file:///E:/Dokumenter/Downloads/78342-Article%20Text-188182-1-10-20170225.pdf MASKINOVERSÆTTELSE

Nogle svampeslægter omfatter både dødeligt giftige arter og værdifulde spiselige arter. Det gælder for Amanita. I Thailand er de mest almindelige giftige svampe Amanita digitosa, A. exitialis, A.gleocystidiosa, A. fuliginea, A. pyriformis, A. virosa, Chlorophyllum molybdites [False Parasol, Vomiter, Green-spored Parasol], Entoloma-arter, Inocybe-arter og Russula emetica [ref.3-6].

Svampeforgiftning er en akut medicinsk situation for lægerne.

Identifikation af svampeprøver og deres giftige stof er nødvendig for at opnå den rette medicinske behandling.

I dag er molekylære metoder blevet vigtige redskaber til hurtig identifikation af arter inden for forskellige grupper af svampe [ref.5, 8-11].'

De mest populære loci er dels et cellekerne-DNA-område, som kaldes den nukleare interne transkriberede spacer (ITS) region og dels cellekerne-DNA-området som kaldes området for den nukleare store underenhed af ribosomalt DNA (nuLSU, nuclear large subunit)

ITS-regionen er udvalgt til at være en universel stregkode-markør for svampe [ref.10]. Denne region viser tydeligt et stregkode-skift mellem variation fra individ til individ inden for samme art (intraspecifik variation) og variation fra art til art (interspecifik variation) [ref.10].

NuLSU-området er mindre variabelt end ITS-områderne. Imidlertid forstærkes NuLSU-området let for en stor gruppe svampe og kan så udgøre en værdifuld kilde til fylogenetisk information, dvs. om beslægtethed [ref.5, 9, 11, 12].

Man har adgang til sekvensdata i GenBank og Barcode of Life Database (BOLD) (dvs. en database om livets stregkoder, etableret af "Consortium for the Barcode of Life"). Desuden kan ma bruge et online identifikationssystem (IDS), som kan sammenligne og identificere de DNA-sekvenser, man har fundet i sin prøve, med DNA-sekvenser i forskellige online-databaser [ref.13].

Man kan også diagnosticere peptidtoksiner ved anvendelse af væskekromatografi-massespektrometri (LC-MS).

METODER
I et studie i Thailand lavede man DNA-test på svampe fra 43 patienter med typisk gastrointestinalt syndrom efter svampeindtagelse, med forgiftningssymptomer, såsom tørst, kvalme, opkastning, mavesmerter, svær diarré, træthed, der skyldtes mave/tarm-irriterende stoffer i parasolhatte, der ligner rabarber-parasolhat.

DNA fra svampen blev isoleret ved anvendelse af DNeasy [varemærke] Plant Mini Kit ifølge ref.43 http://www.jhealthres.org vol.31 no.1 februar 2017 —J Health Res ved at følge producentens vejledning.

En fortynding på 1:10 af det samlede genomiske DNA anvendtes til PCR-opformering (PCR-amplifikation). PCR-profilerne opnås ved at skifte temperatur i prøven (denaturering opnås ved 45 sekunder ved 94°C , annealing opnås med 45 sekunder ved 52-55°C og forlængelsesfasen opnås ved 1,30 minutter ved 72°C.

Hver PCR-reaktion på 25 μl indeholdt en reaktionsblanding med fluorescerende farvestof, de forskellige primere og DNA-template. Prøverne blev sekventeret (sekvensbestemt) ved anvendelse af ITS1F-primer og ITS4-primren [ref.14, 15] for området med nukleær internt transkriberet spacer (ITS) og LROR-, LR5- og LR6-primere [ref.16] for området, som kaldes "den nukleare store underenhed af ribosomalt DNA" (nuLSU ).

Toksiner blev separeret under anvendelse af LC-metoder [19]. Til analyserne anvendtes "omvendt fase LC-MS-metode" (fra firmaet Agilent, USA).
Alfa-amanitin og β-amanitin (fra Sigma-Aldrich, USA) blev anvendt som referencestandardbibliotek.

