Application of direct PCR in a forensic case of yew poisoning

Abstract

Intoxications with yew (Taxus spp.) pose a challenge to forensic toxicology because a variety of Taxus ingredients have been associated with its toxic effects. To provide preliminary evidence in cases where plant material is available, we introduce a novel direct PCR assay for the detection of DNA traces from Taxus spp. This assay has been successfully applied to a forensic case of suicidal poisoning via ingestion of Taxus leaves. PCR primers were designed to target a sequence located in the internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA, which is well conserved among species of the genus Taxus and can, therefore, be exploited to discriminate between Taxus and other conifers. Because ITS1 exists as a multicopy sequence within the plant genome, the assay provides enough sensitivity to work with trace amounts that are below the DNA content of a single cell. Specificity of the assay was tested with DNA extracts from Taxaceae and selected representatives from other related plant families (Cephalotaxaceae, Cupressaceae and Pinaceae). When combined with the commercial Phire® Plant Direct PCR Kit (Finnzymes), the primers allowed application of a two-step cycling protocol (without the annealing step), and because direct PCR requires only little sample pre-treatment, results from PCR could be obtained within 1.5 h after analysis had begun. Direct PCR was performed with diluted gastric content from the forensic case. Amplification products of the expected size were purified and sequenced. Sequence data were subjected to Basic Local Alignment Search Tool analysis and affiliated with ITS1 from Taxus spp.

Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity

“…As most small-scale studies are more likely to suffer from fungus contamination instead of allelic variation, our plant-specific primers were designed to meet such requirements. The newly designed primer pairs are also expected to contribute to other applications such as conservation biology (Cennamo et al 2013), forensic applications (Lee et al 2010;Gausterer et al 2012) and market monitoring (Stoeckle et al 2011;Wallace et al 2012), in addition to the already-prevalent use of ITS for plant DNA barcoding, molecular phylogenetics and biodiversity studies.…”

Suicidal poisoning by ingestion of Taxus Baccata leaves. Case report and literature review

“…The literature mentions different and new methods [17] of confirming the yew poisoning among them being the detection of active compounds from Taxus baccata, most commonly being the identification of 3,5-dimethoxyphenol (3,5 DMP). In our case the poisoning diagnosis has been determined by the corroboration of the personal medical history, the clinical and laboratory investigations with the autopsy findings, a determinant role being played by the botanical exam of the leaves and fragments of leaves taken from the gastric content.…”

“…Other authors, namely Beyer, Drummer and Maurer [19], presented a detailed article regarding the analysis of toxic alkaloids found in different plants, also mentioning the ones detectable in yew tree. Gausterer, Stein and Stimpfl [17], proposed an alternative method of detecting yew intoxication, using direct PCR. We would like to point out that although there are different methods of determining the Taxus compounds, which in many cases imply high financial costs, we should not disconsider the simple and costless botanical examination.…”

Leishmaniasis: Prevention, Parasite Detection and Treatment

“…In addition, background fluorescence may diminish both sensitivity and specificity [200]. The ITS1 region is commonly used target in many different eukaryotic organisms for typing purposes [184,[201][202][203]. It has enough conservation to serve as a PCR target but sufficient polymorphisms to facilitate species typing.…”