QIAcube小型工作站在生物接触类检材提取中的应用研究

目的 探讨QIAcube工作站在生物接触类检材中的提取效果。方法 将90份以脱落细胞粘取膜、实物样本(纱线转移)或棉签擦拭物为载体的生物接触类检材,分别采用QIAcube工作站、EZ1工作站和手工硅珠法进行基因组DNA提取、定量并检测15个常染色体基因座的STR分型。结果 利用QIAcube工作站提取的生物接触类检材平均检出率达到44.4%,其中脱落细胞粘取膜组检出率为56.7%,实物纱线转移组检出率为46.7%,棉签擦拭物组检出率为30.0%。脱落细胞粘取膜组和实物样本(纱线转移)组均获得较好的STR分型结果,所得DNA浓度均优于棉签擦拭物组样本。QIAcube工作站组平均检出率为44.4%,手工硅珠法组平均检出率为43.2%,而EZ1工作站组平均检出率为26.9%,前两者平均检出率明显高于EZ1工作站组。QIAcube工作站组与硅珠法组所得DNA浓度无显著差异,但均明显高于EZ1工作站组。QIAcube工作站组与硅珠法组检测得到的STR分型RFU值及峰值均衡性均优于EZ1工作站组,尤其是大片段基因在前两组中均能够被完整检测到。结论 采用QIAcube工作站提取生物接触类检材检出率高,STR分型结果良好,可应用于法医物证实际案件检验。     

两种DNA提取法对腐败胎盘组织STR分型结果的比较

在法医物证检验工作中.对于新鲜的人体组织检材,一般采用Chelex-100法提取DNA就可得到理想的STR分型结果,但是对于高度腐败的人体组织,单纯地依靠此法检测存在失败的可能.笔者运用两种不同的DNA提取法对高度腐败的胎盘组织进行了DNA提取,并对其STR基因座分型的检出率、DNA含量及纯度进行了比较.     

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。     

衣物类生物检材DNA检验的研究

目的建立对衣物类生物检材STR检验的有效自动提取纯化方法。方法对100例衣物类生物检材用EZ1DNA Investigator试剂盒、EZ1Advanced XL工作站的Large-Volume程序(大体积法)进行提取纯化,AmpFLSTR~Identifiler~Plus荧光STR复合扩增,3500x L型遗传分析仪分析结果。结果 69份检材中检出了单一DNA分型结果,9份检出了混合基因型,22份未检出基因型。结论该方法对衣物类生物检材DNA提取纯化后进行荧光STR检测,可应用于法庭科学实践。     

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。     

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。     

盗窃案件现场检材提取与DNA检测结果的统计分析

目的统计分析1574例盗窃案件中提取的2496个现场生物检材,为提高DNA在盗窃案件侦破中的应用成效提供参考。方法根据2496个生物检材的类型、现场提取方法、重点提取部位、DNA检测结果等进行统计和分析比较,总结常见类型盗窃案件现场生物检材的主要发现提取部位以及不同方法提取的现场生物检材DNA检出率。结果接触类检材已成为盗窃案件最多见的生物检材类型,但检出率仍然较低,对混合分型应进一步分析筛选以提高DNA的认定率;不同方法提取的现场生物检材在DNA检出率方面存在统计学差异,接触类生物检材以植绒拭子和原物提取的方式为首选;现场生物检材的主要发现提取部位根据盗窃案件的类型不同有所侧重。结论现场勘查人员在盗窃案件中发现和提取到有价值的生物检材是提高DNA检出认定能力的关键因素,应着力培养现场勘查人员的微量生物物证意识,提高现场勘查人员提取和处理微量生物检材的能力。     

指甲末端微量物证的DNA检验

1材料与方法1.1材料检材取自近两年35例刑事案件,其中受害人指甲31例(男11例,女20例),嫌疑人指甲4例(均为男性)。1.2方法用0.5cm×0.5cm滤纸两步擦拭法[1]转移指甲末端微量物证,加入5%Chelex-100和蛋白酶K,震荡混匀后置56℃保温1h,99℃8min,提取的DNA用Microcon-100柱纯化处理。用Amp FlSTR(ProfilerPlusTM系统复合扩增试剂盒进行STR基因座复合扩增,扩增产物经ABI 3100或ABI 3130X1型基因分析仪荧光检测分型。2结果与讨论在送检的35例检材中,检出含有他人DNA的案例15例,其中受害人为女性的13例,其中婴幼儿1例;男性2例。具体见表…     

全自动核酸提取仪联合真空浓缩仪在法医学中的应用

目的探讨全自动核酸提取仪联合真空浓缩仪在法医学DNA提取中的应用价值。方法采集人外周静脉血稀释成40、80、120、160、200、240、280和320倍的梯度样本,并对每一梯度的样本分别进行无抑纯化提取,提取后的DNA洗脱液取6μL直接进行扩增,剩余部分使用真空浓缩仪进行浓缩,浓缩前后的DNA均使用Investigator 26plex QS试剂盒进行扩增、检测。结果添加果酸的梯度样本仅在40倍稀释时获得完整STR分型结果,另外5种方式处理的梯度样本在40～160倍稀释时均获得完整的STR分型。DNA浓缩后的STR分型结果显示,平均峰高和基因座检出率均有一定程度的提高,但是效果并不明显。结论全自联合使用可提高法医学检材的检验成功率。     