RESULTATER
Nuclear ITS-baseret identifikation gav den højeste identifikation (BOLD-databasesøgning gav 98,06% – 99,86% identifikation, hvorimod BLAST-databasesøgning gav 99% til 100% identifikation.

Begge databaser udviste identisk artsidentifikation for Chlorophyllum globosum og C. molybdites. ?????

Fylogenetiske undersøgelser af slægten Chlorophyllum blev udført ved anvendelse af ITS- og nuLSU-sekvenserne.

Udseeendet af C. molybdites er karakteriseret ved at hatten har en bule og at der er brune skæl på en hvid overflade, cheilocystidiernes størrelse er 50-60 × 10-17 μm, bredt clavat-formede og basidiosporernes størrelse er 8-9,6 (-12) × 5-6,1 (-7) μm [gennemsnitskvoter (Qm) = 1,6 ± 0,13], ellipsoide, glatte, hyaline og tykvæggede.

Chlorophyllum globosum er kendetegnet ved at hatten err konvekse, dækket af koncentriske skæl, der er brune til mørkebrune på em lysebrun overflade, cheilocystidiernes størrelse er 28-35 × 8-10 μm, clavat-formede og basidiosporernes størrelse er 8-9,8 (-11) × 5-6,5 ( -8) μm [gennemsnitskvotient (Qm) = 1,5 ± 0,14], ellipsoide, glatte, hyaline og tykvæggede.

Alfa-amanitin og beta -amanitin blev påvist i C. globosum, henholdsvis 0,0059 mg/gram tørvægt og 0,0013 mg mg/g tørvægt. Ved forgiftning med C. globosum er latensperioden længere end for C. molybdites. De påviste amanitiner er i så lav koncentration, at det ikke giver dødelig giftvirkning.

Fra svampeforgiftninger fra årene 2007 til 2014 analyseredes 220 svampeprøver. 76% var gastrointestinalt irriterende (GI) svampe, 14% var amanitinholdige svampe og 10% var muscarinholdige svampe.

De fleste GI-svampe tilhørte slægten Chlorophyllum.

Chlorophyllum-slægten har både spiselige og giftige arter. I Thailand er C. humei og C. rhacodes spiselige og ugiftige.

Svampeprøverne, der blev erhvervet fra 2014 til 2015, blev undersøgt igen ved anvendelse af molekylære data, peptidtoxinanalyse og morfologiske data.

Svampeprøver fra otte kliniske forgiftningstilfælde viste sig at falde ud i to grupper, nemlig med C. molybditter og med C. globosum.

Chlorophyllum molybdites adskiller sig fra C. globosum ved at have en større hat og større cheilocystidier.

Forgiftning med C. molybditter medførte kort latentsperiode på 1 til 2 timer efter måltidet, kvalme, opkastning, svær diarré og mavesmerter.

DNA-baseret identifikation er særligt egnet til påvisning og diagnosticering af gastrointestinalt irriterende svampe i slægten Chlorophyllum.

Denne metode kan adskille de arter af Chlorophyllum, der indeholder amatoxiner (som dog kun findes i meget små mængder).

