Mushrooms are widely distributed globally, some of which have high nutritional value and medicinal efficacy (Lei et al., 2020), but some of which are poisonous. There are more than 4,000 mushrooms species in China, among which 966–1,020 are edible (Dai et al., 2010; Wu et al., 2019), 692 are medicinal (Wu et al., 2019), and 435–480 are poisonous (Bau et al., 2014; Wu et al., 2019). Poisonous mushrooms with toxins can make people and animals have toxic reactions and even lead to death. Mushroom poisoning mostly occurs from June to October annually. Mushroom poisoning is mostly common in Southwest China and Central China, followed by Southern China, Eastern China, Northern China, Northeast China, and Northwest China (Chen, 2014; Chen et al., 2016). Although poisonous mushrooms can cause poisoning, they play a certain role in some fields, such as medicine and insecticides, due to their biologically active compounds (Wu et al., 2019; Sheikha, 2021).

Many poisonous mushrooms have similar morphology as edible mushrooms, and it is difficult to distinguish between them through observation (Yang, 2013). Among these mushrooms, Gyromitra infula (Schaeff.) Quél is commonly mistaken as an edible mushroom, and is distributed in different regions of China during summer and autumn. Gyromitra infula is extremely similar to edible Helvella species (such as Helvella macropus) and tend to be confused with the latter, leading to accidental ingestion and poisoning. Gyromitra infula contains the toxin gyromitrin, which is converted to monomethylhydrazine in human body, one of the raw materials of rocket fuel (Pan et al., 2012). Additionally, G. infula may be carcinogenic if accidentally ingested. If G. infula is accidentally ingested, it can lead to epileptogenic neurotoxicity and hemolytic disease (Diaz, 2005; Chen et al., 2016), characterized by early symptoms, such as headache, diarrhea, nausea, and vomiting; 2 days after ingestion, the patient will develop chills, abdominal pain, hepatosplenomegaly, fever, vertigo, delirium, seizures, coma, and hemoglobinuria. In severe cases, patients may die of acute kidney failure. Gyromitrin and the hydrolyzed product monomethylhydrazine in these mushrooms can lyse and destroy erythrocytes, leading to acute hemolysis. Rapid detection and identification of poisonous mushrooms (such as G. infula) when mushroom poisoning occurs is essential and urgent for investigating mushroom poisoning and treatment of poisoning patients (Li et al., 2020). Conventional detection methods such as morphology identification, high-performance liquid chromatography, and polymerase chain reaction (PCR; Ahmed et al., 2010; Yao et al., 2018; Wurita et al., 2019; Cevik, 2020) are not suitable for regions that are economically underdeveloped and have a lack of poisonous mushroom knowledge and equipment limitations. Therefore, there is an urgent need for a new, simple, practical, and cheap rapid detection method for G. infula that can be used for onsite applications at the grassroots level.

Several test methods have been derived from PCR, such as real-time fluorescence PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), and DNA barcoding (Sugano et al., 2017; Sheikha and Hu, 2018; Sheikha et al., 2018; Kondo et al., 2019; Wu et al., 2019). However, these methods depend on expensive equipment, are time-consuming and cannot be used in rapid onsite detection. Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification method developed by Notomi et al. (2000). In LAMP, two pairs of specific primers (one pair of outer primers and one pair of inner primers) are designed for six regions in the target gene, and a strand-displacement DNA polymerase (Bst DNA polymerase) can be used for amplification at a constant temperature (around 65°C), where nucleic acids can be amplified to a magnitude of 10⁹–10¹⁰ within 1 h. The third primer pair (loop primer) can be selectively added to the system to accelerate the reaction and shorten reaction time (Yu et al., 2011). Compared to routine PCR, LAMP has a higher specificity and sensitivity (10 times compared to that of PCR) and does not require template denaturation, long periods of thermal cycling, cumbersome electrophoresis, and ultraviolet observation. Result detection is also extremely simple, where the amplification will simultaneously produce red and yellow changes, of which, a yellow indicates a positive result, and a pink indicates a negative result, which can be observed with only the naked eye, does not require a PCR cycler and other expensive equipment, and only a thermostatic water bath is necessary to conduct the reaction (Xiang et al., 2019). With all abovementioned advantages, LAMP has a high specificity and sensitivity, is easy to operate, does not require expensive reagents and equipment, and is extremely suitable for rapid detection and onsite application at the grassroots level (Kuang et al., 2007). As the technology of LAMP gradually matures, it is widely used in fields such as food safety testing (Zhu and Wang, 2018), testing of pathogenic microorganisms (Lv et al., 2020), environmental testing (Becherer et al., 2020), and clinical disease diagnosis (Gu et al., 2021). In the identification of mushroom species, Vaagt et al. (2013) were the first to employ LAMP to detect Amanita phalloides. 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.

In this study, an accurate, visual, rapid, sensitive and low-cost method was established for the detection of G. infula based on LAMP, which can be used in resource-poor areas without expensive and complex instruments. This method is of great significance for the rapid identification of poisonous mushrooms in poisoning incidents, the targeted treatment after poisoning and the prevention of mushroom poisoning.

