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Hedell R.,Swedish National Forensic Center | Hedell R.,Chalmers University of Technology | Stephansson O.,National Veterinary Institute SVA | Mostad P.,Chalmers University of Technology | Andersson M.G.,National Veterinary Institute SVA
International Journal of Food Microbiology | Year: 2017

Efficient and correct evaluation of sampling results with respect to hypotheses about the concentration or distribution of bacteria generally requires knowledge about the performance of the detection method. To assess the sensitivity of the detection method an experiment is usually performed where the target matrix is spiked (i.e. artificially contaminated) with different concentrations of the bacteria, followed by analyses of the samples using the pre-enrichment method and the analytical detection method of interest. For safety reasons or because of economic or time limits it is not always possible to perform exactly such an experiment, with the desired number of samples. In this paper, we show how heterogeneous data from diverse sources may be combined within a single model to obtain not only estimates of detection probabilities, but also, crucially, uncertainty estimates. We indicate how such results can then be used to obtain optimal conclusions about presence of bacteria, and illustrate how strongly the sampling results speak in favour of or against contamination. In our example, we consider the case when B. cereus is used as surrogate for B. anthracis, for safety reasons. The statistical modelling of the detection probabilities and of the growth characteristics of the bacteria types is based on data from four experiments where different matrices of food were spiked with B. anthracis or B. cereus and analysed using plate counts and qPCR. We show how flexible and complex Bayesian models, together with inference tools such as OpenBUGS, can be used to merge information about detection probability curves. Two different modelling approaches, differing in whether the pre-enrichment step and the PCR detection step are modelled separately or together, are applied. The relative importance on the detection curves for various existing data sets are evaluated and illustrated. © 2016 Elsevier B.V.


Forsberg C.,Swedish National Forensic Center | Jansson L.,Lund University | Ansell R.,Swedish National Forensic Center | Ansell R.,Linköping University | And 2 more authors.
Forensic Science International: Genetics | Year: 2016

Tape-lifting has since its introduction in the early 2000′s become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the “stickiness”, rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p = 0.13 and p = 0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01 ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples. © 2016 The Authors


Hedman J.,Swedish National Forensic Center | Hedman J.,Lund University | Agren J.,Swedish National Veterinary Institute | Ansell R.,Swedish National Forensic Center | Ansell R.,Linköping University
Forensic Science International: Genetics Supplement Series | Year: 2015

A possible alternative to conventional stain recovery by swabbing, taping or cutting, is the M-Vac wet-vacuum instrument (M-Vac Systems Inc.). We have evaluated M-Vac for sampling of dried saliva on porous and non-porous surfaces, shed cells on clothes and touch DNA. M-Vac gave significantly higher DNA yields for dried saliva stains on laminated wood, compared with cotton swabs (average DNA concentrations 1.14 vs. 0.57. ng/μL, p = 0.02). For stains on glass, M-Vac and cotton swabs gave comparable DNA yields. Additionally, M-Vac retrieved three times as much DNA from saliva stains on cotton fabric (T-shirt) compared with saliva on towels (terry cloth), showing that the absorption properties of the surface affect wet-vacuum sampling. M-Vac was also applied for retrieving wearer DNA from clothes, enabling generation of complete DNA profiles from denim jeans, leggings and cotton T-shirt. A mixed DNA profile was retrieved from an "aggressor" pressing a hand against the shoulder area of a worn T-shirt. Since the major component of the obtained mixed DNA profile was from the wearer, M-Vac may not be ideal for touch DNA sampling of clothes. Wet-vacuum sampling requires a fairly large instrument, trained users and DNA extraction procedures handling large sample volumes. The complexity of M-Vac sampling prevents it from being extensively used, but in specific and important cases it can be a valuable sampling tool. © 2015 Elsevier Ireland Ltd.


Forsberg C.,Swedish National Forensic Center | Wallmark N.,Swedish National Forensic Center | Hedell R.,Swedish National Forensic Center | Hedell R.,Chalmers University of Technology | And 5 more authors.
Forensic Science International: Genetics Supplement Series | Year: 2015

Tape-lifting is an efficient method for collecting traces of cellular material from fabrics. Since 2006, an in-house adhesive tape has been used in casework at the Swedish National Forensic Centre, Linköping. Although this tape gives good DNA yields, we aim to replace it with a commercial tape to save cost and labour. In order to enable a fair comparison between different adhesive tapes, we have developed and evaluated a method for production of relevant reference material. One person, known to be a good shedder, wore identical long-sleeved T-shirts under controlled circumstances, and trace recovery was systematically performed with the in-house tape (3 T-shirts, total of 24 samples). Each sample was DNA extracted and quantified to find the normal variation within the reference material. The DNA recovery differed considerably between samples, with DNA concentrations between 0.010-0.48. ng/μL (mean: 0.083, SD: 0.12. ng/μL). Applying such a reference material for comparison between two commercial tapes and our in-house tape resulted in mean DNA recoveries plus/minus one standard deviation of 0.013. ±. 0.006. ng/μL (Scenesafe FAST Box), 0.012. ±. 0.007. ng/μL (Touch tape), and 0.023. ±. 0.013. ng/μL (in-house tape). The in-house tape gave statistically significant higher yield compared to Touch tape (p <. 0.05), but for Scenesafe FAST Box the difference was not significant. The shedding of cells to clothes cannot be fully controlled. Having a systematically prepared, casework-like reference material with known variation is therefore vital for comparative studies of tapes. © 2015.