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MASKINOVERSÆTTELSE AF REFERENCER 1. Sanmee R, Tulloss RE, Lumyong P, Dell B, Lumyong S. Undersøgelser af Amanita (Basidiomycetes: Amanitaceae) i det nordlige Thailand.Svampediversitet.2008 Sep;32: 97-123. 2. Chandrasrikul A, Suwanarit P, Sangwanit U, Lumyong S, Payapanon A, Sanoamuang N, et al.Tjekliste over svampe (Basidiomycetes) i Thailand.Bangkok, Thailand: Kontor for naturressourcer og miljøpolitik og planlægning;2011. p.448. 3. Chaiear K, Limpaiboon R, Meechai C, Poovorawan Y. Fatalsvampforgiftningforårsaget af Amanita virosa i Thailand.Sydøstasiatiske J Trop Med Folkesundhed. 1999 Mar;30 (1): 157-60. 4. Boonpratuang T, Choeyklin R, Promkium-on P, Teeyapan P. Identifikationen af giftige svampe i Thailand i 2008-2012.Thai Champignon.2012; 6-13. 5. Parnmen S, Sikaphan S, Leudang S, Boonpratuang T, Rangsiruji A, Naksuwankul K. Molekylær identifikation af giftige svampe ved anvendelse af nukleare ITS-region og peptidtoksiner: et retrospektivt forsøg på dødelige tilfælde i Thailand.J Toxicol Sci.2016 feb;41 (1): 65-76.doi: 10.2131 / jts.41.65 6. Li GJ, Hyde KD, Zhao RL, Hongsanan S, Abdel-Aziz FA, Abdel-Wahab MA, et al. Svampediversitetnoter253-366: taxonomiske og fylogenetiske bidrag til svamp taxa.Svampediversitet.201678 (1): 1-237.doi: 10.1007 / s13225-016-0366-9 7. Bureau for Epidemiologi.Årlig epidemiologisk overvågningsrapport 2012 [Internet].Thailand;2012. Tilgængelig fra:http://www.boe.moph.go.th/Annual/AESR2012 / index.html8. Dentinger BT, Didukh MY, Moncalvo JM.Sammenligning afCOI og ITS som DNA stregkode markører for svampeog allierede (Agaricomycotina).PLoS One.2011;6 (9):e25081.doi: 10.1371 / journal.pone.00250819. Nugent KG, Saville BJ.Forensisk analyse afhallucinogene svampe: en DNA-baseret tilgang.ForensicSci Int.2004 Mar;140 (2-3): 147-57.doi: 10,1016 / j.forsciint.2003.11.02210. Schoch CL, Seifert KA, Huhndorf S, Robert V, SpougeJL, Levesque CA, et al.Nuclear ribosomal interntranskriberet spacer (ITS) region som en universel DNAstregkode markør for svampe.Proc Natl Acad Sci USA.2012 apr;109 (16): 6241-6.doi: 10.1073 / pnas.111701810911. Vellinga EC.Genera i familien Agaricaceae:Bevis fra nrITS og nrLSU sekvenser.Mycol Res.2004 apr;108 (Pt 4): 354-77.12. Kosentka P, Sprague SL, Ryberg M, Gartz J, May AL,Campagna SR, et al.Evolution af toksinerne muscarinog psilocybin i en familie af svampedannende svampe.PLoS One.2013;8 (5): e64646.doi: 10.1371 / journal.pone.006464613. Ratnasingham S, Hebert PD.fed: Barcode of LifeData System (http://www.barcodinglife.org).Mol EcolNoter.2007 maj 1;7 (3): 355-64.doi: 10.1111 / j.1471-8286.2007.01678.x14. Gardes M, Bruns TD.ITS primere med forbedretspecificitet for basidiomycetes – anvendelse tilidentifikation af mycorrhizae og ruster.Mol Ecol.1993apr;2 (2): 113-8.15. Hvid TJ, Bruns TD, Lee SB, Taylor JW.Amplifikationog direkte sekventering af svampe ribosomale RNA generfor fylogenetika.I: Innis MA, Gelfand DH, SninskyJJ, White T, redaktører.PCR-protokoller: en vejledning til metoderog applikationer.New York: Academic Press;1990.s. 315-22.16. Vilgalys R, Hester M. Rapid genetisk identifikation ogkortlægning af enzymatisk amplificeret ribosomalt DNAfra flere Cryptococcus arter.J Bacteriol.1990aug;172 (8): 4238-46.17. Stamatakis A. RAxML-VI-HPC: maksimal sandsynlighedsbaseredefylogenetiske analyser med tusindvis af taxa- ogblandede modeller.Bioinformatik.2006 nov;22 (21): 2688-90. Doi: 10.1093 / bioinformatics / btl44618. Swofford DL.PAUP-Et computerprogram forfylogenetisk indledning ved brug af maksimal parsimoni.JGen Physiol.1993;102 (6): A9.19. Chung WC, Tso SC, Sze ST.Separation af polarsvampetoksiner ved blandet-mode hydrofil og ioniskinteraktionsvæskekromatografi-elektrosprayioniserings-massespektrometri.J Chromatogr Sci.2007feb;45 (2): 104-11.20. Vellinga EC, De Kok RPJ.Forslag om at bevarenavnet Chlorophyllum Massee mod Endoptychum Czern.(Agaricaceae).Taxon.2002;51: 563-4.21. Bresinsky A, Besl H. En farveatlas af giftige svampe:En håndbog for apotekere, læger og biologer.London: Wolfe Publishing;1990. s.1-295.22. Duffy TJ.Toksisk svampe i vestlige Nordamerika[Internet].Gruppe.MykoWeb;2008. s.166.Tilgængeligfra:http://www.mykoweb.com

