6个Y-STR荧光复合扩增系统的建立及应用

目的建立6个Y-STR荧光复合扩增体系,评价其法医学应用价值。方法设计DYS444,GATA-A7.2,GATA-A10,DYS390,GATA-A7.1,DYS443荧光复合扩增引物,扩增总体积20μL,内含模板DNA0.5～10ng,PCR产物用3130遗传分析仪电泳,GenemapperID v 3.2分析结果,根据等位基因标准命名各等位基因,并评价该系统的特异性、准确性、均衡性、灵敏度及对混合血样的分析能力。结果当dNTP、Mg2+分别为200μmol/L、1.5mmol/L时扩增效果最佳,0.5～10ngDNA模板量均能获得较好的扩增效果,各基因座分型结果清晰,扩增均衡,特异性强,重现性好,基本满足实际应用的性能要求。对湖北汉族群体进行遗传学调查,结果GD值0.580 3～0.722 3,共检出158种单倍型,其多样性为0.995 8。结论本文6个Y-STR扩增系统分型可靠,配合常用Y-STR分型试剂盒,可进一步提高个体识别能力。     

Reduction of Powerplex® Y23 Reaction Volume for Genotyping Buccal Cell Samples on FTATM Cards

PowerPlex^® Y23 is a novel kit for Y‐STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y‐STR analysis. This study examined PCR amplification from 1.2‐mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High‐quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories.     

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。     

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.     

Comparison of operational DNA recovery methods: Swabs versus tapelifts

It is routine among many jurisdictions to recover DNA using tapelifts on porous substrates (e.g. clothing) and swabs on non-porous substrates (e.g. tool handles). Here, we examine this by comparing the efficiency of the NSW jurisdiction’s specific swabbing and tapelift techniques on a range of porous and non-porous substrates. To test DNA recovery efficiency, 30 μl aliquots of 1:50 and 1:100 saliva dilutions were deposited onto the substrates, left to dry overnight, recovered, extracted, quantified and a subset profiled. Tapelifts recovered more DNA and DNA profiles with more detectable alleles than swabs for both saliva dilutions on porous substrates. For non-porous substrates, similar DNA quantities and profiles were generally recovered with both methods for both saliva dilutions. These data underpin current practices to recover DNA using tapelifts for porous substrates and swabs for non-porous substrates. These data also revealed severe degradation of DNA recovered from brass, supporting the on-going need to improve DNA recovery and analysis methods for brass substrates.     

Generating DNA profiles from immunochromatographic cards using LCN methodology

The aim of this research was to obtain DNA profiles from immunochromatographic test devices which have already yielded positive results with body fluids obtained from fourteen volunteers. Three different immunochromatographic cards for the identification of human blood and one for the identification of human saliva were used for this research. Each body fluid was detected using the appropriate immunochromatographic card. The used cards were kept at room temperature for various lengths of time. The membranes were removed at the end of the designated times and the entire strip was extracted using low copy number (LCN) extraction procedure. The extracted DNA was amplified using reduced amplification volume and higher PCR cycle numbers. Autosomal STR profiles were detected using AmpFℓSTR^® Identifiler™ PCR Amplification Kit from Applied Biosystems (AB). Additionally, DNA extracted from the male volunteers was amplified using the AB AmpFℓSTR^® Yfiler™ PCR Amplification Kit. Analysis of the amplified products was carried out by capillary electrophoresis injection on the AB 3130xl Genetic Analyzer. The generated DNA data was analyzed using the SoftGenetics GeneMarker^® HID Version 1.7 software.Autosomal and Y-STR DNA profiles were obtained from most of the cards which were stored at room temperature for up to three months. DNA profile was obtained from all four types of the immunochromatographic cards used in this study. These profiles were concordant with the profiles obtained from the donors’ reference samples.     