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。     

氨基比林血痕预试验处理血痕后样本的DNA定量

目的：探讨氨基比林血痕预试验处理血痕后样本DNA含量的变化及对STR分型检测的影响。方法10名健康无关个体EDTA抗凝血液制成滤纸血痕,氨基比林血痕预试验检测,按试验后血样干燥保存时间分30 min、1 h、3 h、6 h、12 h、24 h共6个实验组,并采用磁珠法、QIAcube DNA纯化法、Chelex-100法三种方法提取样本DNA,应用荧光定量PCR检测样本DNA含量,PCR-STR荧光技术进行STR分型。结果提取方法相同时,氨基比林血痕预试验后血样随干燥保存时间的延长,样本DNA含量呈逐渐降低的趋势。保存时间相同时,不同DNA提取方法间,样本DNA含量差异也有统计学意义。90.56%样本均可获得16个STR基因座明确分型。结论氨基比林血痕预试验对血痕样本DNA有损伤,24 h内多可获有效STR分型。磁珠法提取样本DNA进行STR分型,效果最好。     

短串联重复位点ACTBP2(SE33)的扩增片段长度多态性研究

应用变性聚丙烯酸胺凝胶电泳（dn－PAGE）结合银染色技术对短串联重复（STR）位点ACTBP2（SE33）的扩增片段长度多态性（Amp－FLPs）进行了研究。在210名无关中国个体中观察到了25个等位基因，等位基因频率分布在0．007～0．093之间。基因型的分布符合Hardy－Weinberg定律，个体识别能力（DP）值为0．99，杂合度（H）为98．7％。七个家系分析的结果表明，该位点的遗传符合孟德尔遗传法则，未观察到变异。对几种常见的法医物证检材的分析表明，该分型系统对DNA降解放为严重的检村适用性强，而且灵敏度高（0．5ng），适合于法医学实际应用。     

联合应用常染色体STR和X-STR标记鉴定单亲疑难案例

目的通过对常染色体和X染色体遗传标记的检测,探讨单亲疑难案例的鉴定策略。方法提取3个单亲鉴定案例的6份血样,采用Goldeneye 20A试剂盒和AGCU21＋1试剂盒检测常染色体上39个STR基因座,采用自主研制的16重X-STR扩增系统检测X染色体上16个STR基因座。结果用Goldeneye 20A试剂盒检测后发现每个单亲案例均有一个基因座不符合遗传规律,当常染色体STR基因座增加到39个时,案例1累计出现3个矛盾基因座;案例2和案例3均没有出现新的矛盾基因座。X染色体STR分型结果显示案例1有8个矛盾基因座,案例2和案例3无矛盾基因座,与常染色体分型结论相符。结论对于出现单基因座不符合遗传规律的母女、母子、父女单亲案例鉴定,不仅可以增加新的常染色体STR检测,也可以增加X染色体STR的检测,这样在相互验证的同时也能获得更加可靠的鉴定意见。     

PE‐Swab Direct STR Amplification of Forensic Touch DNA Samples

The PE‐Swab direct STR amplification workflow was developed to process low‐level “touch DNA” samples. In this workflow, a forensic sample is first collected on a 4‐mm PE‐Swab (a novel sample collection device); two 2‐mm punches containing collected samples are then generated from the PE‐Swab and directly amplified for STR typing. Compared to the conventional STR workflow, which involves DNA extraction, purification, and elution volume reduction, the PE‐Swab direct STR amplification workflow does not require sample preparation and takes <60 sec before a touch sample is ready for STR amplification. Because there is no DNA loss due to sample preparation, the PE‐Swab workflow is more sensitive than the conventional STR workflow. The average peak height per sample obtained by the PE‐swab workflow is 3 times higher than that from the conventional workflow with both low‐level single source and two‐contributor mixture samples tested in this study.     

全基因组扩增在微量检材DNA分型中的应用

目的探索全基因组扩增技术对微量检材DNA分型的有效性。方法通过显微操作制备含1~20个细胞的模拟微量检材样本,在常规PCR-STR分型前加入全基因组扩增步骤,从等位基因不平衡、等位基因丢失、基因座丢失、伪等位基因(包含stutter峰)等方面探究PEP和MDA两种全基因组扩增方法对微量检材DNA分型的有效性。结果 MDA扩增效率高于PEP,但等位基因丢失和伪等位基因严重;PEP方法的正确分型率高于MDA,但小片段DNA优势扩增现象较严重。结论 MDA方法并不适合目前以STR分型为主导的法庭科学,当微量检材样本的绝对量相当少时,可以考虑使用PEP方法来扩大样本量,以满足重复检验的要求,但可能面临大片段DNA扩增失败的风险。     

