Rapid Identification Method of Omphalotus japonicus by Polymerase Chain Reaction-Restriction Fragment Length
Yohei Sugano1, Kozue Sakata2, Kosuke Nakamura2
1Hokkaido Institute of Public Health.
Abstract:
Omphalotus japonicus is a poisonous mushroom that grows in Japan. It can be mistaken for edible mushrooms (Shiitake, Hiratake and Mukitake), and if ingested, it causes food poisoning within 30 min to 1 hr. We established a rapid detection method using PCR-RFLP to identify O. japonicus by restriction digestion of the amplified ITS region. By using Sau96I, Bpu10I, SfcI or DrdI/HincII as a restriction enzyme, it was possible to rapidly identify and discriminate O. japonicus based on the fragment length. This study also provided a short PCR-RFLP system comprising amplification and digestion of a short 200-bp DNA fragment within the ITS region. The system could identify and discriminate O. japonicus after in vitro gastric digestion of native and heated mushroom samples as a model of food poisoning. In addition, a confirmatory assay using real-time PCR was developed to achieve more sensitive detection of O. japonicus.
Insights
A new PCR-RFLP method rapidly detects the poisonous mushroom Omphalotus japonicus, preventing misidentification with edible varieties. This technique aids in identifying O. japonicus in food poisoning cases.
Area of Science:
- Mycology
- Molecular Biology
- Food Safety
Background:
- Omphalotus japonicus is a toxic mushroom found in Japan.
- It is often mistaken for edible mushrooms like Shiitake, Hiratake, and Mukitake.
- Ingestion of O. japonicus causes rapid onset food poisoning.
Purpose of the Study:
- To develop a rapid and accurate detection method for Omphalotus japonicus.
- To differentiate O. japonicus from similar edible mushroom species.
- To validate the detection method under simulated food poisoning conditions.
Main Methods:
- Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) targeting the ITS region.
- Use of restriction enzymes (Sau96I, Bpu10I, SfcI, DrdI/HincII) for DNA fragment analysis.
- Development of a short PCR-RFLP assay for a 200-bp DNA fragment.
- Real-time PCR for sensitive confirmatory detection.
Main Results:
- The PCR-RFLP method successfully identified and discriminated O. japonicus based on DNA fragment lengths.
- A simplified short PCR-RFLP system was effective even after simulated gastric digestion.
- Real-time PCR provided a highly sensitive confirmatory assay for O. japonicus detection.
Conclusions:
- PCR-RFLP is a reliable and rapid method for identifying poisonous Omphalotus japonicus.
- The developed short PCR-RFLP assay is suitable for detecting the mushroom in food matrices.
- Real-time PCR enhances detection sensitivity, crucial for food safety applications.
