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- Hedman, Johannes, et al.
(author)
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Validation guidelines for PCR workflows in bioterrorism preparedness, food safety and forensics
- 2018
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In: Accreditation and Quality Assurance. - : Springer Science and Business Media LLC. - 0949-1775 .- 1432-0517. ; 23:3, s. 133-144
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Journal article (peer-reviewed)abstract
- The polymerase chain reaction (PCR) is the backbone of contemporary DNA/RNA analysis, ideally enabling detection of one or just a few target molecules. However, when analysing food or forensic samples the analytical procedure is often challenged by low amounts of poor quality template molecules and complex matrices. Applying optimised and validated methods in all steps of the analysis workflow, i.e. sampling, sample treatment, DNA/RNA extraction and PCR (including reverse transcription for RNA analysis), is thus necessary to ensure the reliability of analysis. In this paper, we describe how in-house validation can be performed for the different modules of the diagnostic PCR process, providing practical examples as tools for laboratories in their planning of validation studies. The focus is analysis of heterogeneous samples with interfering matrices, with relevance in food testing, forensic DNA analysis, bioterrorism preparedness and veterinary medicine. Our objective is to enable rational in-house validation for reliable and swift quality assurance when results are urgent, for example in the event of a crisis such as a foodborne outbreak or a crime requiring the analysis of a large number of diverse samples. To that end, we explain the performance characteristics associated with method validation from a PCR and biological sample matrix perspective and suggest which characteristics to investigate depending on the type of method to be validated. Also, we include a modular approach to validation within the PCR workflow, aiming at efficient validation and a flexible use of methods.
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| 3. |
- Sanga, Malin, et al.
(author)
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A panel of PCR-inhibitory reference materials for quality evaluation of multiplex STR analysis kits
- 2015
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In: Forensic Science International. - : Elsevier. - 1875-1768 .- 1875-175X. ; 5, s. e317-319
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Journal article (peer-reviewed)abstract
- PCR inhibition is a critical parameter in forensic DNA analysis. Substances interfering with amplification of short tandem repeats (STRs) may generate partial and/or ambiguous DNA profiles. We present a strategy for developing a broad panel of PCR-inhibitory reference materials (RMs), representing common casework samples. Our panel, including solutions prepared from for example cigarettes, chewing gum and soil, is a tool for in-house validation and lot testing of STR systems. PowerPlex ESX 16 Fast System tolerated high levels of some RMs, but several substances caused amplification problems. Humic acid had a negative effect on amelogenin, moist snuff hindered amplification of longer fragments, and chewing gum caused generally lowered allele peak heights. Applying a broad panel of RMs ensures that a wide range of inhibitory substances are tested, giving an improved understanding of inhibitor tolerance and effects.
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- Sidstedt, Maja, et al.
(author)
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In-house validation of MPS-based methods in a forensic laboratory
- 2019
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In: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768. ; 7:1, s. 635-636
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Journal article (peer-reviewed)abstract
- Massively parallel sequencing (MPS) methods are increasingly applied in forensic casework. However, adequate validation guidelines are lacking. In this work, we describe our in-house validation of the ForenSeq DNA Signature Prep Kit (Verogen) for analysis of ancestry- and phenotype-informative SNPs. We also discuss in-house validation of MPS assays in general terms. When validating the SNP assay, we focused on the reliability of SNP genotype calls and the compatibility with commonly analysed sample types. Other issues, for example analytical thresholds and accuracy of the data prediction model were considered to be covered by the developmental validation of the kit. Our study included determination of (1) concordance, (2) limit of detection, (3) matrix effects, (4) repeatability, and (5) contamination risk. In conclusion, the MPS-based SNP assay showed overall adequate performance for single-source samples, with correct genotype calls. We welcome a broad discussion on how to perform in-house validation of MPS-based methods, as this is vital to ensure timely implementation of reliable assays in forensic laboratories.
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| 5. |
- Szkuta, Bianca, et al.
(author)
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Assessment of the transfer, persistence, prevalence and recovery of DNA traces from clothing: An inter-laboratory study on worn upper garments
- 2019
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In: Forensic Science International. - : ELSEVIER IRELAND LTD. - 1872-4973 .- 1878-0326. ; 42, s. 56-68
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Journal article (peer-reviewed)abstract
- Among the various items recovered from crime scenes or persons involved in a crime event, clothing items are commonly encountered and submitted for forensic DNA sampling. Depending on the case circumstances and the activity-of-interest, sampling of the garment may concentrate on collecting DNA from the wearer, or from one or more offenders who have allegedly contacted the item and/or wearer. Relative to the targeted DNA, background DNA already residing on the item from previous contacts, or transferred during or after the crime event, may also be collected during sampling and observed in the resultant DNA profile. Given our limited understanding of how, and from where, background DNA is derived on clothing, research on the transfer, persistence, prevalence, and recovery (TPPR) of DNA traces from upper garments was conducted by four laboratories. Samples were collected from several areas of two garments, each worn on separate working or non-working days and individually owned by four individuals from each of the four laboratories, and processed from DNA extraction through to profiling. Questionnaires documented activities relating to the garment prior to and during wearing, and reference profiles were obtained from the wearer and their close associates identified in the questionnaire. Among the 448 profiles generated, variation in the DNA quantity, composition of the profiles, and inclusion/exclusion of the wearer and their close associates was observed among the collaborating laboratories, participants, garments worn on different occasions, and garment areas sampled.
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| 6. |
- Szkuta, Bianca, et al.
(author)
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DNA transfer to worn upper garments during different activities and contacts: An inter-laboratory study
- 2020
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In: Forensic Science International. - : ELSEVIER IRELAND LTD. - 1872-4973 .- 1878-0326. ; 46
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Journal article (peer-reviewed)abstract
- Cellular material derived from contact traces can be transferred via many direct and indirect routes, with the manner of contact and the time of transfer (in relation to the alleged crime-event) having an impact on whether DNA is recovered from the surface and a reportable profile generated. In an effort to acquire information on the transfer and recovery of DNA traces from clothing items worn during scenarios commonly encountered in casework, upper garments were worn during a normal working day before individuals were paired to embrace one another (contact), go on an outing together (close proximity), or individually asked to spend a day in another persons environment (physical absence). Each prescribed activity was repeated by sixteen individuals across four countries, and was the last activity performed before the garment was removed. Samples were collected from several areas of the upper garments and processed from DNA extraction through to profiling within the laboratory of the country in which the individual resided. Activities relating to the garment prior to and during wearing, including the prescribed activity, were recorded by the participant and considered during the interpretation of results. In addition to obtaining reference profiles from the wearer and their activity partner, DNA profiles from the wearers close associates identified in the questionnaire were obtained to assess the impact of background DNA transferred prior to the prescribed activity. The wearer was typically, but not always, observed as the major contributor to the profiles obtained. DNA from the activity partner was observed on several areas of the garment following the embrace and after temporarily occupying another persons space. Particular areas of the garment were more prone to acquiring the hugging partner or office owners DNA than others, and whether they were observed as the major or minor component was activity dependent. For each of the pairs, no DNA from the activity partner was acquired by the garments during the outing, even though both participants were in close proximity. This study provides empirical data on the transfer, persistence, prevalence and recovery of DNA from clothing items, and enables a better understanding of the mechanisms which lead to the transfer and detectability of DNA traces in different scenarios.
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