Loop-mediated Isothermal Amplification(LAMP)-Based Assay For Rapid Detection And Identification Of Lethal Amanitas

In this study, loop-mediated isothermal amplification(LAMP)-based assays for detection and identification of seven species of lethal amanitas including A. rimosa, A. exitialis, A. pallidorosea, A. fuliginea, A. subjunquillea in China and A. phalloides, A. bisporigera in Europe and America and genus Amanita were developed. The LAMP assays for lethal amanitas targeted ITS sequence and the LAMP assay for genus Amanita targeted 5.8s rDNA, partial ITS2 sequence. The in-tube LAMP reactions were occurred at 62 ℃ for 60 min and hydroxynaphthol blue(HNB) was added as a indicator to make the results visualized. The sensitivity and specificity of these LAMP assays were tested and the results were as follows:(1) The lethal amanita contaminations could be detected at the level of 0.1% and the detection limit was 5.37 pg genomic DNA per reaction.(2) Amang the seven sets of specific primers designed, primers for A. rimosa, A. exitialis and A. fuliginea could distinguish the seven species of lethal amanitas while primers for A. pallidorosea and A. bisporigera, A. subjunquillea and A. phalloides only failed to distinguish between each other.(3) A set of universal primers designed could distinguish amanitas from non-amanitas. The LAMP assay for genus Amanita was positive to 23 samples of DNA from amanitas but nagegtive to 19 samples of DNA from non-amanitas.In conclusion, the results suggest that the established LAMP-based assays for rapid detection and identification of lethal amanitas and genus Amanita are simple, rapid, specific, sensitive, visualized, inexpensive and suitable for on-site applications. It is of great significance to investigation for mushroom poisonings and treatment for poisoned patients.