DNATyper^(TM) 15试剂盒直接扩增检验纸质样本的研究

目的检验DNATyperTM15直接扩增系统的有效性。方法使用DNATyperTM15直接扩增系统对我国DNA数据库建库4种常见样本类型进行直接扩增检验。结果获得了血滤纸、公安部物证鉴定中心采血卡、博坤采血卡和FTA卡等样本类型的完整DNA分型。结论 DNATyperTM15直接扩增系统能够用于常见纸质样本的检验。     

AGCU免提取STR荧光检测试剂盒的验证

目的考察AGCU免提取STR荧光检测试剂盒．对保存在滤纸片或FTA卡上血液样本的直接扩增检测情况。方法使用人GCU免提取STR荧光检测试剂盒,对未经提取的滤纸片血液样本、FTA卡血液样本675份进行直接扩增和18个基因座的DNA分型,并对结果的可靠性进行研究。结果18个基因座检测结果与PP16和ID试剂盒分型结果一致,2000年数据库样本成功率92．3％,2001年数据库样本成功率92．6％,2004以后年数据样本及案件样本、亲子鉴定样本成功率在99％以上。结论AGCU试剂盒可以成功地对滤纸片、FTA卡样本的18个STR基因座进行直接扩增检测,检验结果稳定,分型准确。     

荧光标记短片段STR复合扩增系统的法医学应用研究

目的解决严重降解(低于300bp)DNA检验问题,提高降解DNA的检出率。方法重新设计引物,减小扩增产物片段长度,用荧光标记短片段STR复合扩增体系进行DNA检验,扩增结果与用Identifiler试剂盒扩增出的结果进行比较。结果用荧光标记短片段STR复合扩增系统对实际案件中的高度降解DNA检材进行检验,可以获得满意分型。结论该系统可用于严重降解DNA检材的检验工作。     

DNA检验结合临床检查确定男性假两性畸形1例

1案例资料1.1简要案情2012年12月11日,林某（女,42岁）因涉嫌拐卖儿童罪被刑拘,采集血样（编号：3507021202093）作为前科人员建库样本送DNA实验室检验。1.2 DNA分型检验样本采用Identifiler Direct和PowerPlex 21试剂盒进行检验,扩增产物用ABI 3130XL型DNA序列分析仪电泳,GeneMapperID v3.2软件进行结果分析。     

FTA卡直接扩增缓冲增强剂的研制

目的研制一种能够配合非直接扩增试剂应用的PCR缓冲增强剂,实现对FTA卡保存样本的直接扩增。方法基于常规STR复合扩增试剂盒的扩增体系及引物组,加入增强剂对FTA卡类样本进行直接扩增。结果获得样本STR分型结果清晰、完整、平衡性好,无明显抑制现象存在。结论所研制的FTA卡直扩增强剂能达到FTA卡保存样本的直接扩增检验要求,可应用于法医STR检验。     

单细胞检验技术用于混合上皮细胞的分离和检验

目的建立单细胞显微捕获联合低体积扩增技术,用于混合上皮细胞检材分离检验。方法取5名男性口腔上皮细胞拭子浸泡液30μL,分别滴加到5份含同一女性皮肤表皮细胞拭子上,制成5份混合上皮细胞样本为实验组,同时制备同样的5份样本为对照组。实验组样本采用显微捕获单个口腔上皮细胞,并使用低体积扩增技术进行扩增;对照组用M48纯化试剂盒提取DNA,Identifiler试剂盒复合扩增,扩增体系为10μL。所有产物均用ABI 3130遗传分析仪进行STR分型。结果 5份实验组样本均得到男性STR分型结果,5份对照组样本则均仅得到混合分型结果。将该方法应用于1例强奸杀人案例检验,取得了满意效果。结论单细胞显微捕获联合低体积扩增技术可用于混合上皮细胞样本的分离检验。     

AmpF(l)STR(R) Identifiler(R) Plus试剂盒快速直接PCR扩增初探

目的 探索可应用于法医实践的快速直接PCR扩增方法,以缩短扩增时间,提高STR分型速率.方法 联合运用AmpF(l)STR(R) Identifiler(R) Plus试剂盒与TaKaRa快速PCR检测试剂构建快速直接PCR体系,以FTA血卡为模板,采用优化后的扩增程序在快速PCR仪上进行扩增,分型结果与常规直接扩增方法相比较.结果 AmpF(l)fSTR(R) Identifiler(R) Plus试剂盒与TaKaRa的快速PCR检测试剂针对血卡的联合扩增,所得STR分型结果与常规方法一致,扩增用时由常规直接扩增所需的175 min缩短为55 min.结论 采用快速直接PCR方法进行扩增,可得到与常规直接扩增一致的DNA分型,扩增时间明显缩短,DNA分型速率大幅提高.     

