Qualitative Real-Time PCR Method for Poisonous Entoloma rhodopolium-Related Species in Japan: Real-Time PCR Method for Entoloma Mushrooms

Abstract

Qualitative real-time PCR method for three poisonous Entoloma rhodopolium-related species in Japan was established using specific primers and FAM, VIC, Texas Red, Cy5-labeled probes. The use of multicolor probes can extend the method to simultaneous detection of different targets. Standard plasmids were constructed as reference materials. Designed primers and probes in the method detect only a target species, and the detection limit was 12.5 copies or below. This indicates it is highly specific and sensitive enough to detect the poisonous mushrooms in food residues. Next, we applied the method to four food residue samples obtained from food poisoning cases. The real-time PCR method did identify all of four samples as E. subrhodopolium and E. pseudorhodopolium, whereas PCR-RFLP did not. The method established here revealed Entoloma rhodopolium-related species in Hokkaido were different species such as E. eminens and unknown species.