肋软骨10份,采用Identifiler Direct PCR试剂盒进行直接扩增及分型检验,以相同检材采用磁珠法/Chelex-100法提取模板DNA后扩增检验结果作为对照,对两组所得结果进行比较分析。结果棉签擦拭血痕和肋软骨一次检测完整分型率均为100%,分型结果与对照组一致;烟蒂上唾液斑有2份检材第一次未能完整分型,调整方法再次检验后获分型成功。结论实际检案中的棉签血痕、肋软骨和烟上唾液斑,采用直接扩增法检测,方法简单、快速、稳定、检材用量小,可在实际检案中选择使用。     

唾液在毒物的法医学检测中的应用价值

<正> 在对中毒活人体内毒物的法医学检测中(如取血进行滥用药物的检测),有时检材量太少。近年来,已有人提出唾液代替血作为检材或作为血的补充检材。本文综述毒物在唾液中的排泄资料,以探讨唾液在毒物的法医学检测中的应用价值。     

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。     

人唾液中苯丙胺类药物检测方法进展

目前人唾液中苯丙胺类药物的检测研究较少,鉴于唾液检材具备很多的实用价值和优势,本文综述了国内外对唾液检材中苯丙胺类药物的检测研究进展,重点包括免疫分析筛选方法、液-液萃取法、固相微萃取法等萃取方法以及气相色谱/质谱联用法、液相色谱/质谱联用法等确证方法的发展情况。     

唾液、尿液中甲基苯丙胺浓度分布及初筛情况分析

目的获得吸毒者唾液和尿液检材中甲基苯丙胺浓度分布及胶体金试剂条初筛情况。方法液相色谱串联质谱法获得吸毒者唾液和尿液检材中甲基苯丙胺浓度,通过胶体金试剂条检测获得初筛情况。对两者结果进行比对分析。结果采用直接沉淀蛋白法和液质MRM扫描法检测,唾液线性范围是1~100ng/m L,线性相关系数0.9987,检出限是0.1ng/m L,定量限是1ng/m L;尿液线性范围是1~100ng/m L,线性相关系数0.9943,检出限是0.5ng/m L,定量限是1ng/m L。唾液和尿液检材按一定比例稀释,使浓度在线性范围内。采用唾液和尿液四种型号甲基苯丙胺胶体金试剂条初筛,直接点样,目测判断结果。结论胶体金试纸条初筛尿液检出率为79%左右;唾液检出率大概为81%,两种试剂条结合使用,检出率可以提高到93%以上。结合此次初筛结果和仪器确认浓度可以发现:灰区设置和灵敏度的设置对检出率有一定影响,建议提高灵敏度以满足筛查工作需要。     

唾液中滥用物质分析的研究进展

唾液作为非侵入性生物样品具有取材方便无创、感染机会少、适宜大规模人群采样等优势,是近年来法医毒物分析、临床药物监测、鉴定科学等领域的重要研究对象。国际上唾液样品已广泛应用于毒品滥用检测和监管等,与血、尿相比,唾液基底较为洁净,能降低基质效应产生的干扰,但是唾液采集也存在样少量微等困难,需要高效的前处理方法以及准确灵敏的分析技术。以唾液分析的方法学角度,对近十年来唾液中滥用物质的前处理手段和分析技术进行综述,同时对唾液检材的局限性及国内外唾液分析所面临的难点、热点予以讨论。     

D20S161和D8S384两个基因座在法医学中的应用

评估D2 0S16 1和D8S384两个基因座在法医学中的应用价值。用自制的D2 0S16 1和D8S384两个DNA分型试剂盒 ,对人血、人精液、人唾液、动物血、人血与动物血的混合检材和人血痕、人精液斑、人唾液斑、动物血痕、人血与动物血的混合斑痕检材 ,以及陈旧血痕检材进行检测分型 ,并用这两个基因座PCR引物序列与DNA数据库进行联网对比分析。自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血、人精液、人唾液、人血与动物血的混合检材分型 ,而动物血没有PCR产物 ;自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血痕、人精斑、人唾液斑和人血与动物血的混合斑痕检材正确分型 ,而动物血痕没有PCR产物 ;斑痕检材分型结果与对应体液检材分型结果无差异 ;5 0份陈旧血痕检材全部获得阳性分型结果。DNA数据库联网比较提示 ,D2 0S16 1和D8S384基因座引物除了能与各自的模板序列发生特异性扩增外 ,理论上不能与DNA数据库中 6 0 6 36 4种已知序列产生PCR产物。D2 0S16 1和D8S384两个基因座具有高度的种属特异性 ,抗污染能力强 ,不易受降解的影响 ,是解决法医现场生物检材个人识别和亲子鉴定的理想手段     

微量生物检材常规初检后再行PCR检验的方法

在实际检案中，经常遇到案发现场可获取的生物检材量微，在进行必要的种属检验、精斑确证实验、ABO血型检验等常规物证初检后，便无多余的生物检材移送DNA实验室进一步做法医DNA检验。因此，笔者通过对检案中遇到的上述微量物证检材的再行处理利用，在本实验室条件下，对其进行了TH０１、HUMACTBP２、AluVpA、DIS８０等位点的DNA－PCR分析，获得了良好的效果。使其在实际办案中更充分地发挥了证据作用，报告如下。１材料与方法检案中已经种属或ABO血型检验后的血痕、唾液斑（如烟蒂外层纸）浸泡凹板，加入３００μl去离子水，…     

