Swedish National Forensic Center

Linköping, Sweden

Swedish National Forensic Center

Linköping, Sweden
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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.


Hedell R.,Swedish National Forensic Center | Hedell R.,Chalmers University of Technology | Hedman J.,Swedish National Forensic Center | Hedman J.,Lund University | Mostad P.,Chalmers University of Technology
International Journal of Legal Medicine | Year: 2017

Crime scene traces of various types are routinely sent to forensic laboratories for analysis, generally with the aim of addressing questions about the source of the trace. The laboratory may choose to analyse the samples in different ways depending on the type and quality of the sample, the importance of the case and the cost and performance of the available analysis methods. Theoretically well-founded guidelines for the choice of analysis method are, however, lacking in most situations. In this paper, it is shown how such guidelines can be created using Bayesian decision theory. The theory is applied to forensic DNA analysis, showing how the information from the initial qPCR analysis can be utilized. It is assumed the alternatives for analysis are using a standard short tandem repeat (STR) DNA analysis assay, using the standard assay and a complementary assay, or the analysis may be cancelled following quantification. The decision is based on information about the DNA amount and level of DNA degradation of the forensic sample, as well as case circumstances and the cost for analysis. Semi-continuous electropherogram models are used for simulation of DNA profiles and for computation of likelihood ratios. It is shown how tables and graphs, prepared beforehand, can be used to quickly find the optimal decision in forensic casework. © 2017 Springer-Verlag GmbH Germany


Sidstedt M.,Lund University | Sidstedt M.,Swedish National Forensic Center | Romsos E.L.,U.S. National Institute of Standards and Technology | Hedell R.,Swedish National Forensic Center | And 8 more authors.
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. © 2017 American Chemical Society.


Jansson L.,Lund University | Koliana M.,Lund University | Koliana M.,Imperial College London | Sidstedt M.,Lund University | And 3 more authors.
Biotechnology Reports | Year: 2017

The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances. © 2017 The Authors


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.


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.


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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