Sanga M.,Swedish National Forensic Center | Boiso L.,Swedish National Forensic Center | Lindsten H.,Swedish National Forensic Center | Radstrom P.,Lund University | And 4 more authors.
Forensic Science International: Genetics Supplement Series | Year: 2015

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. © 2015 Elsevier Ireland Ltd.


PubMed | Swedish Defence Research Agency, Swedish National Forensic Center and Linköping University
Type: Journal Article | Journal: Drug testing and analysis | Year: 2015

In this work, emergence patterns of synthetic cannabinoids were utilized in an attempt to predict those that may appear on the drug market in the future. Based on this information, two base structures of the synthetic cannabinoid analogues - (1H-indol-3-yl(2,2,3,3-tetramethylcyclopropyl)methanone and 1H-indol-3-yl(adamantan-1-yl)methanone) - together with three substituents - butyl, 4-fluorobutyl and ethyl tetrahydropyran - were selected for synthesis. This resulted in a total of six synthetic cannabinoid analogues that to the authors knowledge have not yet appeared on the drug market. Spectroscopic data, including nuclear magnetic resonance (NMR), mass spectrometry (MS), and Fourier transform infrared (FTIR) spectroscopy (solid and gas phase), are presented for the synthesized analogues and some additional related cannabinoids. In this context, the suitability of the employed techniques for the identification of unknowns is discussed and the use of GC-FTIR as a secondary complementary technique to GC-MS is addressed. Examples of compounds that are difficult to differentiate by their mass spectra, but can be distinguished based upon their gas phase FTIR spectra are presented. Conversely, structural homologues where mass spectra are more powerful than gas phase FTIR spectra for unambiguous assignments are also exemplified. This work further emphasizes that a combination of several techniques is the key to success in structural elucidations. Copyright 2015 John Wiley & Sons, Ltd.


Rosengren-Holmberg J.P.,Linnaeus University | Rosengren-Holmberg J.P.,Swedish National Forensic Center | Andersson J.,Uppsala University Hospital | Smith J.R.,University of Portsmouth | And 8 more authors.
Biomaterials Science | Year: 2015

Heparin-imprinted synthetic polymer surfaces with the ability to attenuate activation of both the complement and the coagulation system in whole blood were successfully produced. Imprinting was achieved using a template coated with heparin, a highly sulfated glycosaminoglycan known for its anticoagulant properties. The N,N′-diacryloylpiperazine - methacrylic acid copolymers were characterized using goniometry, AFM and XPS. The influence of the molecular imprinting process on morphology and template rebinding was demonstrated by radioligand binding assays. Surface hemocompatibility was evaluated using human whole blood without anticoagulants followed by measurement of complement activation markers C3a and sC5b-9 and platelet consumption as a surrogate coagulation activation marker. The observed low thrombogenicity of this copolymer combined with the attenuation of complement activation induced by the molecular imprint offer potential for the development of self-regulating surfaces with important potential clinical applications. We propose a mechanism for the observed phenomena based upon the recruitment of endogenous sulfated glycosaminoglycans with heparin-like activities. This journal is © The Royal Society of Chemistry.


Boiso L.,Swedish National Forensic Center | Sanga M.,Swedish National Forensic Center | Hedman J.,Swedish National Forensic Center | Hedman J.,Lund University
Forensic Science International: Genetics Supplement Series | Year: 2015

Forensic DNA analysis is partly limited by PCR-inhibitory compounds present in the DNA extracts. Generally, these inhibitors disturb amplification, i.e., the production of amplicons. We have found that dithiothreitol (DTT) from the DNA extraction process can cause another type of real-time PCR disturbance, i.e., inhibition of signal detection through fluorescence quenching. DNA extracts containing DTT substantially quenched the passive reference signal in the Quantifiler HP DNA Quantification kit. This quenching resulted in overestimation of DNA concentrations, as target DNA signals are normalized to the passive reference signal. © 2015 Elsevier Ireland Ltd.


Ahlinder J.,Swedish Defence Research Agency | Nordgaard A.,Swedish National Forensic Center | Lindstrom S.W.,Swedish Defence Research Agency
Journal of Chemometrics | Year: 2015

Forensic statistics is a well-established scientific field whose purpose is to statistically analyze evidence in order to support legal decisions. It traditionally relies on methods that assume small numbers of independent variables and multiple samples. Unfortunately, such methods are less applicable when dealing with highly correlated multivariate data sets such as those generated by emerging high throughput analytical technologies. Chemometrics is a field that has a wealth of methods for the analysis of such complex data sets, so it would be desirable to combine the two fields in order to identify best practices for forensic statistics in the future. This paper provides a brief introduction to forensic statistics and describes how chemometrics could be integrated with its established methods to improve the evaluation of evidence in court. The paper describes how statistics and chemometrics can be integrated, by analyzing a previous know forensic data set composed of bacterial communities from fingerprints. The presented strategy can be applied in cases where chemical and biological threat agents have been illegally disposed. © 2015 John Wiley & Sons, Ltd.


PubMed | Swedish National Forensic Center, Lund University and U.S. National Institute of Standards and Technology
Type: | Journal: Analytical chemistry | Year: 2017

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

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