A novel platform for the modular integration of forensic assay setup and medium- to high-throughput purification of nucleic acids

With its extraction and assay setup modules, the QIAsymphony^® provides a highly flexible solution for processing forensic samples in a medium- to high-throughput scale. We tested the sensitivity of extraction, precision of sample processing, and accuracy of automated assay setup of this integrated system. Results attest to QIAsymphony's ability to isolate DNA from a spectrum of common forensic samples and process these samples without cross-contamination. Furthermore, accurate assay setup for downstream applications, like PCR, make this system highly suited for enhancing laboratory workflow.     

Automated processing of forensic casework samples using robotic workstations equipped with nondisposable tips: contamination prevention

An automated process has been developed for the analysis of forensic casework samples using TECAN Genesis RSP 150/8 or Freedom EVO liquid handling workstations equipped exclusively with nondisposable tips. Robot tip cleaning routines have been incorporated strategically within the DNA extraction process as well as at the end of each session. Alternative options were examined for cleaning the tips and different strategies were employed to verify cross-contamination. A 2% sodium hypochlorite wash (1/5th dilution of the 10.8% commercial bleach stock) proved to be the best overall approach for preventing cross-contamination of samples processed using our automated protocol. The bleach wash steps do not adversely impact the short tandem repeat (STR) profiles developed from DNA extracted robotically and allow for major cost savings through the implementation of fixed tips. We have demonstrated that robotic workstations equipped with fixed pipette tips can be used with confidence with properly designed tip washing routines to process casework samples using an adapted magnetic bead extraction protocol.     

DNA提取自动化工作站在法医学领域的应用

自动化工作站是指运用生化技术对生物性检材进行批量处理分析的全自动化工作平台,它可在短时间内批量进行移液,加样,混合等操作,特别适合用于大量生物性检材的PCR模板的制备。本文仅对DNA提取自动化工作站的基本模式、基本方法、法医学应用等进行综述,以期使该技术在法医DNA分析中得到更广泛的应用。     

A simple automated instrument for DNA extraction in forensic casework

The Qiagen BioRobot EZ1 is a small, rapid, and reliable automated DNA extraction instrument capable of extracting DNA from up to six samples in as few as 20 min using magnetic bead technology. The San Diego Police Department Crime Laboratory has validated the BioRobot EZ1 for the DNA extraction of evidence and reference samples in forensic casework. The BioRobot EZ1 was evaluated for use on a variety of different evidence sample types including blood, saliva, and semen evidence. The performance of the BioRobot EZ1 with regard to DNA recovery and potential cross-contamination was also assessed. DNA yields obtained with the BioRobot EZ1 were comparable to those from organic extraction. The BioRobot EZ1 was effective at removing PCR inhibitors, which often co-purify with DNA in organic extractions. The incorporation of the BioRobot EZ1 into forensic casework has streamlined the DNA analysis process by reducing the need for labor-intensive phenol-chloroform extractions.     

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.     

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。     

Direct STR amplification from whole blood and blood- or saliva-spotted FTA without DNA purification

The DNA purification step has been thought to be essential for typing of STR DNA. However, this process is time-consuming, and there is a risk of unexpected cross-contamination during purification. We report a new method for direct short tandem repeat (STR) amplification using a newly developed direct PCR buffer, AnyDirect, which can amplify STR loci from whole blood and blood- or saliva-spotted FTA cards without DNA purification. The autosomal and Y chromosomal STR loci were analyzed for whole blood and blood or saliva spots of random individuals, followed by comparison of the results with those of corresponding purified DNA. The results from whole blood and blood spots showed perfect concordance with those from purified DNA without allele or locus drop-out. However, in the case of saliva spots, no amplification or locus drop-out was observed in some of the samples, which offers a topic for further study. Additionally, some commercial hot-start DNA polymerases other than AmpliTaq Gold DNA polymerase were also found to be compatible with this buffer system. Therefore, this direct PCR buffer was demonstrated to be useful for fast forensic DNA analysis or criminal DNA databases for which there is no need to store DNA samples.     

Automation of the Differential Digestion Process of Sexual Assault Evidence,

Sexual assault evidence samples require the use of a specific process known as a differential digestion to separate sperm from nonsperm cells prior to DNA extraction. An automated differential digestion process was developed using a selective degradation technique, which uses DNase I to digest the remaining nonsperm DNA in the sperm fraction. The use of DNase on pristine samples, as well as aged and degraded samples, was assessed to ensure that the quantity and quality of the sperm DNA were not compromised or adversely affected. Samples processed using the selective degradation technique yielded comparable DNA yield and DNA typing data to the conventional differential digestion process. The automated process utilized 96‐well plates for high throughput and incorporated microscope slide preparations for confirmation of sperm. It reduced processing time by about sixfold and was paramount in the elimination of the Oakland Police Department Criminalistics Laboratory's sexual assault kit backlog.     

