title: Rapid genetic detection of ingested Amanita phalloides.
abstract: Mushrooms are often poorly digested by humans. Thus, their remains (tissues, spores) may persist in the gastrointestinal tract and can be detected in feces several days after mushroom consumption. In this report, we present protocols for the rapid PCR-based detection of fungal traces in a variety of complex samples. Novel primers were designed to amplify portions of ribosomal DNA from deadly poisonous European members of the genus Amanita, namely the death cap (A. phalloides), the destroying angel (A. virosa) and the fool's mushroom (A. verna), respectively. Assay sensitivity was sufficient to discover diluted DNA traces in amounts below the genomic content of a single target mushroom cell. Specificity testing was performed with DNA extracts from a variety of mushroom species. Template amplification was exclusively observed with intended targets and it was not compromised by a vast excess of non-target DNA (i.e. DNA from human and human fecal origin, respectively). A series of experiments was conducted with prepared specimens in order to follow the course of mushroom food processing and digestion. Amplification by direct PCR was successful with raw, fried and digested mixed mushrooms. To improve assay performance with fecal samples, a rapid protocol for sample pre-processing (including water-ether sedimentation and bead beating) and a modified PCR reaction mix were applied. Thereby, it was possible to detect the presence of A. phalloides DNA in spiked feces as well as in clinical samples (vomit, stool) from two independent cases of suspected mushroom poisoning.
authors: FDZ-Forensisches DNA Zentrallabor GmbH, Medical University of Vienna, Sensengasse 2, 1090 Vienna, Austria. Electronic address: christian.gausterer@meduniwien.ac.at.
authors: Christian
authors: C
authors: Gausterer
authors: FDZ-Forensisches DNA Zentrallabor GmbH, Medical University of Vienna, Sensengasse 2, 1090 Vienna, Austria; Department of Health, FH Campus Wien, University of Applied Sciences, Favoritenstraße 226, 1100 Vienna, Austria. Electronic address: martinapenker@hotmail.com.
authors: Martina
authors: M
authors: Penker
authors: Department of Systematic and Evolutionary Botany, Faculty Centre of Biodiversity, University of Vienna, Rennweg 14, 1030 Vienna, Austria. Electronic address: irmgard.greilhuber@univie.ac.at.
authors: Irmgard
authors: I
authors: Krisai-Greilhuber
authors: FDZ-Forensisches DNA Zentrallabor GmbH, Medical University of Vienna, Sensengasse 2, 1090 Vienna, Austria. Electronic address: christina.stein@meduniwien.ac.at.
authors: Christina
authors: C
authors: Stein
authors: Clinical Department of Laboratory Medicine, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. Electronic address: thomas.stimpfl@meduniwien.ac.at.
authors: Thomas
authors: T
authors: Stimpfl
mesh_terms: D000545:Amanita / Q000235:genetics*; D003296:Cooking; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000302:isolation & purification*; D005243:Feces / Q000737:chemistry; D006801:Humans; D009145:Mushroom Poisoning / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D066256:Raw Foods; D017422:Sequence Analysis, DNA; D014839:Vomiting
chemical_list: D017931:DNA Primers; D004275:DNA, Ribosomal
keywords: Amanita phalloides; Direct PCR; Feces; Internal transcribed spacer 1 (ITS1); Mushroom poisoning; Vomit