Successful validation of a fully automated sample lysis workstation

The Netherlands Forensic Institute recently validated a fully automated system for sample lysis, the AutoLys workstation and AutoLys tubes, developed in collaboration with Hamilton Robotics. We have carried out the developmental validation of the AutoLys system and concluded it offers a solution to drastically reduce user-errors, increase DNA yields, gain in productivity, while maintaining quality. This article reports on the study setup and discusses the results of comparing the AutoLys workstation to the conventional (manual) method. The AutoLys system proved to successfully process all commonly found forensic sample types, producing high quality extracts, containing on average 12.1% more DNA than manually extracted samples (as determined by qPCR and STR-profiling). The optimized robotic protocol minimizes the risk on cross-contamination (no cross-contamination detected in over 500 samples tested) and retains the flexibility to adjust chemically relevant variables specific to a laboratory's individual requirements. The fully automated process enables complete sample-tracking, drastically reducing the risk on user-errors. The NFI is currently validating an on-deck magnetic bead-based purification of the lysates.     

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。     

磁珠DNA自动提取系统在法医检验中的应用

磁珠DNA自动提取系统是将磁珠分离技术与DNA自动提取工作站结合运用,以达到高效提取DNA的目的,它具有操作时间短、准确性高、检材用量少等特点,特别适用于大批量生物检材的快速提取,是目前主流的DNA自动化提取方法。本文对磁珠DNA自动提取系统的基本组成、工作原理、操作流程及其在法医检验领域中的应用等进行了综述,并对该系统的发展前景进行了展望。     

Proving liability for highly and fully automated vehicle accidents in Australia

This article considers how liability questions will be resolved under current Australian laws for automated vehicle (‘AV’) accidents. In terms of the parties that are likely to be held responsible, I argue that whether the human driver remains liable depends on the degree to which the relevant AV is automated, and the degree of control the human driver had over the events leading up to the particular accident. Assuming therefore that human drivers would not be held liable for the majority of highly and fully automated vehicle accidents, plaintiffs will have to establish liability on part of those who manufacture, maintain or contribute to the operation of AVs, under the claims available in Australia's product liability regime.This article then turns to the problems of proof that plaintiffs are likely to face in establishing AV manufacturer liability in negligence, or in a defective goods claim under Part 3–5 of the Australian Consumer Law (‘ACL’). Firstly, it may be difficult to determine the cause of the AV accident, due to the technical complexity of AVs and due to ongoing concerns as to the explainability of AI-decision making. Secondly, plaintiffs may struggle to prove fault in a negligence claim, or that the vehicle was defective for the purposes of Part 3–5 of the ACL. Essentially, under both actions, manufacturers will be held to a duty to undertake reasonable testing of their AVs. Given that it is currently impracticable to completely test for, and eliminate all AV errors, and due to the broader social utility the technology is likely to offer, plaintiffs may face evidentiary challenges in proving that the manufacturer's testing was unreasonable.     

A comparison of three automated DNA purification methods in Forensic casework

Manual Chelex^®-100 and organic extractions (phenol/chloroform) are used as routine methods at the Swedish National Laboratory of Forensic Science, SKL. The aim of this study was to find an automated DNA purification system to replace the organic method. The following methods were evaluated and compared to each other and to the organic method used routinely; BioRobot^® EZ1 with EZ1 DNA Investigator Kit and Card (Qiagen), iPrep™ Purification Instrument with iPrep™ ChargeSwitch^® Forensic Kit and Card (Invitrogen), Magnatrix™ 1200 Workstation with the Magnatrix™ gDNA Blood Kit Forensic and two different protocols; Forensic protocol A and B (Magnetic Biosolutions). Blood on fats, cotton swabs, moist snuff, paper towels and leather, post-mortem blood and muscle tissue were extracted with the different methods. DNA concentration and quality of the electropherograms were examined. Individual comparisons between the four extraction methods showed that iPrep™ and Magnatrix™ 1200 gave significantly lower mean quantities compared to BioRobot^® EZ1 and the organic extraction method (p < 0.05). There were no significant differences between the latter two. BioRobot^® EZ1 generated the best results and is in the process of being validated for routine analysis at SKL.     

