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A Simple Chelex Protocol for DNA Extraction from Anopheles spp.
Published on: January 9, 2013
Rapid genetic detection of ingested Amanita phalloides
Christian Gausterer1, Martina Penker2, Irmgard Krisai-Greilhuber3
1FDZ-Forensisches DNA Zentrallabor GmbH, Medical University of Vienna, Sensengasse 2, 1090 Vienna, Austria.
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.
Insights
Detecting deadly mushroom DNA in feces is now faster. New PCR methods can identify traces of toxic Amanita species, aiding in poisoning cases.
Area of Science:
- Mycology
- Molecular Biology
- Forensic Science
Background:
- Human digestion poorly processes mushrooms, leaving detectable traces.
- Accurate identification of ingested toxic mushrooms is crucial for medical treatment.
Purpose of the Study:
- Develop rapid PCR protocols for detecting fungal DNA from toxic Amanita species.
- Validate assay sensitivity and specificity in complex samples.
Main Methods:
- Designed novel primers targeting ribosomal DNA of Amanita phalloides, A. virosa, and A. verna.
- Performed PCR on raw, cooked, and digested mushrooms, as well as spiked and clinical samples.
- Utilized a rapid pre-processing protocol involving water-ether sedimentation and bead beating for fecal samples.
Main Results:
- Achieved high sensitivity, detecting DNA below single-cell levels.
- Demonstrated specificity against various mushroom species and human DNA.
- Successfully amplified target DNA from diverse food preparations and clinical specimens (vomit, stool).
Conclusions:
- Developed a sensitive and specific PCR assay for rapid detection of deadly mushroom DNA.
- The protocol is effective for analyzing complex matrices like feces and clinical samples.
- This method aids in diagnosing mushroom poisoning incidents.
