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

Abstract

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.

Keywords: Chlorophyllum molybdites; ITS; loop-mediated isothermal amplification; on-site rapid detection; poisonous mushroom.

FIGURE 1

Some photographs of mushroom species collected in this study. Among them, Chlorophyllum molybdites was collected in Yunnan, China (24°52′N and 102°49′E), and the other mushrooms were collected from Shanghai, China (31°11′N and 121°29′E). The picture of C. molybdites was taken by ZZ, and the remaining 15 photos of mushrooms were taken by LZ.

FIGURE 2

Positions of the LAMP primer set of Chlorophyllum molybdites designed in the region of the ITS gene. (A) Multiple sequences alignment of homologous species of C. molybdites. (B) Multiple sequences alignment of related species of C. molybdites (not all shown in the figure).

FIGURE 3

Specificity test of the LAMP primer set for Chlorophyllum molybdites designed in this study. The set of primer was amplified for the detection of C. molybdites and 43 other non-target species. 1: C. molybdites; 2: Suillus bovinus; 3: Gymnopus subnudus; 4: Panaeolus subbalteatus; 5: Leucoagaricus rubrotinctus; 6: Macrolepiota dolichaula; 7: Lactarius subbrevipes; 8: Laccaria aurantia; 9: Amanita griseofolia; 10: Rhizocybe alba; 11: Russula variata; 12: Russula senecis; 13: Amanita hemibapha; 14: Boletus kauffmanii; 15: Tylopilus neofelleus; 16: Russula rosacea; 17: Butyriboletus yicibus; 18: Russula velenovskyi; 19: Hydnellum caeruleum; 20: Tricholoma saponaceum; 21: Inocybe mixtilis; 22: Hydnellum concrescens; 23: Amanita spissacea; 24: Tricholoma albobrunneum; 25: Pleurotus eryngii; 26: Flammulina filiformis; 27: Lentinula edodes; 28: Hypsizygus marmoreus; 29: Tricholoma olivaceoluteolum; 30: Amanita citrinoannulata; 31: Russula crustosa; 32: Hebeloma crustuliniforme; 33: Tricholoma imbricatum; 34: Amanita parvipantherina; 35: Amanita verrucosivolva; 36: A. verrucosivolva; 37: Amanita concentrica; 38: Amanita sepiacea; 39: Pleurotus ostreatus; 40: Tylopilus microsporus; 41: Inocybe rimosa; 42: Amanita pseudovaginata; 43: Russula sanguinea; 44: Gymnopus dryophilus; 0: ddH₂O.

FIGURE 4

Sensitivity test of the LAMP assay. Minimum amount of detectable DNA of the LAMP method established in this study was evaluated by using a series of C. molybdites DNA dilutions. For each reaction tube, we carried out three replicates. The color changed to yellow indicated positive amplification. 1: 10 ng; 2: 1 ng; 3: 0.1 ng; 4: 0.01 ng; 5: 1 pg; 6: 0.1 pg; 7: 0.01 pg; 8: 1 fg; 9: 0.1 fg; 0: ddH₂O.

FIGURE 5

Suitability assessment of the LAMP assay. Feasibility analysis of the LAMP method in on-site detection was evaluated by simulating the course of processing and digestion of mushrooms. (A) LAMP assay used for the detection of boiled Chlorophyllum molybdites. 1: C. molybdites; 2: 50% C. molybdites + 50% Pleurotus eryngii; 3: 10% C. molybdites + 90% P. eryngii; 4: 1% C. molybdites + 99% P. eryngii; 0: ddH₂O. (B) LAMP assay used for the detection of digested C. molybdites after boiled. 1: C. molybdites; 2: 50% C. molybdites + 50% P. eryngii; 3: 10% C. molybdites + 90% P. eryngii; 4: 1% C. molybdites + 99% P. eryngii; 5: human saliva; 0: ddH₂O.