Developmental validation of a real-time quantitative PCR assay for automated quantification of human DNA

Our laboratory has developed an automated real-time quantitative PCR assay for detecting human DNA. The assay utilizes an in-house, custom-designed TaqMan-MGB sequence-specific probe (CFS-HumRT) and the ABD 7900HT SDS platform. Developmental validation has followed TWGDAM (1) guidelines and demonstrates that the assay is primate specific, is highly sensitive, yields consistent results, and works with human DNA extracted from a variety of body fluid stains. When operating within the dynamic range of the system using high-quality DNA samples. the technique yields similar quantification results to our current QuantiBlot assay with the added benefit of time saving through automation. Furthermore, the QPCR assay identifies how much amplifiable DNA is in a sample and thus has the potential to predict PCR success in downstream applications such as STR analysis.     

国产磁珠结合自动化工作站批量提取生物检材DNA的应用

目的建立国产磁珠结合自动化工作站批量提取案件中生物检材DNA的方法。方法采用国产磁珠结合Bio-Robert Universal System自动化工作站对案件中常见的生物样本进行DNA提取,检测Identifiler系统16个STR基因座,在ABI3130XL遗传分析仪上进行STR分型。其中210份样品同时在ABI7500型荧光定量PCR仪上进行定量。结果9100份10类生物检材应用国产磁珠结合自动化工作站,大部分可提取到足够的DNA进行STR检验。STR检验成功率最高的为口腔拭子、肌肉,达100%,接触细胞检材的成功率较低,为50.0%。结论国产磁珠结合自动化工作站可用于案件中常见的大部分生物样本的DNA提取。     

DNA forensics and the poaching of wildlife in Italy: a case study

DNA molecular techniques were used in a forensic investigation involving the poaching of wildlife in a national park of Italy. A poacher, after having snared a wild boar (Sus scrofa) sow, knifed it to death. The animal was retrieved by conservation officers at the scene before the poacher could remove the carcass. Subsequently, the suspect denied the charges. During a search of his home, a bloodstained knife was confiscated. A method to identify the species from the DNA extracted from the stains revealed the blood to be that of the non-domestic form of Sus scrofa. Further DNA typing for individual identity using species-specific single tandem repeats or microsatellites (STRs) showed that the DNA on the knife matched that of the poached boar. Based upon the forensic evidence obtained, the suspect was convicted of poaching and of cruelty to animals.     

免疫磁珠技术和精子抗原研究进展与法医学应用

免疫磁珠技术在生物学领域应用广泛,通过制备精子特异性抗体,并与磁性微球偶联,可达到对精阴混合斑中精子和女性阴道上皮细胞分离。本文简述了免疫磁珠技术的研究进展及磁珠主要制备方法,同时结合精子表面抗原的研究进展,提出磁珠抗体法提取精子DNA的可能性,并展望了磁珠抗体结合技术在法医学应用前景。     

Chelex法和两种磁珠法提取接触DNA效果的比较

目的比较Chelex法、DNA IQ磁珠法、EQ国产磁珠法对接触DNA的提取效果。方法将稀释为10ng、100ng的标准品DNA,分别采用Chelex法、DNA IQ磁珠法、EQ国产磁珠法处理;对30例烟蒂和30例牙刷分别采用Chelex法、DNA IQ磁珠法和EQ国产磁珠法提取DNA,然后进行PCR定量和STR检测。结果Chelex法对DNA的提取无损失,DNA IQ磁珠法、EQ国产磁珠法对DNA的提取均有不同程度的损失;烟蒂、牙刷等检材采用Chelex法提取的接触DNA量和IPC CT值显著高于IQ磁珠法、EQ国产磁珠法,但STR检验成功率却低于IQ磁珠法、EQ国产磁珠法。2种磁珠法提取的DNA量、IPC CT值和STR检验成功率无显著性差异。结论污染轻、杂质少的接触DNA检材,用Chelex法提取最为方便快捷;IQ磁珠法、EQ国产磁珠法更适合污染接触DNA检材的提取及自动化操作。     

