A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpFlSTR^® Idenitfiler^® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.     

Human chorionic gonadotropin detection by means of enzyme immunoassay: a useful method in forensic pregnancy diagnosis in bloodstains

This paper reports on human chorionic gonadotropin (HCG) detection in bloodstains using a commercial kit based on enzyme immunoassay. The specificity and sensitivity of the method is tested, as well as HCG stability over time in the samples. Detection of this hormone in bloodstains is of special interest for pregnancy diagnosis in forensic science applications. The experimental series comprises 60 bloodstains prepared in our laboratory and obtained from blood samples taken from 30 pregnant women and 30 healthy, nonpregnant individuals. The bloodstains were studied at different stages of aging over a 6-month period, each sample being stored at 2 different temperatures throughout the process. The experimental evidence proves HCG to be a useful and stable diagnostic indicator of pregnancy in bloodstains. In addition, the technique used is fast, as well as highly sensitive and specific.     

混合血迹中不同个体成份逐一分离识别的研究

目的研究法庭科学混合血迹物证中不同个体成份逐一分离、识别的问题,建立适合混合血迹个体识别的分析技术。方法采用PCR-SSCP及测序技术,选择m tDNA D-loop区的HVI 16030~16481区域452 bp片段作为分析目标,对中国汉族两无关个体、三无关个体混合血迹进行分析。结果100份两个体混合血迹样品m tDNA 452bp的PCR产物经SSCP电泳分离,结果有95份样品完全分离开,分离成功率达95%;30份三个体混合血迹样品452 bp片段经SSCP电泳分离,结果有26份样品有1~3个个体完全分离开,分离成功率达84%。对其中3份两个体混合血样、2份三个体混合血样SSCP电泳分离后的谱带进行回收、测序分析,两个体混合血样每一份均可准确获得其中单一个体序列及以另一个体主要成份(峰值比达4∶1以上)的序列结果;三个体混合血迹中不同个体成份可以达到初步分离,1份可准确确定单一个体序列。对两个体不同比例混合样品SSCP分析,结果可以检测到较少成份的最低比例为20∶80。结论本研究建立的PCR-SSCP及测序分析混合血迹综合技术,是对混合血迹中不同个体成份逐一分离、识别的一种有效技术手段。     

PuriTyper~(TM)法医学纯化试剂盒的性能验证

目的验证PuriTyperTM纯化试剂盒各项性能指标和法医学应用价值。方法收集及制备抗凝血液、常见案件检材(唾液、烟头、精液、毛发、指甲、骨骼及组织块)、斑痕样本(血斑、唾液斑、精斑)以及模拟添加抑制剂和模仿自然环境中放置的血斑。采用PuriTyperTM纯化试剂盒提取纯化并进行DNA定量,IdentifilerTM复合扩增试剂盒扩增,产物经ABI 3130遗传分析仪进行检测,Genemapper软件分析结果,对该试剂盒灵敏度、稳定性、重复性、检材适应性进行测试。结果采用该试剂盒提取0.1～40μL血液分别获得0.042～26.45ng/μL的DNA。3种斑痕样本DNA产量平行试验结果稳定。不同类型检材重复检验所获IPC的CT平均值在27.60至28.03之间。常见案件检材所得分型与已知结果均一致。结论 PuriTyperTM纯化试剂盒能够满足法医DNA检验的要求,对法医学实践具有重要的应用价值。     

Adaptation and evaluation of the PrepFiler™ DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security

Within the initial step of the forensic DNA analysis process, the DNA extraction efficiency and especially the removal of potential PCR inhibitors is crucial for subsequent steps, e.g. quantification by real-time PCR and amplification of short tandem repeats (STRs). The protocol of the PrepFiler™ Forensic DNA Extraction Kit was optimized for the application on a Tecan liquid handling workstation Freedom EVO^® 150. This modified application of the PrepFiler™ technology was compared with respect to DNA yield, sensitivity and the ability to remove potential PCR inhibitors to an established routine method working on the same liquid handling workstation based on ChargeSwitch^® Technology (CST) from Invitrogen.     

MiniFiler~(TM)试剂盒在LCN-STR分型中的应用

目的评估MiniFilerTM试剂盒在LCN-STR分型中的法医学应用价值。方法采用MiniFilerTM与IdentifilerTM试剂盒,对49份常量与39份LCN检材,包括血迹、精斑、骨骼等7类常见检材的检验结果进行比较,并对两种试剂盒的检测灵敏度进行比较。结果在49份常量检材的检验结果中,两种试剂盒的STR分型结果全部一致;在39份LCN生物检材的检验结果中,MiniFilerTM试剂盒获得全部STR分型的有30份,部分分型的5份,阴性的4份;IdentifilerTM试剂盒获得部分STR分型的22份,阴性的17份。MiniFilerTM试剂盒检验成功率明显高于IdentifilerTM试剂盒;MiniFilerTM试剂盒的检测灵敏略高于IdentifilerTM。结论MiniFilerTM试剂盒可显著提高LCN生物检材的检验成功率,适合应用于法庭科学实际检案中LCN生物检材的检验鉴定。     