应用自动化工作站提取常见生物样本DNA

目的建立使用自动化工作站提取法医案件生物样本DNA的方法。方法选用Biomek 3000自动化工作站,采用DNA IQTM系统及Chelex法对法医案件中常见生物样本进行DNA提取,荧光定量技术进行定量,PCR扩增16个STR基因座并与手工提取方法比较。结果与手工提取方法进行比较,选用自动化工作站结合使用DNAIQTM系统及Chelex法提取DNA可获得满意的STR检验结果。结论自动化工作站可用于法医案件中常见生物样本的DNA提取。     

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.     

法医DNA实验室的DNA污染和防范

DNA污染是产生DNA鉴定结论错误的重要因素,法医DNA实验室要努力去解决这一问题。DNA污染有自身污染、交叉污染、PCR污染3种。法医DNA实验室要采取实验室分区、严格检验操作步骤、对试剂及消耗材料进行质量控制等方法防止发生DNA污染,采取设置对照样本、核查DNA结果、建立DNA排查数据库等方法监测和发现DNA污染。     

Optimization and validation of a fully automated silica-coated magnetic beads purification technology in forensics

Automated procedures for forensic DNA analyses are essential not only for large-throughput sample preparation, but are also needed to avoid errors during routine sample preparation. The most critical stage in PCR-based forensic analysis is DNA isolation, which should yield as much highly purified DNA as possible. The extraction method used consists of pre-treatment of stains and samples, cell lysis using chaotropic reagents, binding of the DNA to silica-coated magnetic particles, followed by elution of the DNA. Our work focuses mainly on sample preparation, obtaining the maximum possible amount of biological material from forensic samples, and the following cell lysis, to create a simple standardized lysis protocol suitable for nearly all forensic material. After optimization and validation, the M-48 BioRobot((R)) workstation has been used for more than 20,000 routine lab samples. There has been no evidence of cross contamination. Resulting DNA from as small as three nuclear cells yield reliable complete STR amplification profiles. The DNA remains stable after 2 years of storage.     

96孔过滤板在接触性DNA自动化检验中的应用

目的将96孔过滤板用于自动化工作站,对接触性DNA进行检验。方法收集553份现场提取的附着在烟蒂、饮料瓶、门窗把手、电源网线头、作案工具、手套上的微量接触性生物检材,在96孔过滤板上进行裂解后整体离心,分离载体与裂解液;应用自动化核酸提取纯化仪结合M48磁珠纯化试剂盒提取纯化DNA,采用IdentifilerPlus试剂盒进行扩增,3500XL测序仪电泳分型,ID-X专家系统分析结果。结果在553份检材中,成功获得STR分型的有271份,检验成功率在31.6%~97.3%之间,烟蒂、饮料瓶检材的成功率均在90%以上,提取纯化过程约90min。结论将96孔过滤板用于自动化工作站,可在实现批量接触性DNA的快速、自动化提取的同时,有效提高接触性DNA检出率。     

批量陈旧血样DNA的自动化检验

目的建立批量陈旧血样DNA自动化提取检验的方法。方法采用普通磁珠法和本文建立的磁珠法经TECAN Freedom EVO150—8型自动化工作站分别提取540份陈旧血样模板DNA,采用Sinofiler^TM试剂盒进行荧光标记复合扩增。结果采用普通磁珠法和本文所建DNA提取方法,在540份样本中获得全部基因座STR分型的样本分别为217份和488份,检验成功率分别为40．2％和90．3％。结论本文所建方法可显著提高大批量陈旧血样自动化检验成功率。     

Evaluation of a semi-automated, magnetic bead-based DNA extraction method for genetic fingerprinting of forensic casework samples

In order to cope with the demanding workload for DNA profiling of forensic casework samples a concept for a semi-automated processing system was developed at the Landeskriminalamt (Office of Criminal Investigation) Baden-Württemberg, Germany [K. Vollack, et al., Implementation of a semi-automated processing system for DNA profiling of forensic casework samples, this issue]. The applied magnetic bead extraction method is based on ChargeSwitch^® Technology (CST) from Invitrogen and was established on a liquid handling workstation Freedom EVO^® 150 from Tecan.     

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.     

胍盐裂解液对自动化提取工作站DNA回收率的影响

目的研究不同稀释度胍盐裂解液对自动化提取工作站DNA回收效率的影响。方法制备不同浓度的DNA样本,并将EQ1000法医DNA提取试剂盒中的胍盐裂解液梯度稀释,在本实验室配置的自动化提取工作站上,运行＂工作站磁珠法＂提取程序,AB-7500型荧光定量PCR仪检测提取前后的样本DNA浓度。选取2～7号梯度组DNA回收效率数据进行统计学检验,比较不同稀释度胍盐裂解液的处理对样品DNA回收效率的影响。结果 90%～60%稀释度胍盐裂解液组样品回收效率在66%左右;50%～20%稀释度组样品DNA回收效率在41%左右;稀释度在50%以下与60%以上组样品回收效率之间差异极显著。结论应用＂工作站磁珠法＂提取生物检材DNA,其DNA回收效率受胍盐裂解液浓度的影响。     

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。