Optimization and validation of 10 mitochondrial DNA SNPs using SNaPshot Kit

In some forensic cases, nuclear DNA is degraded and cannot be analyzed. In such a case mitochondrial DNA (mtDNA) is usually used in forensic cases for identification because of its special features as high number of copies per cell, maternal inheritance and high mutation rate. Single nucleotide polymorphisms (SNPs) represent the most abundant class of human polymorphisms. The aim of this study was optimization of 10 mtDNA SNPs by using SNaPshot minisequencing technique on ABI310 genetic analyser in forensic molecular genetics laboratory. At the end of this study, the optimization of minisequencing technique was done by changing some assay parameters. Also, during the optimization of 10 mtSNP loci in our laboratory.     

磁珠直接吸附法对体态检材游离DNA的提取

目的采用磁珠直接吸附法对人体尿液、唾液、血液3种体态生物检材中的游离DNA进行提取检验,为法医物证中游离DNA的研究及检验工作提供参考。方法对3种生物检材采取离心吸取上清液的方法分离游离DNA,然后采用磁珠直接吸附法进行提取纯化,Identifiler-Plus试剂盒进行复合扩增后常规STR检测。结果在3种检材中均检出了游离DNA,其中血液中游离DNA检出率为100%,唾液为90%,尿液为70%。结论人体体态生物检材中存在游离DNA,同时磁珠直接吸附法可高效、快捷的提取生物检材中的游离DNA。     

Optimization and validation of CEDIA drugs of abuse immunoassay tests in serum on Hitachi 912

Due to sensitive limits of detection of chromatographic methods and low limit values regarding the screening of drugs under the terms of impairment in safe driving (§ 24a StVG, Street Traffic Law in Germany), preliminary immunoassay (IA) tests should be able to detect also low concentrations of legal and illegal drugs in serum in forensic cases. False-negatives should be avoided, the rate of false-positive samples should be low due to cost and time. An optimization of IA cutoff values and a validation of the assay is required for each laboratory. In a retrospective study results for serum samples containing amphetamine, methylenedioxy derivatives, cannabinoids, benzodiazepines, cocaine (metabolites), methadone and opiates obtained with CEDIA drugs of abuse reagents on a Hitachi 912 autoanalyzer were compared with quantitative results of chromatographic methods (gas or liquid chromatography coupled with mass spectrometry (GC/MS or LC/MS)). Firstly sensitivity, specificity, positive and negative predictive values and overall misclassification rates were evaluated by contingency tables and compared to ROC-analyses and Youden-Indices. Secondly ideal cutoffs were statistically calculated on the basis of sensitivity and specificity as decisive statistical criteria with focus on a high sensitivity (low rates of false-negatives), i.e. using the Youden-Index. Immunoassay (IA) and confirmatory results were available for 3014 blood samples. Sensitivity was 90% or more for nearly all analytes: amphetamines (IA cutoff 9.5 ng/ml), methylenedioxy derivatives (IA cutoff 5.5 ng/ml), cannabinoids (IA cutoff 14.5 ng/ml), benzodiazepines (IA cutoff >0 ng/ml). Test of opiates showed a sensitivity of 86% for a IA cutoff value of >0 ng/ml. Values for specificity ranged between 33% (methadone, IA cutoff 10 ng/ml) and 90% (cocaine, IA cutoff 20 ng/ml). Lower cutoff values as recommended by ROC analyses were chosen for most tests to decrease the rate of false-negatives. Analyses enabled the definition of cutoff values with good values for sensitivity. Small rates of false-positives can be accepted in forensic cases.     

