Universal identification of lethal amanitas by using Hyperbranched rolling circle amplification based on α-amanitin gene sequences

摘要

Hyperbranched rolling circle amplification (HRCA) with a padlock probe (PLP) targeting the alpha-amanitin (alpha-AMA) gene, as a screening tool for the universal identification of lethal amanitas, was established in this study. With the isothermal HRCA assay, all of the lethal Amanita species tested from Phalloideae (10) were positive, while the non-Phalloideae Amanita species (15) and three amanitin-containing Lepiota and Galerina species were negative. Furthermore, the PLP based on alpha-AMA sequences from lethal Amanita species was effective for Amanita alpha-AMA, but not Amanita beta-AMA or non-Amanita alpha-AMA. HRCA sensitivity was 100-fold higher than conventional PCR with a detection limit of 100 copies (recombinant plasmid containing alpha-AMA), and 0.2% lethal amanitas could be detected in dry mushroom blends. The HRCA method presented provided a rapid, specific, sensitive and low-cost identification tool for lethal amanitas.