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.

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

“…Therefore, the present LAMP method with abovementioned advantages is superior to other methods and is suitable for identifying G. infula. However, we need to pay attention to controlling the content of the DNA template in the reaction system because the larger amount of DNA template can have an inhibitory effect on the LAMP reaction (Njiru et al, 2008;Wang et al, 2021) presumably due to the negative influence of polyphenols and polysaccharides contained in mushrooms (Newbury and Possingham, 1977;Fang et al, 1992).…”

“…He et al (2019) used LAMP to detect 10 highly toxic species of Amanita. Besides the Amanita genus, Wang et al (2021) used LAMP to detect Chlorophyllum molybdites. However, there is a lack of effective and rapid identification methods for G. infula.…”

Development of a loop-mediated isothermal amplification assay for the rapid detection of Russula subnigricans and Russula japonica

“…In the detection of Mycoplasma ovipneumoniae , the reaction times of real-time recombinase polymerase amplification (RT-RPA) and lateral flow strip recombinase polymerase amplification (LFS-RPA) are shorter than LAMP via a lateral flow dipstick (LAMP-LFD) assay, but the effectiveness of RT-RPA and LFS-RPA in the detection needs to be further validated (Zhang et al, 2019 ; Wang et al, 2020 ). Furthermore, LAMP primers for detecting poisonous mushrooms all are based on ITS regions, and according to various families and genera, we can design specific primers based on other gene fragments, such as nrLSU, rpb1 , and rpb2 (Vaagt et al, 2013 ; He et al, 2019a ; Wang et al, 2021 , 2022 ).…”

“…LAMP has been used in the detection of lethal mushrooms belonging to Amanita, R. senecis , and C. molybdites in the field of mushroom identification to date (Vaagt et al, 2013 ; He et al, 2019a ; Wang et al, 2021 , 2022 ). In this study, LAMP, as a rapid and effective method, is applied to detect the deadly poisonous species R. subnigricans and the widely distributed toxic species R. japonica .…”

Rapid identification of Amanita citrinoannulata poisoning using colorimetric and real-time fluorescence and loop-mediated isothermal amplification (LAMP) based on the nuclear ITS region

“…The sequences were analyzed using the National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) and also compared with published sequences to confirm the mushroom species. Finally, the obtained sequence was uploaded to GenBank database (accession number in Table 1 ) ( Wang et al, 2021 ).…”