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。     

两无关个体单亲亲子鉴定不排除1例

1案例资料2004年4月5日,黄埔分局送来在某医院提取的两名涉嫌被拐卖男童(均约6个月大)的口腔拭子,要求进行DNA检验,以确定是否有血缘关系。用Chelex-100法提取该两名儿童的口腔拭子DNA。先后用Identifiler试剂盒、PowerPlex 16试剂盒、FFFL试剂盒和PowerPlex Y试剂盒进行扩增。扩增产物用AB I 3100型遗传分析仪检测分型,并进行m tDNA L15997~L16401片段序列测定。经Identifiler系统15个常染色体STR基因座检验,两男孩的基因型不同,但每个基因座均至少有1个相同的等位基因,见表1。再使用Power Plex16和FFFL试剂盒检验,在增加的…     

葡萄胎的DNA检验

目的探讨一例强奸案中的葡萄胎样本的DNA检验及结果分析。方法用Identifiler试剂盒对葡萄胎样本和受害人血样进行荧光复合STR基因座扩增并分型。结果该例的葡萄胎在15个STR基因座上分型均为纯合子,应属于单精子受精的完全性葡萄胎。结论葡萄胎类型多种,对应着不同的DNA分型,在实际案件的检验中应引起注意。     

Autosomal short tandem repeat analysis of ancient DNA by coupled use of mini- and conventional STR kits

Abstract: Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpF?STR^® MiniFiler? kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs (n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpF?STR^® MiniFiler? and Identifiler? kits. Whereas only 21.87% of larger‐sized loci profiles could be obtained with the Identifiler? kit, 75% of the same loci profiles were determined by MiniFiler? kit analysis. This very successful amplification of large‐sized STR markers from highly degraded aDNA suggests that the MiniFiler? kit could be a useful complement to conventional STR kit analysis of ancient samples.     

STR Data for the AmpFlSTR Identifiler loci in three ethnic groups (Malay, Chinese, Indian) of the Malaysian population

Allele frequencies for the 15 STR loci in the AmpFlSTR Identifiler kit were determined and compared for the three main ethnic groups of the Malaysian population comprising 210 Malays, 219 Chinese and 209 Indians. Blood was placed on FTA paper and DNA was purified in-situ.     

DNA Typer~(TM)15 plus直扩试剂盒在DNA数据库建设中的应用

目的测试DNA TyperTM15 plus直扩试剂盒的技术性能指标,评价其在DNA数据库建设中的应用价值。方法采用DNA TyperTM15 plus试剂盒,并使用IdentifilerTM和DNA TyperTM15试剂盒进行比较,设定不同体系和引物量、不同退火温度和循环次数以进行方法验证;设定不同模板量标准品、不同比例混合样本,取猪、狗、兔等动物的血液样品,血痕、骨骼、唾液斑等常见检材样本以及不同建库样本,以验证试剂盒灵敏度、特异性、稳定性以及混合样本、常见检材及建库样本的检测能力。结果直扩试剂盒分型结果准确,重复性好,灵敏度可达0.125ng,不同批次间试剂检测结果稳定,对不同检材有很好的适应性。10μL扩增体系时FTA卡和加强型血液采集卡取样直径应为0.5mm,而血滤纸、血液采集卡样本和经典型血液采集卡取样直径应为1.0mm。结论 DNA TyperTM15 plus直扩试剂盒的性能可以满足DNA数据库建设及检案的需要,可在相关实验中选择使用。     