Analysis of human fecal material for autosomal and Y chromosome STRs

Human stool samples from eight volunteers were stored under various conditions and extracted by three different procedures. Fecal material and tissue paper soiled with fecal material obtained from a crime scene were also extracted. Extracted DNA was amplified using the AmpFlSTR Profiler Plus, AmpFlSTR COfiler, and the AmpFlSTR Identifiler PCR amplification kits for the detection of the autosomal STR allelic patterns. DNA extracted from the male volunteers and from the soiled tissue paper evidence sample was also amplified using the Y-PLEX 6 and Y-PLEX 5 amplification kits. Analysis of the amplified products was carried out by capillary electrophoresis on the ABI PRISM 310 Genetic Analyzer. Autosomal and Y-STR profiles obtained from the fecal material were concordant with the results from the donors' buccal swabs.     

24个Y-STR基因座荧光标记复合扩增体系的建立

目的构建包含24个Y-STR基因座的荧光标记复合扩增体系。方法选择DYS531、DYS630、DYS622、DYS552、DYS510、DYS449、DYS459a/b、DYS446、DYS443、DYS635、DYS587、DYS527a/b、DYS460、Y-GATA-A10、DYS520、DYS557、DYS522、DYS481、DYS570、DYS385a/b、DYS444 24个Y-STR基因座,构建荧光复合扩增体系。检测该体系的特异性、同一性、灵敏度、扩增均衡性、抗干扰性和准确性,调查其在广东地区人群的基因多样性。结果建立的复合扩增体系在检测的非人类及女性样本中未发现谱带,同一个体不同组织检测结果一致,0.1 ng以上标准品9948可检测获得完整分型结果。常见抑制物中体系内加入120~200μmol/L血红素、1.5~2.0 mmol/L钙离子时出现等位基因丢失,对靛蓝、腐殖酸、EDTA具有较强抗干扰性。通过146例无关个体与Yfiler系统平行检测比对,24 Y-STR检测体系分型准确。广东地区人群单倍型多样性(HD)为0.999 72,优于Yfiler系统(HD=0.998 58)。结论本研究建立的24个Y-STR基因座荧光标记复合扩增体系法医学应用前景广阔,可用于案件检验、亲权鉴定与Y-STR数据库建设。     

Low-cost direct PCR for aged and processed wildlife sample analysis

Direct PCR has been used successfully in wildlife forensic DNA analysis from several types of biological samples using specialized, commercial direct PCR kits. This is attributed to the proprietary chemicals provided in the kits such as pre-PCR buffer and modified DNA polymerases. These reagents can be expensive, thereby limiting their widespread adoption in developing countries, where wildlife crimes are often encountered. We report on a study to evaluate the possibility of using low-cost direct PCR assay for degraded and processed wildlife sample analysis. Phire^® and Q5^® polymerases were used, due to their relatively low cost, for direct amplification of six aged and processed sample types (dried skin, 30-year old hair, muscle tissue, bone, trace blood mixed in vodka, and dried soft antler). The result indicated that Phire^® Hot Start II DNA polymerase and Q5^® DNA polymerase performed similarly to commercially available direct PCR kit. The low-cost amplification could efficiently identify species origin from all aged and processed samples. We observed a rate of more than 80% amplification success and high PCR product concentrations sufficient for further sequencing. The assay proved to be cost effective and robust; thus, we expect it to be adopted by wildlife forensic laboratories in developing countries.     

Validation of a 17-locus Y-STR multiplex system

Internal validation of a commercial 17-locus Y-STR system (AmpFlSTR^® Yfiler™, Applied Biosystems) has been performed on the ABI PRISM^® 3130 Genetic Analyzer for use in forensic cases. The Yfiler™ kit was validated according to SWGDAM guidelines. Our results show that it is possible to obtain full profiles with as little as 30 pg of male DNA even in the presence of 20,000-fold amounts of female DNA. Reaction volume was optimized for 10 μl. Male-male mixtures yielded full profiles of the minor contributor with 10-fold excess of the major contributor. Stutter values for each locus were determined from data generated for the population study which included Y-STR profiles from 156 caucasian males from the Montreal and Lac St.-Jean areas of Québec, Canada. The study recorded 141 different haplotypes of which 131 were unique with a haplotype diversity of 0.9965. A number of non-probative forensic samples from rape kit epithelial fractions and fingernail scrapings were also successfully tested.     

