Multiplex TaqMan real-time PCR assay for high-throughput identification of highly toxic mushroom species-induced foodborne poisoning

The highly toxic Amanita species, Russula subnigricans, and Lepiota brunneoincarnata are the major mushroom types causing mushroom poisoning fatalities worldwide, which seriously endanger human life and health. However, the detection method for the simultaneous identification of these highly toxic mushroom types is scarce. In this study, a one-tube multiplex TaqMan real-time PCR assay was established for simultaneous detection of the highly toxic mushroom species from three genera. The developed assay could simultaneously detect at least 6 highly toxic Amanita species and specially identify R. subnigricans and L. brunneoincarnata without the cross-reaction in 37 mushroom species. The method detection limits were 0.1 pg/& mu;L, 1 pg/& mu;L, and 0.1 pg/& mu;L DNA for highly toxic Amanita species, R. subnigricans, and L. brunneoincarnata with high amplification efficiency. Moreover, the multiplex real-time PCR assay was successfully applied to the cooked mushroom. The developed method is expected to become an effective tool for high-throughput identification of foodborne poisoning associated with these highly toxic mushroom species.