DNATyper^TM15与Identifiler^TM试剂盒遗传学调查应用比较

目的比较DNATyper^TM 15和Identifiler^TM试剂盒的遗传学调查应用结果，以评价DNATyper^TM15试剂盒的指标和性能。方法用DNATyper^TM 15和Identifiler^TM试剂盒同时对290份北方汉族群体的血样本进行扩增检测，比较遗传学统计数据。结果DNATyper^TM15和Identifiler^TM荧光检测试剂盒中各基因座在汉族群体中的基因型分布均符合Hardy—Weinberg平衡，累积个人识别率和累计非父排除率：DNATyper^TM15试剂盒分别为2．66×10^-18和0．9999997；Identifiler^TM试剂盒分别为1．28×10^-17和0．9999984。且两个试剂盒对相同基因座的频率调查数据一致。结论DNATyper^TM15具有较高的个体识别和亲权鉴定能力，对法医学检案和DNA数据库建设具有应用价值。     

Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits

The AmpFlSTR MiniFiler polymerase chain reaction amplification kit developed by Applied Biosystems enables size reduction on eight of the larger STR loci amplified in the Identifiler kit, which will aid recovery of information from highly degraded DNA samples. The MiniFiler Kit amplifies CSF1PO, FGA, D2S1338, D7S820, D13S317, D16S539, D18S51, and D21S11 as well as the sex-typing locus amelogenin. A total of 1308 samples were evaluated with both the MiniFiler and Identifiler STR kits: 449 African American, 445 Caucasian, 207 Hispanic, and 207 Asian individuals. Full concordance between Identifiler and MiniFiler Kits was observed in 99.7% (10,437 out of 10,464) STR allele calls compared. The 27 differences seen are listed in Table 1 and encompass the loci D13S317 (n = 14) and D16S539 (n = 10) as well as D18S51 (n = 1), D7S820 (n = 1), and CSF1PO (n = 1). Genotyping discrepancies between the Identifiler and MiniFiler kits were confirmed by reamplification of the samples and further testing using the PowerPlex 16 kit in many cases. DNA sequence analysis was also performed in order to understand the nature of the genetic variations causing the allele dropout or apparent repeat unit shift.     

Isolation of DNA from saliva of betel quid chewers using treated cards

Betel quid (BQ) chewing, a common tradition in tropical areas, often poses a problem during collection and DNA analysis of buccal samples from many indigenous communities for population genetic studies and in forensic analysis of chewed BQ residues. This study evaluated the use of FTA card, a chemically treated filter paper, in collecting buccal samples from long-term BQ users and subsequent PCR-based analysis using nine STR markers. A low overall success rate of amplification was observed in the samples extracted using a standard organic extraction procedure (7%) as compared with those prepared using the FTA card (89%). The presence of inhibitors in liquid DNA samples was verified when control DNA failed to amplify in the presence of an equal volume of liquid BQ samples. The use of the FTA card is more practical during field sampling than handling tubes containing buccal swabs.     

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。     

Application of BioRobot M48 to forensic DNA extraction

The development of a nucleic acid extraction method based on magnetic separation has opened up possibilities forl automation of DNA extraction. The BioRobot M48 is one of robotic stations applicable to automated DNA extraction in forensics. However, each new method should be thoroughly validated before application to routine casework. Our aim was to compare the effectiveness of the currently utilized organic/Microcon 100 based extraction procedure and magnetic extraction with BioRobot M48. The DNA concentration of DNA extracts obtained from different kinds of typical forensic material was evaluated followed by amplification with the SGM Plus or Identifiler kit and capillary electrophoresis using ABI 3100 Avant. We can conclude that BioRobot M48 is a very effective instrument for DNA extraction from most specimens and can be successfully applied in forensic laboratories.     

DNA数据库建设中批量样品不同DNA提取方法的比较

目的比较和选择自动化工作平台提取DNA的方法,并用于DNA数据库建设。方法用手工Chelex-100法、Biomek3000自动化工作平台结合Chelex-100法及DNA-IQTM磁珠法对实验室收集的建库滤纸血样进行DNA提取,荧光定量技术对上述3种方法提取的模板DNA进行测定;扩增产物用3100基因分析仪检测并用基因分析软件分析。结果手工Chelex-100法、自动化Chelex-100法及DNA-IQTM磁珠法提取的DNA模板浓度分别为0.593ng±0.131ng/μl、0.579ng±0.096ng/μl、0.447ng±0.056ng/μl;成功率分别为100%、98.9%、99.5%。结论本文建立的自动化Chelex-100法可用于大规模DNA数据库建设。     

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。