A Comparison of DNA Extraction Using AutoMate Express™ and EZ1 Advanced XL from Liquid Blood,Bloodstains, and Semen Stains

In this study, DNA was extracted using an AutoMate Express? and an EZ1 Advanced XL from liquid blood, fresh and aged bloodstains, and fresh and aged semen stains. Extracted DNA was quantified by real‐time PCR using the D17Z1 locus. Short tandem repeat typing was performed using an AmpF?STR^® Identifiler kit. The yields of DNA obtained by the AutoMate Express? were higher from fresh bloodstains and fresh semen stains, almost the same from aged bloodstains and aged semen stains, but slightly lower from liquid blood compared with those obtained by the EZ1 Advanced XL. The addition of dithiothreitol or the use of PrepFiler? lysis buffer improved the EZ1 Advanced XL results from fresh bloodstains, but not for liquid blood and aged bloodstains. Our results demonstrated that the PrepFiler? lysis buffer is the main contributor to the higher DNA yields of the AutoM ate Express? for fresh bloodstains.     

PCR-based detection of salivary bacteria as a marker of expirated blood

Distinguishing between bloodstains caused by a spatter pattern or by expirated blood may be crucial to a forensic investigation. Expirated blood is likely to be contaminated with saliva but current techniques have limited sensitivity, especially with small bloodstains. We report that a PCR assay, designed to detect salivary bacteria, can amplify streptococcal DNA from saliva stains applied to fabrics for at least 62 days after seeding. Bacterial DNA was detected when 0.01 µl of saliva was present in the stain and the amplification was not affected by contamination with blood. These findings indicate that PCR amplification of salivary microbial DNA may have application in the identification of expirated bloodstains in forensic case-work.     

Influence of the luminol chemiluminescence reaction on the confirmatory tests for the detection and characterization of bloodstains in forensic analysis

Preliminary tests for the detection of stains at crime scenes aim to focus the police work making them more efficient in the combat of criminality. The application of the luminol chemiluminescence reaction (3-aminoftalhidrazida) in presumptive tests for the detection of bloodstains is known for more than 40 years in forensic science. This reaction is based on the emission of light through the chemical reaction of luminol mixed with hydrogen peroxide and a hydroxide in the presence of a catalytic molecule (iron from the hemoglobin) (Laux [1]).This work evaluates the luminol interference and its effect on subsequent serological and DNA testing. Samples prepared with blood and different concentrations of luminol solution containing luminol, peroxide of hydrogen and sodium carbonate, were analyzed. Additionally, samples of serial dilutions of standard DNA mixed with luminol solution were also analyzed. Although presumptive tests with luminol do not establish the characterization and identification of stains at crime scenes, preliminary results indicated that it is suitable for the detection of invisible bloodstains for forensic analysis, with few detrimental effects on the serological tests and subsequent DNA recovery and typing.     

Utility validation of extraction of genomic DNA from hard tissues, bone and nail, using PrepFiler™ Forensic DNA Extraction Kit

STR profiling using hard tissues obtained from a severely decomposed body is sometimes a laborious work. There is now on a market a new DNA extraction kit, PrepFiler™ Forensic DNA Extraction Kit (AppliedBiosystems), and we tested it for missing persons. Postmortem intervals ranged from weeks to several years. Fifteen bone fragments and eleven nails were used in this report. Genomic DNA was quantified by QuantiFiler^® DUO Quantification Kit (AppliedBiosystems), and STRs were analyzed using AmpFlSTR^® Identifiler^® PCR Amplification Kit (AppliedBiosystems). The profiling of 16 STR loci was successful in all nail samples. However, STR profiling was successful in only 6 of 15 bone materials. Nine cases failed to analyze STR polymorphisms using another DNA extraction kit, the QIAamp DNA Mini Kit (QIAGEN). For bone samples, it seems that STR profiling depends on the quality of samples.     

被洗织物上潜血痕迹发现方法的比较

目的探索被洗涤后衣物上潜血痕迹的发现方法。方法用联苯胺法、鲁米诺法和新型仪器TL-445激光生物检材发现仪寻找不同织物上不同洗涤强度的潜在血迹。结果联苯胺仅能发现部分纯棉织物上潜血,鲁米诺能显示全部纯棉织物上血迹,TL-445激光生物检材发现仪能显示所有纯棉、纯化纤织物上血迹。结论用TL-445激光生物检材发现仪检测相比联苯胺法和鲁米诺法更适合洗涤后织物上潜血痕迹的发现。     

Validation studies of an immunochromatographic 1-step test for the forensic identification of human blood.