Gyromitra infula is distributed in different regions of China in summer and autumn and is similar to some edible saddle mushrooms, such as Helvella macropus. Humans may confuse G. infula with them, thereby developing mushroom poisoning. The technology of LAMP can be used for rapid detection of G. infula and is a simple method. The species-specific amplification region is an essential challenge in designing efficient LAMP primer sets (Tomita et al., 2008). Schoch et al. (2012) amplified SSU, LSU, ITS, RPB1, RPB2, and MCM7 fragments during fungal molecular identification and found that ITS sequence was highly consistent in intraspecies, and significantly different in interspecies, and it was easy to conduct PCR amplification. Finally, they proposed using the ITS gene as the target gene fragment for fungal amplification. Subsequently, they obtained extensive support, and ITS was used for fungal DNA barcoding studies (Garnica et al., 2016; Badotti et al., 2017; Wang et al., 2019). Simultaneously, in consideration that sequences of many Gyromitra species are present in the NCBI GenBank database, we selected the ITS gene sequence as an amplification target to design G. infula LAMP-specific primer sets. In the selection of LAMP primer design sites, to ensure the specificity of the primers, there should be a certain number of mismatches between the sequence bases of the target species and the closely related species to reduce nonspecific amplification. For example, Xiong et al. (2020) found mismatch sites between Oncorhynchus mykiss and closely related species when designing LAMP primers for O. mykiss so that the primers were highly specific for O. mykiss. The G. infula LAMP primer set I designed in this study had some mismatches with other Gyromitra species at the 3' end of G.mol-F3/B3 and the 5' end of G.mol-F1c/B1c, showing that the G. infula LAMP primers designed in this study had high specificity. Whereas, the control primer set II presented non-specificity, and the main reason is there are not sufficient numbers of mismatches with closely related species, resulting in presence of binding sites with non-target species.

Recently, various LAMP visualizable detection methods have continuously emerged, and the most commonly used visualizable method is turbidimetry and colorimetry (Tomita et al., 2008; Denschlag et al., 2013). Turbidimetry is simple to operate and low-cost, but the sensitivity is low. Furthermore, it is difficult to see the results when the low reactant concentration (Njiru et al., 2008). Currently, colorimetry is widely accepted. Examples include methods with results that can be observed with the naked eye, such as the calcein fluorescence method, hydroxynaphthol blue/eriochrome black T staining method, pH indicator method. During the reaction, the reaction tubes do not need to be opened, which reduces aerosol contamination and false positive rate (Goto et al., 2009). In this study, the pH indicator method was used (Daskou et al., 2019, 2021; Shi et al., 2021), such that a chromogenic agent was added to the LAMP reaction system (such as Warm Start LAMP master mix), and the changes in the colorimetric result were used for interpretation to observe whether red-yellow color reaction occurred during product amplification. A yellow color means that the result is positive, that the target species DNA or the target species component is present in the tested sample. A red color means that the result is negative, that the target species DNA is absent. The pH indicator of phenol red was used as a dye in the reaction system. As LAMP amplification occurred, Bst conducted polymerization, and the number of protons in the reaction mix was changed, thereby changing the pH and causing the solution color to change from pink to yellow.

Currently, in addition to LAMP method established in this study, G. infula can be identified by other methods, such as traditional morphology identification, routine PCR, and so on. Extensive morphology features and professional mycologists are required to accurately identify species in traditional morphology method. Whereas, in mushroom poisoning incidents, the morphology of mushrooms will change greatly because of procession, such as cooking or digesting, which is hard to get correct identification results based on morphology. Moreover, compared to routine PCR, the LAMP method in this study has a higher specificity and sensitivity due to its specific primer set designed based on six specific binding sites of G. infula. Additionally, the LAMP method does not require complex heating and cooling processes, cumbersome electrophoresis, and ultraviolet observation, which have distinct advantages over routine PCR. Furthermore, although some other methods, such as real-time fluorescence PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), and the liquid chromatography-mass spectrometry (LC–MS, used for the detection of toxins) might be used for identification of G. infula, they rely on expensive instruments, a long detection cycle, high detection costs, and high sample pretreatment, which are not suitable for application in resource-poor areas and on-site detection. 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).

In this study, we established a visual and rapid LAMP method for the identification of G. infula. The results showed that this method can accurately and specifically identify poisonous G. infula within 90 min. The test results can be directly observed with the naked eyes, which can be used for rapid onsite detection. Meanwhile, high sensitivity for the LAMP method with the detection limit of 1 ng was confirmed in this study. The method can be conducted without expensive and sophisticated instruments, which is suitable for resource-poor areas. Furthermore, the boiled and/or digested G. infula were successfully detected in this study, indicating its strong adaptability. Therefore, the LAMP method established in this study is accurate, visual, rapid, sensitive, adaptable, and low-cost for the identification of G. infula. Using our method, an on-site detection kit of G. infula can be developed for clinical treatment and forensic analysis. In the future, increasing methods that are more rapid, more efficient, simpler and more low-cost will be explored for identifying varied poisonous mushrooms, further used for diagnosis, targeted treatment and prevention of poisoning.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

ET designed the experiments and morphologically identified the species collected in this study. YF provided some specimens and photos of Gyromitra, Morchella, Helvella, and Verpa species. XX, RD, BL, CG, and YZ completed the analysis ITS sequences of all specimens and LAMP assay. XX and ET wrote the manuscript. XX, BL, YF, RD, CG, YZ, and ET contributed to collection of specimens used in this study. All authors contributed to the article and approved the submitted version.

This research was supported by the National Key Research and Development Program of China (2019YFC1604703) and National Natural Science Foundation of China (no. 31860009).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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