Performance evaluation of two multiplexes used in fluorescent short tandem repeat DNA analysis

The performance of two commercial multiplex kits that together amplify the 13 core short tandem repeat (STR) loci currently in use by forensic laboratories and the U.S. national Combined DNA Indexing System (CODIS) were evaluated. The typing systems examined were AmpFlSTR Profiler Plus and AmpFlSTR COfiler (PE Applied Biosystems, Foster City, CA). Electrophoretic separation and detection of the fluorescent PCR products was achieved by capillary electrophoresis (CE) using an ABI Prism 310 Genetic Analyzer. The studies addressed the on-site validation of the instrument, the software, and each typing system. These studies included instrument sensitivity, resolution, precision, binning, peak height ratios, mixtures, stutter, and the amplification of non-probative and simulated forensic samples. Other additional developmental-type work is also reported herein, such as species specificity testing and amplification of environmentally insulted samples. Amplification conditions were found to be robust and the primer sets shown to be specific to human DNA. Stutter and peak height ratios fell within limits published by the manufacturer and other laboratories. The data demonstrate that the CE instrument can consistently resolve fragments differing in length by one base and that the +/-0.5 base bin used by the Genotyper software is acceptable for making accurate allele calls. Correct typing results were obtained from non-probative and simulated case samples, as well as samples exposed to outdoor environmental conditions. The results support the conclusion that DNA extracted from biological samples routinely encountered in the forensic laboratory can be reliably analyzed with AmpFlSTR Profiler Plus and COfiler using CE.     

DNA IQ磁珠法结合Maxwell~（TM） 16自动仪提取接触DNA

目的研究DNA IQ磁珠法结合MaxwellTM 16自动仪对接触DNA提取的应用价值。方法 151份案件接触DNA检材95℃裂解后,采用DNA IQ磁珠法结合MaxwellTM 16自动仪提取DNA,然后进行DNA定量和STR分型检测,统计各种类型的接触DNA含量I、PC CT值和STR分型成功率。结果 151份案件接触DNA检材中,除果核平均DNA获得量为9.51ng以外,其它接触检材的平均DNA获得量均大于10ng,烟蒂检验成功率最高为93%,果核检验成功率较低,为60%。所有DNA样品的IPC CT值均在27左右,纯度高。结论大部分接触DNA检材采用DNA IQ磁珠法结合MaxwellTM 16自动仪可提取到足以进行STR分型的DNA。     

Fungal DNA challenge in human STR typing of bone samples

The present study focuses on possible cross-reaction of fungal DNA with human STR primers that may affect subsequent forensic DNA analysis of forensic samples. Specificity of human STR markers namely HUMAMEL, HUMCSF1PO, D8S306, HUMTH01, HUMvWA, HUMFES/FPS, HUMF13A01, HUMDHFRP2, HUMFGA and HUMTPOX was tested using DNA of 24 different filamentous fungal isolates obtained from exhumed bone samples. The specificity of these ten STR markers for human DNA was demonstrated. Presence of non-human DNA in five bone samples analyzed did not alter scoring of detected alleles. Notably, amplification was inhibited in the presence of a high proportion of fungal DNA compared to human DNA (1000 ng: 1 ng) in DNA mixture experiments. The results of the present study underscore the importance of carefully analyzing the presence of non-human biological contaminants that may affect DNA typing of environmentally challenged forensic samples to avoid spurious data interpretation.     

STR and Y-STR genotyping of 30–50-year-old semen stains

This study performed the semen discrimination test, short tandem repeat (STR) typing, and Y-chromosome specific-STR (Y-STR) typing on five, 30–50-year-old semen stains. All samples reacted positively with the SM test reagent, and we observed sperm heads in all samples microscopically. The quantity of DNA extracted from the 43- and 50-year-old samples was much lower than from the other samples. STR typing of the 30-, 32-, 32-, 43-, and 50-year-old semen samples detected a maximum of 13, 15, 15, 11, and 6 of 15 loci, respectively, while Y-STR typing detected 16, 16, 16, 10, and 10 of 16 loci. These results suggest that the semen discrimination test and STR and Y-STR typing can detect extremely old semen stains and are useful for forensic practice.     

Systematic analysis of stutter percentages and allele peak height and peak area ratios at heterozygous STR loci for forensic casework and database samples

To assist the interpretation of STR DNA typing results from forensic casework samples containing mixtures, the range of heterozygous allele peak height and peak area ratios (HR) and stutter percentages (stutter %) for the loci comprised in the AmpFlSTR Profiler Plus (PP) kit were assessed on 468 database and 275 casework single source samples. Stutter % medians were similar for database and casework samples, ranging from 2% to 7%. The upper limit of the stutter value range was 16%, calculated as median +3 SD, although lower locus-specific values could be used. HR medians were 93 +/- 6.5% for database samples, 88 +/- 12% for casework samples. For casework samples, the maximum signal imbalance noted was 52%, calculated as median -3 SD. No significant difference was observed between peak height and peak area calculated values. This study shows the importance of selecting the proper reference database for the establishment of HR threshold values.