ORIGINAL ENGELSK TEKST:
The most popular loci are the nuclear internal transcribed spacer (ITS) region and the nuclear large subunit (nuLSU) ribosomal DNA. The ITS region has been chosen as universal barcode marker for fungi [10]. This region clearly showed a barcode gap between intra- and interspecific variation [10]. The nuLSU is less variable than the ITS regions; however, this area is readily amplified from a large group of mushrooms and contain a valuable source of phylogenetic information [5, 9, 11, 12]. In addition, the availability of sequence data in GenBank and the Consortium for the Barcode of Life has constructed the Barcode of Life Database (BOLD) together with an online identification system (IDS), which can be compared and identified the sequences of interests with the online databases [13]. Thus, the aim of this study was to identify gastrointestinal irritant mushrooms in the genus Chlorophyllum using the ITS regions and the nuclear large subunit of ribosomal DNA sequences as a species marker based on phylogenetic approaches. We also aimed to diagnose peptide toxins using liquid chromatography-mass spectrometry (LC-MS) Total genomic DNA was isolated using DNeasyTM Plant Mini Kit according to 17 http://www.jhealthres.org Table 1 Clinical manifestation of patients and mushroom samples used in this study Case Year Region of Thailand No. of patients Latent period Symptom Mushroom samples ID 1 2014 South 4 2.30 hr Intense thirsty, nausea, vomiting, abdominal pain and severe diarrhea DMSC09255 2 2014 West 4 2 hr Thirsty, nausea vomiting and severe diarrhea DMSC09538 3 2014 Northeast 16 1.30 hr Nausea and vomiting, abdominal pain, fatigue and diarrhea DMSC11138 4 2015 East 2 1.30 hr Abdominal pain, nausea, vomiting and diarrhea DMSC04364, DMSC04365 5 2015 Northeast 5 1.30 hr Nausea, vomiting, diarrhea and fatigue DMSC07290, DMSC07291 6 2015 Northeast 8 1-7 hr Abdominal pain, severe vomiting and watery diarrhea DMSC 09391 7 2015 Northeast 1 2 hr Vomiting, diarrhea and fatigue DMSC13590 8 2015 North 3 2 hr Spontaneous vomiting, watery diarrhea and fatigue DMSC14088 and Russula virescens [1, 2]. Some mushroom genera include both deadly poisonous species and valued edible species such as Amanita and Russula. Here is the most common poisonous mushrooms including Amanita digitosa, A. exitialis, A. gleocystidiosa, A. fuliginea, A. pyriformis, A. virosa, Chlorophyllum molybdites, Entoloma sp., Inocybe sp. and Russula emetica [3–6]. These poisonous mushrooms are sometimes misidentified as resemble edible species. Mushroom toxicity presents after ingestion of toxic substances. These symptoms can vary from gastrointestinal irritants (GI) to severe cytotoxic effects resulting in death of patients. According to the data provided by the Bureau of Epidemiology (Thailand), the annual mortality rate of mushroom poisoning has been increasing particularly in the rainy season [7]. Mushroom poisoning is an emergency medical situation for physicians. Thus, identification of mushroom samples and their toxic substance is needed for appropriate medical treatments. Nowadays molecular methods have become important tools for rapid species identification in various groups of fungi [5, 8–11]. The most popular loci are the nuclear internal transcribed spacer (ITS) region and the nuclear large subunit (nuLSU) ribosomal DNA. The ITS region has been chosen as universal barcode marker for fungi [10]. This region clearly showed a barcode gap between intra- and interspecific variation [10]. The nuLSU is less variable than the ITS regions; however, this area is readily amplified from a large group of mushrooms and contain a valuable source of phylogenetic information [5, 9, 11, 12]. In addition, the availability of sequence data in GenBank and the Consortium for the Barcode of Life has constructed the Barcode of Life Database (BOLD) together with an online identification system (IDS), which can be compared and identified the sequences of interests with the online databases [13]. Thus, the aim of this study was to identify gastrointestinal irritant mushrooms in the genus Chlorophyllum using the ITS regions and the nuclear large subunit of ribosomal DNA sequences as a species marker based on phylogenetic approaches. We also aimed to diagnose peptide toxins using liquid chromatography-mass spectrometry (LC-MS).

