NCJ Number
245535
Journal
Forensic Science International: Genetics Volume: 9 Dated: March 2014 Pages: 66-71
Date Published
March 2014
Length
6 pages
Annotation
Since mushroom remains (tissues and spores) may be detected in feces several days after mushroom consumption, this report presents protocols for the rapid PCR-based detection of fungi traces in a variety of complex samples.
Abstract
Applying a short protocol that combines stool pre-processing with a modified PCR reaction mix, the authors proved the presence of highly diluted A. phalloides traces in spiked feces and in a clinical stool sample in a case that involved suspected mushroom poisoning. Novel primers were designed to amplify portions of ribosomal DNA from poisonous European members of the genus Amanita, i.e., the "death cap" (A. phalloides); A. virosa, known as "the destroying angel," and A. verna, called the "fool's mushroom." 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 the intended target, and it was not compromised by an excess of non-target DNA, i.e., DNA from human and human fecal origin. A series of experiments was performed with prepared specimens, so as to monitor the process of mushroom food processing and digestion. Amplification by direct PCR was successful with raw, fired, and digested mixed mushrooms. In order 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. Results from PCR, including documentation on agarose gel, could be obtained within 2 hours. 49 references