利用唾液斑DNA上12S rRNA基因片段进行动物种属鉴定

目的通过检测唾液斑DNA确定案件所涉及动物的种属。方法通过提取动物唾液斑DNA,扩增其线粒体DNA上的12S rRNA基因片段,并进行DNA测序,测序结果在GenBank上进行BLAST搜索,再利用DNA MAN软件进行同源性分析。结果从唾液斑中成功地提取到了基因组总DNA,并成功扩增出了用于动物种属鉴定的12SrRNA基因片段。结论所报道的方法能用于动物种属鉴定。     

粪便DNA提取及检验

目的　研究人类粪便DNA的提取和检验方法。方法　 8人份粪便样本 ,磁珠法提取DNA后 ,进行STR复合扩增和mtDNAHVI区测序分析。结果　用 2种方法提取的粪便DNA ,STR复合扩增检验均未获成功 ;方法1提取的粪便DNA有 6个样本、方法 2有 7个样本获得了清晰可读的mtDNAHVI区序列 ,并与唾液对照样本DNA的序列完全一致。结论　用本文建立的方法提取粪便DNA ,不适于STR分析 ,可通过mtDNA测序分析进行检验。     

Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva.

Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at -20 degrees C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.     

唾液中Lewis血型物质的定量研究

应用时间决定性荧光免疫测定法（TR－FIA法），对129例健康成人唾液中Lweis及H1血型物质进行定量检测。Le阳性个体均不同程度地检出了Lea和Leb物质。Lea物质含量：Le（a＋b-）型＞Le（a－b＋）型；Leb物质含量：Le（a－b＋）型＞Le（a＋b－）型。Le（a＋b－）型的Lea物质＞Leb，Le（a－b＋）型的Leb物质＞Lea物质。Le阴性的部分个体未能检出Lewis物质，其余个体也仅检出微量。根据Lea和Leb物质在唾液中的相对含量，可以推测红细胞Lewis型，并提示Leb物质可能由Lea物质转化而形成。     

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.     

Forensic evaluation of the QIAshredder/QIAamp DNA extraction procedure

The potential to recover genetic profiles from evidence samples has substantially increased since robust and sensitive amplification kits are commercially available. Nevertheless, even the best amplification kits cannot succeed when the extracted DNA is of poor quality. In this study we compared the efficiency of silica (QIAamp DNA Mini Kit), Chelex and Phenol-Chloroform (PC) based protocols to recover DNA from different categories of samples (blood and saliva on cotton swabs, muscles, cigarette butts, saliva on foods and epidermal cells on clothes). The efficiency of the QIAamp system was improved when samples were treated with QIAshredder homogenizing columns. Overall, conventional Chelex or PC protocols allowed to recover conclusive SGM Plus profiles for 61% of the samples considered in this study. Contrastingly, 82% of them were successfully genotyped after being treated with a combination of QIAshredder and QIAamp systems. Our results further suggested that the QIAshredder/QIAamp protocol was particularly helpful to analyze evidence samples with few DNA and/or that were collected on substrates containing PCR inhibitors.     

A rapid method to detect dried saliva stains swabbed from human skin using fluorescence spectroscopy

Saliva on skin is important in forensic trace evidence. If areas where saliva is present can be outlined, this may lead to DNA analysis and identification. This study describes a rapid and non-destructive method to detect dried saliva on the surface of the skin by fluorescence spectroscopy. Eighty-two volunteers deposited samples of their own saliva on the skin of their ventral forearm. A control sample of water was deposited at three different sites on the contralateral arm. Saliva and water control were then allowed to air-dry. Swab samples were taken from dried saliva and control sites and were dissolved in 0.1M KCl solution. Emission spectra were obtained from the solution and were characterized by a principal maximum at 345-355nm with excitation at 282nm. The fluorescence emission intensity was greater than background readings obtained from the control swab site in 80 of 82 volunteers (approximately 97.6%). The fluorescence profile of saliva samples were similar to those obtained from aqueous samples of pure amylase and tryptophan, an endogenous fluorophore in alpha-amylase. The presence of an emission peak at 345-355nm with excitation at 282nm could provide a strong presumptive indication of saliva deposition.     

The effect of freezing,thawing and long-term storage on forensic DNA extracts

Following forensic DNA profiling (extraction, quantification and STR typing) the remaining extract is generally stored frozen. Our routine at the Swedish National Forensic Centre is to immediately after analysis freeze the sample. If a subsequent reanalysis is needed the sample is thawed and then refrozen. In this study the effects of freezing and thawing as well as long-term storage of DNA extracts in refrigerator or freezer have been investigated. The following sample types were extracted: two levels of blood and saliva, saliva on cigarette filter paper, saliva on cotton swabs and a combination of saliva and semen to mimic samples from sexual assaults. All extraction methods used were Chelex-based, DNA quantification was performed using PowerQuant System and STR profiling with PowerPlex ESX 16 Fast System. The study was divided into three parts: 1) freezing and thawing the extracts up to ten times, 2) storage in refrigerator or freezer up to four weeks and 3) long-term storage in refrigerator or freezer for 3, 6, 9, 12 and 35 months. Generally, the quantification and STR typing results show no indication of degradation after repeated freezing and thawing or long-term storage in refrigerator or freezer.