METHODS Case reports Details of the case reports of Chlorophyllum mushroom poisoning and samples obtained from eight clinically reported cases during 2014 to 2015 were summarized (Table 1). Mushroom samples were harvested by the local epidemiologists and delivered to toxicology center. In some case, the patient brought samples of the mushrooms that they ate to the hospital. A total of 43 patients with typical gastrointestinal syndrome after mushroom ingestion were revealed in the Table 1. Mushroom samples and molecular methods Total genomic DNA was isolated using DNeasyTM Plant Mini Kit according to 43 http://www.jhealthres.org J Health Res  vol.31 no.1 February 2017 manufacturer’s guide. A dilution of 1:10 of the total genomic DNA was used for PCR amplifications. Samples were PCR amplified and/or sequenced using the ITS1F and ITS4 primers [14, 15] for the nuclear internal transcribed spacer (ITS) region and the LROR, LR5 and LR6 primers [16] for the nuclear large subunit of ribosomal DNA (nuLSU). PCR reactions were conducted on GeneAmp® PCR System 9700 Thermal Cycler (Applied Biosystems®, USA) and reactions were carried out for 34 cycles with PCR profiles of 45 sec at 94°C (denaturation), 45 sec at 52-55°C (annealing) and 1.30 min at 72°C (extension) with final extension of 72°C for 10 min. Each PCR reaction of 25 µl contained 9.5 µl of OnePCRTM (GeneDirex®, Korea) reaction mixture with fluorescence dye, 2.5 µl of 10 µM each primer, 1 µl of genomic DNA template and 9.5 µl nuclease-free water. Amplification products will be cleaned using either QIAquick PCR Purification Kit (QIAGEN) or QIAquick Gel Extraction Kit (QIAGEN) and eluted with 35 µl of elution buffer. DNA sequencing analyses were performed by Macrogen Inc. in Korea. Molecular identification Specimens and sequences used for the molecular analysis are showed in Appendix 1. Sequence alignment was done using Geneious Pro 5.4.3 (http://www.geneious.com/) and edited conflicts manually. Nucleotide similarity was performed using the BLAST server in GenBank (http://blast.ncbi.nlm.nih.gov) and the Barcode of Life Database (BOLD) [13]. Phylogenetic trees were performed using maximum likelihood (ML) and maximum parsimony (MP). Maximum likelihood analyses were analyzed in RAxML 7.2.6 using the GTRGAMMA model [17]. Maximum parsimony analyses were performed using PAUP* version 4.0b [18]. The settings for MP were as follows. Outgroup was defined. Heuristic searches setting optimality criterion with parsimony were employed. All characters are of type unordered and have equal weight. Initial MaxTrees setting equaled 100. Branches collapsed (creating polytomies) if maximum branch length is zero. MulTrees option is in effect. The topological constraints were not enforced. Gaps were treated as missing. Starting tree(s) was obtained via stepwise addition. Branchswapping algorithm was used the tree-bisectionreconnection (TBR) method. Support was then estimated by performing 1000 bootstrap pseudoreplicates. Only clades that received bootstrap support equal or above 70 % under ML and MP were considered as strongly supported. Phylogenetic trees were visualized using the program FigTree (http://tree.bio.ed.ac.uk/ software/FigTree). Toxins detection Five gram of mushroom samples were blended and extracted with 20 ml of methanol. The extract was incubated at 60 °C for 10 min, followed by centrifugation at 8000g for 5 min. The clear supernatant liquid was decanted to dryness under a stream of nitrogen. Toxins including α-amanitin and β-amanitin (Sigma-Aldrich, USA) used as a references standard library. Toxins were separated using LC methods as described by Chung et al. [19]. The analyses