免疫磁珠法与差异裂解法检验混合样本的效果观察

目的分析应用免疫磁珠分离技术和差异裂解法对混合样本的DNA检验效果,评价其在法医学应用中的价值。方法制备含有不同数量精细胞与阴道上皮细胞混合悬液和植绒混合斑拭子,分别采用MOSPD3抗体、SPAG8抗体免疫磁珠法及差异裂解法对样本进行检验,观察分析不同比例的混合细胞悬液,以及不同保存时间植绒混合斑拭子DNA分型的正确率。结果当混合细胞悬液内精细胞与阴道上皮细胞比例约1∶10时,免疫磁珠法的正确率明显高于差异裂解法(P0.01);当比例等于或高于1∶1时,两种方法正确率的差异无显著性意义(P0.05)。植绒拭子混合斑的免疫磁珠法正确率明显低于差异裂解法(P0.01);随混合斑保存时间的延长,差异裂解法正确率无明显下降(P0.05)。结论免疫磁珠法适用于新鲜混合样本中精细胞的分离检验,且在精细胞含量较低时优势更明显,差异裂解法较适合陈旧混合斑的分离检验。     

Optimization and validation of a fast amplification protocol for AmpFlSTR Profiler Plus for rapid forensic human identification

The goal of this work was to optimize and validate a fast amplification protocol for the multiplex amplification of the STR loci included in AmpFlSTR^® Profiler Plus^® to expedite human DNA identification. By modifying the cycling conditions and by combining the use of a DNA polymerase optimized for high speed PCR (SpeedSTAR™ HS) and a more efficient thermal cycler instrument (Bio-RAD C1000™), we were able to reduce the amplification process from 4 h to 26 min. No modification to the commercial AmpFlSTR^® Profiler Plus^® primer mix was required. When compared to the current Royal Canadian Mounted Police (RCMP) amplification protocol, no differences with regards to specificity, sensitivity, heterozygote peak height ratios and overall profile balance were noted. Moreover, complete concordance was obtained with profiles previously generated with the standard amplification protocol and minor alleles in mixture samples were reliably typed. An increase in n − 4 stutter ratios (2.2% on average for all loci) was observed for profiles amplified with the fast protocol compared to the current procedure. Our results document the robustness of this rapid amplification protocol for STR profiling using the AmpFlSTR^® Profiler Plus^® primer set and demonstrate that comparable data can be obtained in substantially less time. This new approach could provide an alternative option to current multiplex STR typing amplification protocols in order to increase throughput or expedite time-sensitive cases.     

Optimization and validation studies of the Mentype Argus X-8 kit for paternity cases

The use of ChrX-STRs is enormous in forensic case as these have proven to be powerful tools, mainly in deficiency paternity cases when the disputed child is female, and also some special cases involving blood relatives, incest cases, fetal typing in abortion material. The Mentype^® Argus X-8 kit is a commercial multiplex system which contains Amelogenin for gender determination as well as gonosomal STR markers (DXS8378, HPRTB, DXS7423, DXS7132, DXS10134, DXS10074, DXS10101 and DXS10135). Validation studies were being performed on blood obtained from the volunteers in Turkish population. In this study, some parameters were taken under consideration for validation like DNA extraction using different protocols, quantitated by using commercially available Invitrogen Qubit Fluorometer, reaction volume validation of Master Mix and the analysis of female/male, female/female and male/male mixtures were performed. The conditions were optimized and validated using GenAmp 9700 and reducing reaction volume from 25 μl to 12.5 μl and 6.5 μl. After reducing the total volume of the reaction, the results were same and there was no effect on peak height and quality when analyzed on ABI 310 genetic analyzer. 2 paternity cases were also performed which gave the same power of discrimination as has been mentioned in Mentype^® Argus X-8 kit.     