Simplified Direct PCR Method for Reference Buccal Samples Using a Non-FTA Card by Omitting the Pretreatment Step

When using non-FTA cards in commercial multiplex STR kits for direct PCR, pretreatment steps with specific buffers are recommended. Here, we designed a rapid direct PCR method utilizing a non-FTA card, Oral Cell Sampling Kit, by omitting the pretreatment step involving Prep-n-Go™ Buffer, and it showed compatibility with the GlobalFiler™ Express PCR Amplification Kit, GlobalFiler™ PCR Amplification Kit, and PowerPlex^® Fusion system. To optimize the PCR conditions, we tested the method with different final PCR volumes and cycles. Finally, we conducted a performance test using 50 Korean buccal samples and confirmed the high performance of the method, detecting more than 90% of the samples with full profiles when using GlobalFiler™ PCR Amplification Kit and PowerPlex^® Fusion system at 29 cycles in a 10 μL final PCR volume. Thus, we report a simple direct PCR set-up to analyze reference samples collected using a non-FTA card manufactured in Korea.     

DNA investigative lead development from blood and saliva samples in less than two hours using the RapidHIT™ Human DNA Identification System

The RapidHIT™ Human DNA Identification System produces full STR DNA profiles (sample in, results out) in 90 min. Here we present results from a new protocol on the RapidHIT System, designed for crime scene samples. Data are presented for blood and saliva samples, with results for a range of samples and substrates. Success rates and sensitivity display a high level of performance for the sample types evaluated in the study and demonstrate the efficacy of the new protocol for crime scene samples.     

荧光STR直接复合扩增试剂缓冲体系的研制

目的研制适用于数据库样本荧光STR直接复合扩增体系。方法针对常规血卡、FTA和903血卡样本,配制扩增缓冲液基准母液,采用不同配方的扩增缓冲体系进行直接扩增及检测。考察不同种类增强剂、4种商业化DNA聚合酶、不同复性温度和终延伸时间对检材的检测效果,并验证优化体系的适应性。结果采用本文所建体系对各类血卡样本进行检验,均可获得样本清晰、完整的STR分型。体系选择BSA\Tween20\DMSO\甘油等增强剂组合、Typer热启动聚合酶1.5U/10μL、57～59℃复性温度、30～50min终延伸时间,采用10μL体系即可对直径1.2mm FTA卡血样进行有效分型。结论本文所研制的缓冲体系能够满足常规血卡、FTA和903血卡样本直接扩增检验的需要。     

Internal Validation of Human Mitochondrial DNA Quantification Using Real‐Time PCR

The quantity of mitochondrial DNA (mtDNA) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data. Validation of a human mtDNA real‐time quantitative PCR (qPCR) assay demonstrated its high degree of reproducibility and precision as well as an extremely sensitive threshold of detection (0.0001 pg/μL or approximately six human mtDNA copies/μL). A study of 35 nonprobative bone and teeth evidence samples revealed that 20 pg of mtDNA template is recommended for successful HV1 and HV2 sequence analysis; however, as little as 0.013 pg can generate a full mtDNA profile when using enhanced amplification reactions. The assay can also detect PCR inhibition and is useful for identifying samples that may benefit from re‐purification. Overall, the assay is an excellent method to quantify mtDNA and is useful for determining the best analytical approach for successful sequencing.     

