ABSTRACT
Omphalotus japonicus is a major toxic mushroom in Japan. When food poisoning caused by O. japonicus occurs, quick and accurate identification using a method that does not rely on morphological discrimination is required. Because the loop-mediated isothermal amplification (LAMP) method meets these requirements, we developed a LAMP method for detecting O. japonicus. Amplification occurred within 60 min, and the presence or absence of O. japonicus was confirmed within 2 h, including the DNA extraction protocol. The LAMP method did not show cross-reactivity with 13 species of edible mushrooms, had high specificity toward O. japonicus, and had sufficient detection sensitivity even in a mixed mushroom sample containing 1% O. japonicus. Additionally, O. japonicus could be detected in simulated food poisoning samples of heated and digested mushrooms, and in actual food poisoning residual samples.
ABSTRACT
The mouse bioassay (MBA) for paralytic shellfish toxins (PSTs) in bivalves has been used as an official method in Japan. It is necessary to develop an alternative method to animal experiments in PSTs assay because 3Rs (Replacement, Reduction, and Refinement) of animal experiments are required from the animal welfare point of view. Various methods such as HPLC-FL, receptor binding assay, LC-MS/MS and ELISA have been established to detect PSTs without performing animal experiments. The present study was undertaken to develop a screening method using oligonucleotide lateral flow immunoassay (OLFIA) for detecting PSTs in bivalves. The screening level was defined as positive at 2 MU/g of MBA that is the half regulation limit of PSTs monitoring in Japan. All 20 positive (equal to or more than 2 MU/g) samples judged from MBA showed a positive reaction in the OLFIA. No positive samples resulted in a false negative reaction. The OLFIA exhibited high accuracy at 2 MU/g of screening criteria. The authors demonstrated here that the OLFIA can be useful for rapid detection of PSTs in bivalves.