Development and validation of a next generation STR ESS-pentaplex

We constructed a simple STR pentaplex of new loci recommended as next generation markers for the European Standard Set (ESS) comprising normal-amplicon STRs: D12S391 and D1S1656, plus mini-amplicon STRs: D2S441, D10S1248 and D22S1045. Validation of the pentaplex included evaluation of its ability to amplify DNA from a variety of degraded forensic casework samples. Although the ESS-pentaplex was designed in the first instance to generate allele frequency data to supplement existing databases of established STRs, the multiplex proved to be a valuable tool for the analysis of challenging DNA when certain markers of Identifiler or MiniFiler occasionally failed.     

TWGDAM validation of a nine-locus and a four-locus fluorescent STR multiplex system

The Gene Print PowerPlex 1.1/Amelogenin and FFFL Fluorescent STR Systems have been validated following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). The PowerPlex 1.1/Amelogenin System supports simultaneous amplification of eight short tandem repeat loci and the Amelogenin gender identification marker. The loci D16S539, D7S820, D13S317, and D5S818 are labeled with fluorescein (FL) while the loci CSF1PO, TP0X, TH01, vWA and Amelogenin are labeled with carboxy-tetramethylrhodamine (TMR). The FFFL Multiplex System is composed of the loci F13A01, FESFPS, F13B, and LPL, each labeled with fluorescein. We have observed no overlap of alleles across loci labeled with an individual fluorescent dye. Samples of each system were amplified and labeled in a single reaction, separated by electrophoresis through a denaturing polyacrylamide gel, and amplified alleles detected using a Hitachi FMBIO Fluorescent Scanner. Alterations from the standard amplification protocols in cycle number and annealing temperature generally produced excellent results. In experiments testing sensitivity as little as 0.2 ng of DNA template could be detected. As expected, different body fluids from the same individuals generated identical DNA profile results. Template DNA derived from blood-strains deposited on a variety of matrix supports displayed robust amplification except for material derived from deposits on wood and Japanese orchid leaves. Mixtures of DNA templates could be interpreted with the minor component present in as little as ten percent of the total sample. Monoplex and multiplex amplifications produced identical amplified allele patterns, indicating that STR multiplex systems save template and increase efficiency in the amplification procedure without loss of quality. Analyses of genotype frequencies in African-American, Caucasian-American and Hispanic-American populations using all twelve loci were used to determine matching probabilities smaller than 1 in 1.14 x 10(8) and 1 in 2658 for the PowerPlex 1.1 and the FFFL Multiplex Systems, respectively. The matching probability achieved with the two systems combined is smaller than 1 in 3.03 x 10(11). The independence of alleles within loci was generally demonstrated by applying the exact test to demonstrate Hardy-Weinberg Equilibrium. All of the studies performed indicate that the PowerPlex 1.1/Amelogenin and FFFL Multiplex Systems are powerful, robust, and reliable investigative tools that can be used in the analysis of forensic samples.     

Method validation of a survey of thevetia cardiac glycosides in serum samples

A sensitive and specific liquid chromatography tandem mass spectrometry (HPLC-ESI(+)-MS/MS) procedure was developed and validated for the identification and quantification of thevetin B and further cardiac glycosides in human serum. The seeds of Yellow Oleander (Thevetia peruviana) contain cardiac glycosides that can cause serious intoxication. A mixture of six thevetia glycosides was extracted from these seeds and characterized. Thevetin B, isolated and efficiently purified from that mixture, is the main component and can be used as evidence. Solid phase extraction (SPE) proved to be an effective sample preparation method. Digoxin-d3 was used as the internal standard. Although ion suppression occurs, the limit of detection (LOD) is 0.27 ng/ml serum for thevetin B. Recovery is higher than 94%, and accuracy and precision were proficient. Method refinement was carried out with regard to developing a general screening method for cardiac glycosides. The assay is linear over the range of 0.5-8 ng/ml serum. Finally, the method was applied to a case of thevetia seed ingestion.