Direct amplification of STRs from blood or buccal cell samples

Forensic databasing laboratories routinely analyze blood or buccal cell samples deposited on FTA^® paper. Prior to PCR amplification of the STRs, the FTA^® samples must undergo multi-step sample purification protocols to remove the PCR inhibitors present within the sample and from the FTA^® paper. The multi-step sample purification protocols are laborious, time-consuming and increase the potential for sample cross-contamination.To eliminate the need for DNA purification, we conducted studies to optimize the PCR buffer and thermal cycling parameters to allow for direct amplification of STRs from blood or buccal samples on FTA^® paper. We evaluated the effect of various factors on the DNA profile including: FTA^® disc size, blood sample load variation, and buffer formulation. The new STR assay enables the direct amplification of DNA from single source samples on FTA^® discs without sample purification. The new STR assay improves the workflow by eliminating tedious steps and minimizing sample handling. Furthermore, the new STR assay reduces cost by eliminating the need for purification reagents and expensive robots.     

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。     

Y-STR analysis on DNA mixture samples—Results of a collaborative project of the ENFSI DNA Working Group

The ENFSI (European Network of Forensic Science Institutes) DNA Working Group undertook a collaborative project on Y-STR typing of DNA mixture samples that were centrally prepared and thoroughly tested prior to the shipment. Four commercial Y-STR typing kits (Y-Filer, Applied Biosystems, Foster City, CA, USA; Argus Y Nonaplex, Biotype, Dresden, Germany; Powerplex Y, Promega, Madison, WI, USA; and DYSplex-3, SERAC, Bad Homburg, Germany) were used for the amplification of the mixture samples. The results of the study showed a striking inter-laboratory difference of kit performance as determined from the peak heights of the obtained Y-STR genotypes. Variation in quantity and quality of the shipped DNA can be excluded as reason for the observed differences because both samples and shipping conditions were found to be reproducible in an earlier study. The results suggest that in some cases a laboratory-specific optimization process is indicated to reach a comparable sensitivity for the analysis of minute amounts of DNA.     

5种免提取试剂盒检验滤纸血痕结果的比较

目的探讨5种免提取试剂盒对滤纸血痕样本检验的效果。方法陈旧滤纸血痕(存放时间12～14个月)及新鲜滤纸血痕(存放时间小于1个月)各920份,分别随机分为5组。应用AGCU 17+1、Goldeneye 20A、Powerplex16HS、Identifiler Plus、Identifiler Direct 5种免提取试剂盒进行检验,对比各组检验结果。结果陈旧滤纸血痕5种试剂盒的检验成功率为98.91%～100%,各组间无差异(P>0.05);新鲜滤纸血痕,Identifiler Plus和Identifiler Direct试剂盒检验成功率高于AGCU17+1、Goldeneye 20A及Powerplex 16HS试剂盒(P<0.01);将样本做陈旧化处理后再用成功率较低的3种试剂盒进行检验,成功率分别升至100%、99.46%、99.46%;Identifiler Plus试剂盒扩增循环27次效果优于28次。结论本文5种试剂盒均可用于滤纸血痕的直接扩增检验,但使用AGCU17+1、Goldeneye 20A及Powerplex 16HS试剂盒需将新鲜血痕做陈旧化处理;Identifiler Plus试剂盒需将循环次数降为27次。     

Ultrafast Amplification of DNA on Plastic Microdevices for Forensic Short Tandem Repeat Analysis

The majority of microfluidic devices used as a platform for low‐cost, rapid DNA analysis are glass devices; however, microchip fabrication in glass is costly and laborious, enhancing the interest in polymeric substrates, such as poly (methyl methacrylate) (PMMA), as an inexpensive alternative. Here, we report amplification in PMMA polymerase chain reaction (PCR) microchips providing full short tandem repeat profiles (16 of 16 loci) in 30–40 min, with peak height ratios and stutter percentages that meet literature threshold requirements. In addition, partial profiles (15 of 16 loci) were generated using an ultrafast PCR method in 17.1 min, representing a ~10‐fold reduction in reaction time as compared to current amplification methods. Finally, a multichamber device was demonstrated to simultaneously amplify one positive, one negative, and five individual samples in 39 min. Although there were instances of loci dropout, this device represents a first step toward a microfluidic system capable of amplifying more than one sample simultaneously.     

Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas^® Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5–32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime.