European Standard Set Research Articles

Use of the Investigator ESSplex SE QS Kit (QIAGEN) at Half PCR Reaction Volumes for the Analysis of Forensic Samples

The Investigator ESSplex SE QS Kit (Qiagen) is a next-generation polymerase chain reaction (PCR) kit that, in 60 min, amplifies 17 Short Tandem Repeat (STR) markers, including the five European Standard Set (ESS) loci (D10S1248, D12S391, D1S1656, D22S1045, D2S441), the SE33 marker, and the locus Amelogenin for sex determination. Two quality sensors (QS1 and QS2) are also co-amplified to check PCR performance. Since forensic laboratories carry out hundreds of DNA typings annually, we verified the kit’s performance using half reaction volumes with the aim of improving the number of samples that may be amplified with a single kit and consequently reducing laboratory costs. In the present study, intended as a technical note rather than internal validation, some control samples (oral swabs) with known DNA profiles and 40 real casework samples were analyzed. We observed that reducing the total reaction volume, while keeping all component ratios unaltered, yields DNA profiles comparable to those obtained using standard reaction volumes and with allele peaks higher than those with regular volumes. Using half volumes for PCR amplification enables the analysis of a larger number of samples compared to the standard protocol, thereby reducing laboratory costs without compromising the quality of the analysis.

Recommendations for the successful identification of altered human remains using standard and emerging technologies: Results of a systematic approach

Successful DNA-based identification of altered human remains relies on the condition of the corpses and varies between tissue types. Therefore, the aim of this prospective multicenter study was to generate evidence-based recommendations for the successful identification of altered remains. For this, 19 commonly used soft and hard tissues from 102 altered human bodies were investigated. The corpses’ condition was categorized into three anatomical regions using a practical scoring system. Besides other data, DNA yields, degradation indices, and short tandem repeat (STR) profile completeness were determined in 949 tissue samples. Additionally, varying degrees of alteration and tissue-specific differences were evaluated using the Next Generation Sequencing (NGS) platform MiSeq FGx™. Selected challenging samples were sequenced in parallel with the Ion S5™ platform to assess platform-specific performances in the prediction of the deceased’s phenotype and the biogeographic ancestry. Differences between tissue types and DNA extraction methods were found, revealing, for example, the lowest degradation for vertebral disc samples from corpses with initiating, advanced and high degrees of decomposition. With respect to STR profile completeness, blood samples outperformed all other tissues including even profoundly degraded corpses. NGS results revealed higher profile completeness compared to standard capillary electrophoresis (CE) genotyping. Per sample, material and degradation degree, a probability for its genotyping success, including the “extended” European Standard Set (eESS) loci, was provided for the forensic community. Based on the observations, recommendations for the alteration-specific optimal tissue types were made to improve the first-attempt identification success of altered human remains for forensic casework.

Internal Validation of Global Filer PCR Amplification Kit in DNA Section of Punjab Forensic Science Agency, Lahore

Background: Global Filer PCR Amplification Kit is a 6-dye amplification assay, which amplifies 24 genetic loci simultaneously in a single reaction. The amplification system encompasses both Combined DNA Index System loci and European standard set loci along with a highly polymorphic short tandem repeat (STR) locus SE33. The addition of a Y-STR marker and an insertion deletion marker of Y chromosome has provided additional aid in the gender identification of Amelogenin-Y null males. Global Filer PCR Amplification Kit has provided understandably more discrimination power compared with Identifiler Plus PCR Amplification Kit currently being used in Punjab Forensic Science Agency (PFSA). Methodology: In this study, Global Filer PCR Amplification Kit was internally validated in PFSA following the Scientific Working Group on DNA Analysis Methods laid principles to establish quality assurance parameters and interpretation guidelines. Known nonprobative DNA samples were utilized in this study to determine the sensitivity, establishment of proper thresholds for analysis, mixture analysis to determine detectable mixture ratios, contamination assessment, and reproducibility for the new method. Conclusion: The results of this internal validation strongly suggest the use of Applied Biosystems Global Filer PCR Amplification Kit in forensic casework analysis.

Ergonomic design standards: harmonization with international and European

The authors notes that the current format for creating new types of marketable products requires a rethinking of the basic approaches and criteria of design-ergonomic designing, adapting with modern international and European standards. And the transition to the principles of voluntariness in the use of regulatory requirements requires a change in the principles of their application in design activities. The article proves that without realization of design-ergonomic standards, harmonized with the international and European, world, in particular, the European market, for domestic commodity products will be closed, it will be impossible to create modern conditions of human life. From these positions the content and specificity of the main set of international and European standards, harmonized and included in the national regulatory framework and currently in force in Ukraine, are analysed. In particular, to consider standards developed by the EU Directives, which set design-ergonomic indicators and standards for the following processes and products in its designing, manufacture, operation and disposal, namely: standards for human anthropometric parameters required for the design of jobs, dimensions of openings for access to equipment, safe distances, etc. standards for standardization of workplace parameters, principles, recommendations and requirements for their designing; standards for regulating the parameters of the working environment, especially the environment of control centres, (requirements for thermal, lighting, acoustic components, etc.); standards of functional zones of control centres, their planning; standards for design-ergonomic requirements for the designs of indicators and controls, principles for human interaction with information display and controls; international standards harmonized with national standards, which establish the design-ergonomic principles of designing work equipment, work systems; a large set of standards for audio and visual danger signals, creation of sign information, guidelines for the design of signs, their colours, criteria for the perception of visual signals, video terminals, requirements for danger signals for public areas and work places, etc. The scope of the main groups of international and European standards, harmonized and in force in Ukraine, is also analysed.

A study of skeletal remains using GlobalFiler™

DNA analysis from human remains is of immense relevance in missing persons identification and disaster victim identification (DVI). DNA degrades gradually in hard tissues, such as bones and teeth and remains somewhat safe from the effects of high temperature, humidity, pH, geochemical properties of the soil, and microorganisms. . The GlobalFiler™ kit simultaneously amplifies and detects 21 autosomal loci including CODIS extended set of STR loci. The kits was designed to combine all 21 autosomal STR loci along with a novel male specific Y insertion/deletion marker the sex-determining marker, amelogenin. Thus, GlobalFiler™ combines the CODIS extended set of loci and includes seven autosomal STR loci from the expanded European Standard Set of Loci (ESSL). The kit also includes the highly discriminating SE33 locus. The aim of this study was to profile the old skeletal remains using GlobalFiler™ for use in disaster victim identification that were previously profiled using Identifiler Plus kit using 3500 (CE). This study investigated the success rate of short tandem repeat (STR) typing from different types of bone samples and observed a higher STR success rate using GlobalFiler™ PCR kit, which contains more than 10 loci under 220 bp size. Thus, GlobalFiler™ could potentially be a good choice for the limited amounts of degraded DNA obtained from challenging skeletalsamples. The performance of the GlobalFiler™ kit and the profile quality was examined for the degraded bone samples to determine the robustness of genetic profiling.

Effect of sodium hypochlorite decontamination on the DNA recovery from human teeth.

Genetic identification of skeletal human remains is often realized by short tandem repeat (STR) genotyping of nuclear DNA. Dental DNA is preferred to DNA from bone for the better protection of the endogenous DNA. Especially if whole tooth grinding is intended to access the DNA, contaminations with exogenous DNA have to be avoided. The immersion of the tooth in sodium hypochlorite (NaOCl, known as bleach) is one common procedure to clean the outer surface from extraneous DNA and PCR inhibitors. To investigate the impact of bleaching on endogenous DNA and the decontamination success, 71 recently extracted teeth were differently treated with sodium hypochlorite (2.5 or 5.0% NaOCl for 30 or 60 s, 5.0% NaOCl for 10 min, and control group) in the beginning of the extraction process, whereas equally handled afterwards. Quantitative and qualitative evaluation of the extracted DNA was performed. There was a great variation for the DNA concentration of the extracts even within a group of the same NaOCl treatment. Complete DNA profiles from single persons with alleles for the 16 ESS (European Standard Set) STR loci were obtained for all regarded teeth. A statistically significant difference between the DNA yields of the treatment groups was not determined. Moreover, a negative effect of NaOCl (2.5% and 5.0%) on the DNA recovery could not be observed. Significant larger amounts of DNA were extracted from anterior teeth in contrast to posterior teeth.

Development and validation of a 21-plex STR panel

Short tandem repeat (STR) analysis is the most common and informative type of DNA profiling widely used in paternity testing, missing persons’ cases, and forensic applications. Several commercial kits based on STR technology were developed to meet current international standards, such as the European Standard Set (ESS) and the Combined DNA Index System (CODIS). Here, we report the development of the in-house STR panel ixMark PLUS that contains 21 markers: the 13 original CODIS STR loci with four non-overlapping loci from the expanded ESS, as well as SE33 and D6S1043, one Y-STR DYS391, and the sex-determining marker Amelogenin. The full list of markers in the panel are as follows: vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, DYS391, D2S441, D22S1045, THO1, FGA, Amelogenin, D5S818, D13S317, D7S820, SE33, D3S1358, D1S1656, D12S391, and D6S1043. The novel 21-plex assay ixMark PLUS includes primers, the internal size standard of 14 fragments, the allelic ladder of 241 fragments, and the matrix standard set, all designed and developed in-house. The results of developmental validation showed the kit’s robustness, reliability, and suitability as a valuable addition to the current commercial kits for human DNA identification.

Validation study and forensic applications of investigator 24plex QS Kit

The Investigator 24plex QS Kit (Qiagen) is used for multiplex PCR in forensic human identity and paternity testing. The PCR simultaneously amplifies 22 polymorphic STR markers: the Combined DNA Index System (CODIS) core loci, the European Standard Set (ESS) loci, SE33, DYS391 and Amelogenin. The Investigator 24plex QS Kit is specifically designed for rapid and reliable generation of DNA profiles from blood, buccal swabs, and forensic stains. The kit utilizes QIAGEN’s fast-cycling PCR technology, allowing amplification in around 60 min and provides highly robust results with inhibitor-resistant chemistry. The Investigator 24plex QS Kit Primer Mix contains two innovative internal PCR controls (Quality Sensor QS1 and QS2), which are amplified simultaneously with the polymorphic STR markers, to yield helpful information about the efficiency of the PCR and the presence of PCR inhibitors. The Investigator 24plex QS Kit User Guide describes instrument setup and sample preparation of PCR products using the Applied Biosystems 3500 Series Data Collection Software and the GeneMapper ID-X Software, but an internal validation is always needed.In this study, we report the successful analysis of amplicons obtained with Investigator 24plex QS Kit on the 3500 Genetic Analyzer. We performed internal validation studies following the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling Group (EDNAP) guidelines, testing several critical areas of kit performance such as sensibility, sensitivity, DNA mixtures and inhibited samples.

Forensic and population genetic analysis of Serbian population using 21 STR loci of GlobalFiler™ PCR amplification kit

Autosomal short tandem repeats (STRs) have been widely used in forensic investigations. Prior to the application of any DNA based identification method, it is essential to estimate the allele frequencies and forensic statistical parameters of targeted STR loci in each population in order to provide a more precise reference database for forensic investigation. The GlobalFiler™ Kit is a multiplex assay that combines the 13 original CODIS loci with 7 non-overlapping loci from the expanded European Standard Set (ESS), as well as the highly discriminating SE33 locus, two Y-based loci and the sex determining maker, Amelogenin. The full complement of loci in the GlobalFiler™ Kit are: D13S317, D7S820, D5S818, CSF1PO, D1S1656, D12S391, D2S441, D10S1248, D18S51, FGA, D21S11, D8S1179, vWA, D16S539, TH01, D3S1358, AMEL, D2S1338, D19S433, DYS391, TPOX, D22S1045, SE33 and a Y-specific insertion/deletion locus (Yindel). The 6-dye GlobalFiler™ PCR Amplification kit (ThermoFisher Scientific) comprises 21 autosomal STRs have already been proven to be able to provide reliable DNA profiling results and enhance the power of discrimination between individuals. In this study, we are presenting an analysis of GlobalFiler STR loci on 209 unrelated individuals from Serbia.

Development of a multiplex forensic identity panel for massively parallel sequencing and its systematic optimization using design of experiments

The application of massively parallel sequencing (MPS) in forensic sciences enables high-resolution short tandem repeat (STR) genotyping for the characterization of biological evidence. While MPS supports multiplexing of a large number of forensic markers, the performance of an MPS-STR panel depends on good primer design and optimal PCR conditions. However, conventional strategies for multifactorial assay optimization are labor-intensive and do not necessarily allow the experimenter to identify optimum factor settings.Here we describe our new multiplex PCR assay, monSTR, which supports the simultaneous amplification of 21 forensic markers followed by targeted sequencing on the Illumina MiSeq. The selection of STR markers adapts on the expanded European Standard Set (ESS), including the highly polymorphic locus SE33, for compatibility with existing forensic DNA databases. Primer engineering involved bioinformatics tools to create a multiplex-compatible primer set. Primer quality was evaluated in silico and in vitro. We demonstrate the systematic optimization of multiplex PCR thermocycling conditions using Design of Experiments (DOE) methodology. The objective was to yield a specific, balanced, low-noise amplification of forensic targets. A central composite face design of experiments enabled an efficient simultaneous investigation of multiple critical process parameters and their interactions. Optimal multiplex PCR conditions were predicted using software-aided modelling based on DOE data. Verification experiments suggested a balanced, reproducible amplification of all markers with reduced formation of artefacts. Fully concordant STR profiles were obtained for the investigated reference samples even with challenging input DNA concentrations. We found that application of DOE principles enabled an experimentally practical and economically justifiable assay development and optimization, even beyond the field of forensic genetics.

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Developmental validation of GlobalFiler\u2122 PCR amplification kit: a 6-dye multiplex assay designed for amplification of casework samples

The GlobalFiler™ PCR Amplification Kit is a single multiplex assay that amplifies a set of 24 markers, which encompass the European Standard Set and CODIS (Combined DNA Index System) recommended composite set of loci. In addition to more loci and a 6-dye chemistry format, the Master Mix has been formulated to allow higher sample loading volume for trace DNA samples. The GlobalFiler™ Kit has been optimized to deliver high performance on casework samples, while also delivering fast thermal cycling, with an amplification time of approximately 80 min. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

Analysis of 16 autosomal STR loci in Uyghur and Kazakh populations from Xinjiang, China

The AmpFℓSTR® NGM SElect™ Kit is a highly robust multiplex kit developed for human identification. This kit contains the expanded European Standard Set of Loci and the highly polymorphic SE33 locus. The high power of discrimination makes it one of the most informative kits for forensic applications. In order to evaluate the kit and get population data for Chinese Uyghurs and Kazakhs, the genetic polymorphisms of 16 autosomal STR loci included in the NGM SElect™ Kit: D3S1358, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, FGA, D10S1248, D22S1045, D2S441, D1S1656 D12S391, SE33, and Amelogenin were studied in 324 unrelated Uyghur and 124 unrelated Kazakh individuals from the Xinjiang Autonomous Region of China. Allele frequencies and forensic relevant parameters were calculated using the Arlequin software and DNAVIEW. The genotype frequency distribution of each locus did not deviate from Hardy-Weinberg Equilibrium in any of the two populations. The forensic parameters indicated that the kit is suitable for personal identification, paternity testing, and complex kinship analysis in Uyghur and Kazakh populations. Allele frequency data for the STR loci were compared with other previously published population data.

A preliminary evaluation study of new generation multiplex STR kits comprising of the CODIS core loci and the European Standard Set loci

The GlobalFiler™ (Life Technologies), Investigator® 24plex QS (Qiagen), and PowerPlex® Fusion 6C (Promega) kits are the latest generation 6-dye fluorescent chemistry STR-PCR amplification kits. These kits allow for the simultaneous amplification of the CODIS core loci and the European Standard Set loci, as well as a few Y-STR loci in addition to the standard sex-determining marker Amelogenin. The present study was designed to be a preliminary evaluation of the three STR-PCR kits in terms of sensitivity, profile recovery from degraded DNA samples, tolerance to PCR inhibitors, and detection of minor components in DNA mixtures. The results showed that the three STR-PCR kits had relatively similar performance with each kit faring better for the different aspects studied. The PowerPlex® Fusion 6C and the Investigator® 24plex QS kits were shown to tolerate inhibitors better, while the GlobalFiler™ kit appeared to have a higher mean percentage recovery of alleles from low template DNA samples and for minor components in DNA mixtures.

Developmental validation of a 6-dye typing system with 27 loci and application in Han population of China

In this study, a novel 27-locus system (now known as the SureID PanGlobal system), including 24 autosomal STRs (D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D2S441, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D16S539, D22S1045, SE33, D10S1248), two Y-chromosome markers (DYS391 and Y-indel) and the sex determining marker, Amelogenin was developed with six fluorescent dyes labeling. The included STR loci belonged to the core loci in the Combined DNA Index System (CODIS) and the European Standard Set (ESS) as well as some additional loci commonly used in commercial kits and national DNA databases. This paper describes the validation studies conducted with the SureID PanGlobal system using Applied Biosystems 3500 XL Genetic Analyzer for fragment length detection that included the analysis of the following parameters and aspects: PCR conditions, sensitivity, species specificity, inhibition, precision, stutter, DNA mixtures, and stability studies with crime scene samples. The studies demonstrated that the SureID PanGlobal system is reproducible, accurate, sensitive and robust for forensic application and databasing. Additionally, the whole cycling time of the system can finish within 65 minutes, which was developed specifically for rapid and reliable generation of DNA profiles obtained from blood, buccal swabs and forensic stains.

Evaluation of the genetic parameters for 10 common and five new ESS core autosomal STR loci in seven major geographic regions and the largest metropolitan province of Turkey

Background: Situated at the crossroads of Asia, Middle East and Europe, Turkey has an ethnically diverse population of over 78 milllion people.Aim: To investigate the population genetics and potential differences in the autosomal short tandem repeat (STR) polymorphisms across all the major geographic regions and largest metropolitan province of Turkey within the context of the Near Eastern/European genetic landscape.Subjects and methods: Samples from a total of 5299 unrelated individuals were analysed at 10 common [D2S1338, D3S1358, D8S1179, D16S539, D18S51, D19S433, D21S11, FGA, TH01, vWA] and five new European Standard Set (ESS) core autosomal STR loci [D1S1656, D2S441, D10S1248, D12S391, D22S1045].Results: Allele frequencies, statistical parameters of forensic interest and population differentiation tests were calculated for nine population datasets corresponding to the seven major geographic regions, the largest metropolitan province, and a combined dataset for the entire country. Cumulative results confirmed the presence of significant differences among these nine autosomal datasets themselves and with those from the nearby populations, therefore justifying the differential use of these separate datasets on a case-by-case basis in forensic investigations.Conclusion: This collection of autosomal STR population datasets comprises the largest and most comprehensive of its kind from Turkey so far.

Difficulties in Analytical Fire Safety Assessment of Steel Bridges, Using the Provisions of EU Design Standards and Legislation

Today, there is a complete lack of appropriate regulatory provisions governing the fire safety assessment of bridges, in particular, specifying the load criteria under fire exposure, with which primary and secondary load-bearing elements should comply. Similarly, the new harmonized bridge structure design standards do not put forward in their decisions any substantive provisions which would allow conducting this type of analysis. In accordance with the preamble of the latest set of European structural design standards “The Member States of the EU and EFTA recognize that Eurocodes serve as reference documents for the following purposes: – as a means to prove compliance of building and civil engineering works with the essential requirements of Council Directive 89/106/EEC, particularly Essential Requirement No 1 (Mechanical resistance and stability), and Essential Requirement No 2 (Safety in case of fire);[…]”. In fact, it appears that, with regard to Essential Requirement No 2 this clause - in particular in relation to objects other than buildings - is dead and does not translate to any specific guidelines, which would allow the achievement of the objective. The authors shall carry out a thorough analysis of legislation and design standards in force in the EU Economic Area, indicating their flaws and lack of coherence. They shall provide a proposal for how to carry out a load assessment of bridge structure elements under fire exposure and specify criteria which, in their opinion, strongly justify the need to conduct such analyses.

Missing population studies on the new European Standard set loci (ESS) and its impact on population statistics

We have encountered a decrease in the number of population studies published in recent years. Allelic frequencies for the new European standard set of loci (D12S391, D1S1656, D2S441, D10S1248, and D22S1045) are not published for many countries. This can lead to bias in statistical evaluation of DNA typing results.

Development of a one-step immunochromatographic assay with two cutoff values of aflatoxin M1

Aflatoxin M1 (AFM1) in milk has two standard limits, namely, the European standard set up at 0.05 ng/mL and the other one (including America, China, and other countries) at 0.5 ng/mL. A one-step immunochromatographic assay (ICA) with two cutoff values was developed to satisfy these two standard limits, wherein test zones including test 1 and 2 (T1 and T2) lines were formed by dispensing AFM1-BSA antigen onto a nitrocellulose membrane. The migrating AFM1 analyte in the sample and capture reagent immobilized on two test lines competed for the limited binding sites of AFM1 antibody, which conjugated with colloidal gold. Three red lines appeared at T1 and T2 lines and control (C) line when the sample was negative or the AFM1 analyte concentration was lower 0.05 ng/mL. However, only two red lines appeared at T1 and C lines when the AFM1 analyte concentration was 0.05 ng/mL or higher but lower 0.5 ng/mL. One red line appeared at C line when AFM1 analyte concentration was 0.5 ng/mL or higher. Results showed that the novel immunochromatographic assay was superior to traditional ICAs because of satisfying two standard limits at one step.

Usefulness of the Powerplex® Fusion System as typing methodology for CODIS and ESS genetic markers in an immigrant population from Cabo Verde in Lisboa

Cabo Verde is located on the West African coast, was discovered uninhabited by Portuguese explorers, colonized by European and African slaves and is culturally characterized by a mixture of their settlers. Cabo Verde is one of the countries with the highest number of emigrants living in Portugal.In order to validate the genetic markers included in the Combined DNA Index System and in the European Standard Set, samples from 500 unrelated individuals from Cabo Verde, currently living in Lisboa, were studied with the PowerPlex® Fusion System. To implement the genotyping method at the Serviço de Genética e Biologia Forenses (SGBF) we determined the minimum final volume of the PCR reaction, the proper number of PCR cycles and the minimum DNA concentration to obtain a complete genetic profile. Stutter identification and its proportion and allelic imbalance within markers were also determined.Results were compared with genetic profiles obtained by the current PCR kits used in SGBF routine testing verifying concordance between common loci. There were no off-ladder alleles detected and it was possible to type microvariants which were not present in the allelic ladders of other PCR kits.PowerPlex® Fusion System allows for the simultaneous characterization of Combined DNA Index System and in the European Standard Set genetic markers, in an economically way, with greater sensitivity and solid results.