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。     

磁珠法自动化纯化现场检材DNA方法研究

目的利用TE-MAGS在TECAN工作站上结合磁珠试剂盒,建立自动化工作站批量纯化现场检材DNA的方法,并探讨其在法医物证检案中的应用。方法灵敏度测试:标准品使用0.1ng/μL 9947A,用200μL TES稀释制备DNA总量0.1ng~1ng共10种的标准样品,采用本文方法提取纯化,使用IdentifilerTM试剂盒扩增,用3130XL型测序仪检测,Gene Mapper ID-X分析,分析STR图谱质量;纯化能力测试:在1ng总量的标准样品中加入腐殖酸、血红素,采用本文方法提取纯化、扩增检测,分析STR图谱质量;实际案件应用对比:收集304份现场检材,分别采用本方法和硅珠法进行提取纯化,经扩增检测,统计对比两种提取纯化方法 STR分型成功率。结果灵敏度测试:0.1ng~0.2ng总量标准样品提取的DNA模板,扩增后可检测到部分基因座STR图谱,0.3ng~1ng总量标准样品提取的DNA模板,扩增后可以得到完整的STR图谱;纯化能力测试:对混合有一定浓度的腐殖酸、血红素的标准样品的提取产物检测图谱未见明显抑制;实际案件应用对比测试:304份现场检材工作站磁珠法检出成功率(50%)高于硅珠法(40.8%)。结论本文所建立的方法缓冲范围较大,回收率高,纯化能力强,提取产物STR分型成功率高,适合现场检材批量化DNA检验。     

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。     

ABO基因分型与多重STR联合检测在法医学中的应用

目的建立ABO基因型和Goldeneye16A试剂盒联合检测的方法,并评价其在法医学实践中的应用价值。方法将6种ABO基因型(A/A,A/O,B/B,B/O,A/B,O/O)的序列特异性引物(PCR-SSP)检测方法与Goldeneye16A试剂盒相整合进行同步分型。通过对460份男性个体血痕样本、9947A DNA及90份案件样本进行检测,考察方法的一致性、灵敏度及对法庭科学检材的适用性。结果应用本文方法可同时检出6种ABO基因型和15个常染色体STR基因座及性别决定基因座,检测灵敏度为125pg,其中ABO基因检测灵敏度达63pg。460份男性血痕和90份案件检材证实该联合分型方法用于各类检材结果准确、稳定。结论本文ABO基因分型与多重STR联合检测方法,适用于各类含有核细胞的生物检材,在法庭科学DNA鉴定中有较好的应用前景。     

Evaluation of two DNA/RNA co-extraction methods for body fluid identification in forensics

Body fluid identification has become a field of interest in forensic casework as it can add value to particular investigative scenarios. Identifying the source of the biological material is not always an upfront task using conventional methods; therefore, profiling of specific mRNA markers can provide the answer. The implementation of RNA based analyses in forensic casework must focus on the quality and sensitivity of methods, starting with nucleic acid extraction, and without loss of DNA for STR profiling. In this work, two methods for DNA and RNA co-extraction were tested and compared: a commercial kit that uses a spin, mini column methodology, and a quick, simple nucleic acid isopropanol precipitation based protocol. Both methods simultaneously extract DNA and RNA, crucial for forensic casework and were tested in semen samples. Nucleic acid quantifications as well as purity assessment ratios (OD₂₆₀/OD₂₃₀ and OD₂₆₀/OD₂₈₀) were obtained by both methods to infer on the use of extracts in downstream applications such as PCR. The performance of the two tested protocols was further evaluated by analyzing two semen mRNA specific markers, PRM1 and SEMG1. When compared to the commercially developed kit, results suggest that the literature adapted protocol allowed more carryover of contaminants absorbing at 230 nm and 280 nm as the purity ratios were below the accepted standard ranges. Negative results for mRNA profiling supported the QC results obtained by spectrophotometry. On the hand, PRM1 and SEGM1 were positive in RNA samples extracted with the commercial kit.     

MiniFiler试剂盒运用于法医微量物证的检验

目的应用MiniFiler试剂盒对法医微量物证进行DNA分析。方法采用MiniFiler试剂盒对样本进行DNA分型,确定样本9个STR基因座的等位基因型。结果10pg DNA模板经MiniFiler试剂盒扩增后,能够进行DNA分型,但40pg以上的DNA方可得到稳定可靠的分型结果。结论MiniFiler试剂盒可以用于法医微量物证的STR分析。     

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.     

Developmental validation of reduced-size STR Miniplex primer sets

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.     

Applicability of DNA analysis on adhesive tape in forensic casework

Adhesive tape is commonly used in crimes and is often the subject of forensic evaluation. DNA analysis of adhesive tape can provide DNA profiles of suspects. The object of this study was to evaluate the applicability of DNA analysis on adhesive tape samples in forensic casework. We retrospectively reviewed all cases involving adhesive tape or similar items received by our institute for DNA analysis during the past 11 years. From 100 forensic cases reviewed, 150 adhesive tape samples were examined. A total of 98 DNA profiles were obtained from these samples. Sixty-two of the profiles provided feasible case-relevant information. In conclusion, DNA profiling of adhesive tape samples can be useful in a variety of forensic cases.     

Chelex-100提取生物检材DNA实时PCR定量研究

目的研究Chelex-100法提取的生物检材DNA用量与复合STR分型成功率的关系。方法113份各种生物检材采用Chelex-100法提取DNA，应用Quantifiler人类DNA定量试剂盒在ABI 7500荧光定量PCR仪上进行实时PCR定量，同时用Identifiler复合扩增系统在ABI 3100遗传分析仪上对这些DNA样品进行STR分型。结果各种生物检材提取的DNA浓度分别为：37份滤纸、纱布血痕0．042～5．28ng／μl，16份口腔拭子1．15—4．21ng／μl，18份烟头0．016～1．46ng／μl，10份肋软骨0．531—14．40ng／μl，8份肌肉5．75—24．80ng／μl，7份指甲0．788—11．50ng／μl，17份精斑0．79～99．50ng／μl。在建立的8μl扩增体系中，根据上述结果，调整用于复合STR扩增的DNA模板量在0．5—3ng之间，大部分样品可获得完全的STR分型。结论Chelex-100法提取的检材DNA模板用量在0．5—3ng之间可得到有效STR扩增，浓度为0．5ng／μl以上的DNA样品，用小体积模板（1μl）比大体积（3μl）模板扩增效果好。     

Assessing the performance of quantity and quality metrics using the QIAGEN Investigator® Quantiplex® pro RGQ kit

The Quantiplex® Pro RGQ kit quantifies DNA in a sample, supports the detection of mixtures and assesses the extent of DNA degradation based on relative ratios of amplified autosomal and male markers. Data show no significant difference in the accuracy and sensitivity of quantification between this and the Promega PowerQuant® System, both detecting the lowest amount of DNA tested, 4 pg. Laboratory controlled mixed male:female DNA samples together with mock sexual assault samples were quantified across a range of mixture ratios. Analysis software detected mixed DNA samples across all ratios for both quantification kits. Subsequent STR analysis using the Investigator® 24Plex QS Kit was able to corroborate mixture detection down to 1:25 male:female DNA ratios, past which point mixtures appeared identical to single-source female samples. Analysis software also detected laboratory degraded DNA samples, with data showing a positive trend between the Degradation Index (DI) and length of time of sonication. When used on ancient remains the assay was able to triage samples for further analysis, and STR profiles were concordant with DNA quantification results in all instances. STR analyses of laboratory-controlled sensitivity, mixture, and degradation studies supports the quality metric obtained from quantification. These data support the use of the Quantiplex® Pro RGQ kit for sample screening and quantification in forensic casework and ancient DNA studies.     

Validation of PrepFiler™ forensic DNA extraction kit (Applied Biosystems)

The PrepFiler™ is a new kit recently introduced by Applied Biosystems for DNA extraction from a wide range of forensic samples. In the present study we tested the performance of PrepFiler™ kit against other commonly used commercially available kits on a variety of real forensic casework samples: bloodstains on different substrates, washed bloodstains, semen stains, saliva stains, hairs, bones, tissues, nails, prints after chemical treatments, skin swabs.     

法医物证DNA自动化检验技术体系的研究

目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO100．4、75—2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,进行比较测试。在“全国公安机关DNA数据库应用系统”中建立并应用实验室信息管理系统（LIMS）模拟实施规范化DNA检案。结果1552份各类检材,采用工作站-磁珠法提取DNA效果最佳,STR检测成功率为95％,工作站-Chelex法为88％;二者分别与其手工提取法比较,成功率无明显差异。92个样本同期检测,自动化工作站较手工操作DNA检案时间可缩减1．25倍。结论工作站域珠法提取涉案检材DNA,可获得满意的STR分型结果。应用LIMS管控,可有效防控污染,明显提高检案效率及鉴定质量。     

Development of two new Mini-STR multiplex assay for typing archival Bouin's fluid-fixed paraffin-embedded tissues

Short amplicon autosomal short tandem repeat (Mini-STR) assay has proved to be a highly useful tool in forensic applications, especially for highly degraded DNA samples that typically result in partial profiles and total loss of information from regular STR amplicons.In this study two new quadruplex systems were designed to get nuclear DNA profile from degraded forensic casework samples. In order to obtain PCR products less than 120 bp in size, primer pairs of eight STR markers, included in available commercially multiplex PCR kits, were redesigned and assembled in two PCR-multiplexes: D8S1179, D3S1358, TPOX, D16S539 and CSF1P0, TH01, D13S317, D5S818.After validation, these two Mini-STR quadruplex were employed in paternity testing case that involved DNA extraction from archival postmortem Bouin's fluid-fixed paraffin-embedded tissue where commercial kit yielded low success. The results obtained with the present Mini-STR PCR-multiplexes proved clearly demonstrating their usefulness in analyzing degraded DNA samples.     

Implementation and Validation of the Teleshake Unit for DNA IQ™ Robotic Extraction and Development of a Large Volume DNA IQ™ Method

Abstract: Automated platforms used for forensic casework sample DNA extraction need to be versatile to accommodate a wide variety of sample types, thus protocols frequently need modification. In this study, DNA IQ? methods previously developed for the Biomek^® 2000 Automation Workstation were adapted for the Teleshake Unit using normal volumes and all deepwell extraction, and a large volume DNA IQ? method developed. DNA purification without detectable contamination of adjacent reagent blanks is reported in the extraction of tissue samples containing several micrograms of DNA. Sensitivity and contamination studies demonstrated similar performance with the manual organic extraction method for bloodstain dilution samples. Mock casework samples demonstrated the effectiveness of the Teleshake and Teleshake large volume methods. Because of the performance and increased versatility of the DNA IQ? extraction with these modifications, the Teleshake Unit has been implemented in both normal and large volume automated DNA extractions at the Virginia Department of Forensic Science.     

Real-time polymerase chain reaction quantification of canine DNA

The accurate quantification of target DNA is an important step in the short tandem repeat analysis of forensic biological samples. By utilizing quantification data to control the amount of template DNA in the polymerase chain reaction (PCR), forensic scientists can optimize testing and minimize the consumption of limited samples. The ability to identify and quantify target DNA in mixed-species samples is crucial when it may be overwhelmed by nontarget DNA, as in cases of dog attack. We evaluated two quantitative real-time PCR assays for dynamic range, species specificity, and inhibition by humic acid. While both assays proved to be highly sensitive and discriminating, the Melanocortin-1 Receptor (MC1R) gene Taqman assay had the advantages of a shorter run time, greater efficiency, and safer reagents. In its application to forensic casework, the MC1R assay has been advantageous for quantifying dog DNA in a variety of mixed-species samples and facilitating the successful profiling of individual dogs.     

Effectiveness of Single‐Nucleotide Polymorphisms to Investigate Cattle Rustling

Short tandem repeats (STR)s have been the eligible markers for forensic animal genetics, despite single‐nucleotide polymorphisms (SNP)s became acceptable. The technology, the type, and amount of markers could limit the investigation in degraded forensic samples. The performance of a 32‐SNP panel genotyped through OpenArrays^TM (real‐time PCR based) was evaluated to resolve cattle‐specific forensic cases. DNA from different biological sources was used, including samples from an alleged instance of cattle rustling. SNPs and STRs performance and repeatability were compared. SNP call rate was variable among sample type (average = 80.18%), while forensic samples showed the lowest value (70.94%). The repeatability obtained (98.7%) supports the used technology. SNPs had better call rates than STRs in 12 of 20 casework samples, while forensic index values were similar for both panels. In conclusion, the 32‐SNPs used are as informative as the standard bovine STR battery and hence are suitable to resolve cattle rustling investigations.     

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).     

Application of direct PCR in forensic casework

Direct PCR is fast becoming a popular method in forensic science due to the advantages of saving time and money in the lab while increasing the probability of obtaining substantial results has a positive rippling effect. A laboratory is able to reduce the time spent on processing trace DNA samples, which can lead to investigators receiving important information in a timely manner that may not have been possible using standard methods. This study highlights the benefits of direct PCR in forensic casework by analysing trace and touch DNA on a range of substrates and exploring the loss of initial DNA due to extraction.     

Internal validation study of the PowerPlex® Y23 system

The PowerPlex Y23 System is a 23-loci, 5-color Y-STR multiplex designed for genotyping forensic casework samples, database samples and paternity samples. The kit contains: all 12 loci in the current PowerPlex Y System, the additional 5 loci found in AmpFlSTR Y-filer, plus 6 new loci. An internal validation study of the PowerPlex Y23 kit was therefore conducted including the following aspects: sensitivity, mixture studies of male–male and female–male DNA, performance with simulated inhibition and stutter calculations. 100 Caucasians living in Switzerland were also typed using the PowerPlex Y23 kit.