An immunochromatographic 1-step test for the detection of fecal occult blood was evaluated for applicability for the forensic identification of human blood in stained material. The following experiments were conducted: 1) determination of the sensitivity and specificity of the assay; 2) evaluation of different extraction media for bloodstains (sterile water, Tris buffer pH 7.5 provided in the test kit, 5% ammonia); 3) analysis of biological samples subjected to a variety of environmental insults; and 4) evaluation of casework samples. This immunochromatographic 1-step occult blood test is specific for human (primate) hemoglobin and is at least an order of magnitude more sensitive than previous methods for detecting human hemoglobin in bloodstains. The antigen is insensitive to a variety of environmental insults, except for exposure to certain detergents and household bleaches and prolonged exposure to certain preparations of luminol. The entire assay can be conducted in field testing conditions within minutes. When in the laboratory the supernatant from a DNA extraction is used for the assay, there is essentially no consumption of DNA for determining the presence of human hemoglobin in a forensic sample. The data demonstrate that this test is robust and suitable for forensic analyses.     

Developmental validation of the AmpF?STR SEfiler Plus™ PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples

Since its introduction in 2002, the AmpF?STR^® SEfiler™ kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus^® Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfiler™ kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples.The new AmpF?STR^® SEfiler Plus™ kit contains the same loci and primer sequences as the SEfiler™ kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfiler™ kit.Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples.     

Species identification in routine casework samples using the SPInDel kit

The identification of species in casework samples is of fundamental importance for forensic investigations. Laboratories are increasingly compelled to provide accurate and fast identifications in trace materials left on crime scenes, wildlife poaching, illegal trade of protected species, fraudulent food products cases, etc. However, the field of nonhuman forensic genetics is still working on the standardization of typing methods and practices. Here we describe the successful implementation of the Species Identification by Insertions/Deletions (SPInDel) method in routine casework analyses in 11 laboratories worldwide. The SPInDel was developed to detect human DNA, at the same time that identifies common animal species. The fragment size analysis of six mtDNA regions allows identification in suboptimal DNA samples, including mixtures, with no need for sequencing. The samples were collected from 2013 to 2018 and included hair, blood, meat, saliva, faeces, bones, etc. The SPInDel kit successfully identified >95% of the samples, being dog, human and pig the most frequently detected species. The six SPInDel loci were successfully amplified in mixtures and degraded samples (river water, sand, stains in clothes, etc.). Interestingly, several species that were not originally targeted by SPInDel primers were also identified (e.g., red fox, brown bear, fallow deer and red deer). In conclusion, the SPInDel kit was successfully used in crime scene investigations (often involving human DNA detection) and in cases of poaching, environmental contamination and food fraud. It is now becoming a useful tool for the routine analysis of nonhuman DNA samples within the high quality standards of forensic genetics.     

Gm/Km determination in sperm traces

In the forensic laboratories of the Federal Republic of Germany and West-Berlin 23 different semen stains and in our own laboratory 20 semen stains were typed in the gm/km-system doing 125 and 61 (own) test respectively. Examination was carried out by means of the haemagglutination method, which has been used successfully in typing bloodstains. Our critical assessment based on earlier experiences with semen stains was now confirmed and statistically evaluated: typing was successful in about 35-50% of the tests, but besides false-negative results, there was also a considerable percentage (4-10%) of false-positive ones. Therefore for the present it seems best to exclude the gm/km-typing of secretion stains from forensic investigations in order to avoid false incriminations or exonerations of suspects.     

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。     

Effect of fabric washing on the presumptive identification of bloodstains

The purpose of this study was to investigate the retention of blood stains on twelve different types of fabrics after washing at various drying times. The findings of this study, supported by chi-square analysis, indicate that the retention of bloodstains on washed fabrics depends upon the particular fiber composition of the fabric, the specific blood screening test used, and whether or not a detergent was used in the wash. The results of this research did not reveal a significant effect of the drying time on the retention of bloodstains, as tested during the 48-h limit of this experiment. The author concludes that the forensic serologist should consider the factors investigated in this study before rendering an opinion on the retention of bloodstains on washed garments.     

Use of “AnyDirect PCR buffer” for PCR amplification of washed bloodstains: A case report

AnyDirect PCR buffer (BioQuest) permits to perform direct PCR from different kinds of forensic samples (blood, saliva, sperm, etc.) without any DNA purification step.This is very useful in particular when working with small quantity of sample since it avoids the risk of loosing sample step by step as often happens with traditional DNA extraction procedures.In the present casework we analyzed some bloodstains found by luminol test onto washed clothes and linen: all samples were analyzed either using the AmpFlSTRs Identifiler kit (Applied Biosystems).     

The detection of Rh antigens (D,C,c,E,e) on bloodstains by a micro-elution technique using low ionic strength solution (LISS) and papain-treated red cells

Ninety experimental bloodstains, were examined, with the intention of detecting the principal Rh antigens, by using a micro-elution method improved by the use of low ionic strength solution (LISS) and papain-treated red cells. This method makes it possible to employ most commercially produced sera in routine forensic haematology laboratory work.The antigens could regularly be detected in stains of the following ages: D, C and c in stains of at least 6 months, E in stains of at least 4 months, and e in stains of at least 2 months.