Identification of lethal species in amanita section Phalloideae based on nucleotide signature and specific TaqMan-MGB probe and primer.

Amanita section Phalloideae consists of lethal toxic mushroom species, causing many fatal poisoning incidents worldwide. Molecular techniques of nucleotide signatures and single nucleotide polymorphism (SNP) detection could be used to develop a specific method for identifying lethal section (sect.) Phalloideae species. A comparison of 38 sequenced and 228 validated sequences from sect. Phalloideae species showed a 17-base pair nucleotide signature and an SNP site between the lethal and non-lethal species. A specific minor groove binder probe was designed based on them. The results indicated that this method exhibited excellent specificity for the lethal subgroup, good detection in samples subjected to simulated gastric digestion (60 min boiling and 120 min digestion), and a 10 pg./µL detection limit. This method enables accurate detection of target species in samples under complex conditions and can provide evidence for poisoning incidents caused by lethal sect. Phalloideae species to assist in targeted treatment strategies.

Identification of Gyromitra infula: A Rapid and Visual Method Based on Loop-Mediated Isothermal Amplification.

With mushroom poisoning emerging as one of the most serious food safety problems worldwide, a rapid identification method of poisonous mushrooms is urgently required to investigate the source of poisoning. Gyromitra infula, a kind of poisonous mushroom, contains gyromitrin toxin, which causes epileptogenic neurotoxicity and hemolytic disease. This study aimed to establish a rapid and visual method of G. infula identification based on loop-mediated isothermal amplification (LAMP). A set of specific LAMP primers was designed, and its specificity in G. infula was confirmed against various mushroom species, including its closely related species and other macrofungi. The sensitivity assay showed that the minimum concentration of genomic DNA detected by LAMP was 1 ng/µl. The method's applicability was conducted by preparing mushroom samples that were boiled and digested in artificial gastric juice. The results showed that the content as low as 1% G. infula can be successfully detected. This method can be completed within 90 min, and the reaction results can be directly observed by the naked eyes. Hence, the identification method of G. infula established based on LAMP in this study is accurate, rapid, sensitive, and low-cost, which is required for clinical treatment or forensic analysis when mushroom poisoning occurs.

Rapid Russula senecis identification assays using loop-mediated isothermal amplification based on real-time fluorescence and visualization.

Russula senecis, a common poisonous mushroom, is widely distributed in China. Mushroom poisoning is becoming a major threat to human health and its rate is increasing worldwide. For the first time, we developed a set of loop-mediated isothermal amplification (LAMP) assays based on a real-time fluorescence and a visualization method to detect R. senecis, and the visual LAMP reaction system was optimized to further shorten the reaction time. Both real-time LAMP and visual LAMP could detect as low as 3.2 pg of genomic DNA. In addition, fried and digested mushrooms were used to validate the proposed LAMP method, and mushroom mixtures with as low as 1% of the target species could be successfully detected, indicating that the LAMP assays established in this study had good applicability and could be used for clinical sample detection and forensic identification. Furthermore, the LAMP assays were proven to be comparable to the real-time PCR method. KEY POINTS: • A set of loop-mediated isothermal amplification (LAMP) assays based on real-time fluorescence and visualization to detect Russula senecis was developed. • Both real-time LAMP and visual LAMP can be used to detect genomic DNA at concentrations as low as 3.2 pg. • By simulating mushroom processing and digestion in gastric juice, LAMP assays were proved to have good applicability and could be used for clinical diagnosis and forensic analysis.

Rapid and Visual Identification of Chlorophyllum molybdites With Loop-Mediated Isothermal Amplification Method.

Chlorophyllum molybdites is a kind of common poisonous mushroom in China that is widely distributed in different areas. Food poisoning caused by accidentally eating C. molybdites has become more frequent in recent years. In 2019, there were 55 food poisoning incidents caused by eating this mushroom in China. Mushroom poisoning continues to be a common health issue of global concern. When mushroom poisoning occurs, an effective, simple, and rapid detection method is required for accurate clinical treatment or forensic analysis. For the first time, we established a loop-mediated isothermal amplification (LAMP) assay for the visual detection of C. molybdites. A set of specific LAMP primers was designed, and the specificity was confirmed against 43 different mushroom species. The LAMP method could detect as low as 1 pg of genomic DNA. Boiled mushrooms and artificial gastric-digested mushroom samples were prepared to test the applicability of the method, and the results showed that as low as 1% C. molybdites in boiled and digested samples could be successfully detected. The LAMP method can also be completed within 45 min, and the reaction results could be directly observed based on a color change under daylight by the naked eye. Therefore, the LAMP assay established in this study provides an accurate, sensitive, rapid, and low-cost method for the detection of C. molybdites.

A phylogenetic assessment of Pholiota and the new genus Pyrrhulomyces.

Multigene data sets were assembled to evaluate the phylogeny of species attributed to the genus Pholiota sensu A.H. Sm. & Hesler. This effort included generation of just more than 200 new sequences from 19 type collections of Pholiota and recent samples from East Asia. Phylogenetic analyses reinforced the autonomous phylogenetic positions of pholiotoid taxa in the genera Flammula (Hymenogastraceae) and Kuehneromyces (Strophariaceae). Samples of Pholiota astragalina from diverse geographic regions split into two species-level lineages but occupied an isolated phylogenetic position apart from Pholiota sensu stricto. The new genus Pyrrhulomyces is described to accommodate P. astragalina and a new cryptic species from the Southern Appalachians, Pyrrhulomyces amariceps. Pyrrhulomyces is distinguished from other genera of Strophariaceae by the blackening basidiomata with a bitter taste, smooth basidiospores without a germ pore under light microscopy, presence of pleurochrysocystidia, an ixocutis, rugulose spore ornamentation under scanning electron microscope (SEM), and association with late stages of conifer wood decay. Pholiota subochracea was found to be sister to a clade containing samples of Hypholoma and Bogbodia, but this portion of the Strophariaceae will require further taxon and gene sampling to resolve relationships between these three taxa. Pholiota sensu stricto comprised at least two major groups, but several residual poorly placed lineages were also noted depending on the data set analyzed. New combinations are made in the genera Flammula, Kuehneromyces, and Stropharia for three species of Pholiota-P. abieticola, P. obscura, and P. scabella, respectively, based on molecular annotation of type collections. Overall, 20 new synonymies are proposed, mostly in Pholiota. Illustrations of Pyrrhulomyces are provided along with a key to genera of Strophariaceae and Hymenogastraceae.