were performed using a reversed phase LC-MS method on Agilent technologies 1100 series LC/MSD system (Agilent, USA). RESULTS Molecular identification Twenty-two new sequences were generated for this study (Appendix 1). Nuclear ITS basedidentification using BOLD revealed the highest identity for all samples tested with scores ranging from 98.06% to 99.86%, while BLAST search yielded 99% to 100%. Both databases exhibited identical species identification for Chlorophyllum globosum and C. molybdites. Phylogenetic studies of the genus Chlorophyllum was carried out using the nuclear ITS and nuLSU sequences. A matrix of 1688 unambiguously aligned nucleotide positions was constructed (763 in nuITS and 925 in nuLSU) and 1124 characters were constant. The topology of the trees from MP and ML analyses did not show any conflict and hence only the ML tree is shown here (Figure 1). Most of mushroom samples are clustered in clades I and II (Figure 1). Clade I comprise only a member of Chlorophyllum molybdites (Figure 2E). Morphologically, C. molybdites is characterized by pileus umbonate with brown squamules, surface white, cheilocystidia 50−60 × 10−17 μm, broadly clavate and basidiospores 8−9.6(−12) × 5−6.1(−7) μm [average quotient (Qm) = 1.6±0.13], ellipsoid, smooth, hyaline and thick-walled. Clade II contains a sample of Chlorophyllum globosum (Figure 2A). This species is characterized by pileus convex, covered with concentrically arranged patches of brown to dark brown colored squamules, surface pale brown, cheilocystidia 28−35 × 8−10 μm, clavate and basidiospores 8−9.8(−11) × 5−6.5(−8) μm [average quotient (Qm) = 1.5±0.14], ellipsoid, smooth, hyaline and thick-walled. Toxins detection The standard reference material and two purified compounds obtained from the mushroom samples were analyzed with MS and the corresponding molecular weights were calculated based on their molecular ion peaks. The molecular weights of these compounds were identical to that of the standard peptide toxins. Of the 11 mushroom samples assayed, only a sample of C. globosum contained amatoxins (Figure 2). The MS method generated positive results for alpha-amanitin (m/z 919.3, RT = 13.865 min) and beta-amanitin (m/z 920.3, RT = 11.046 min) (Figure 3). All alphaamanitin and beta -amanitin were detected in C. globosum sample with the amount of toxins (per gram of mushrooms, dry weight) indicated as 0.0059 and 0.0013 mg, respectively.

DISCUSSION AND CONCLUSION Based on our Toxicology Center database, we analyzed toxic substances following poisonous mushroom ingestion from the years 2007 to 2014. A total of 220 mushroom samples were analyzed. Most of samples were identified to 76% gastrointestinal irritant (GI) mushrooms, 14% amanitin-containing mushrooms and 10% muscarine-containing mushrooms, respectively. The number of GI mushroom ingestions was found at a very high percentage. Most GI mushroom belongs to the genus Chlorophyllum. In this study, we focused on mushroom samples that caused gastrointestinal irritation during a 2-year period. A mushroom samples obtained from eight clinically reported cases were used. The remnant samples of mushrooms harvested by the patients were delivered to our laboratory. Based on their morphology, most samples were primarily identified as Chlorophyllum molybdites. This genus is a common mushroom genera in Agaricaceae [20]. Within the genus Chlorophyllum is containing both edible and poisonous species. In Thailand, there are currently two recognized species including C. humei, C. molybdites and C. rhacodes [2]. The toxic species inhibited is C. molybdites, whereas C. humei and C. rhacodes are no general information for mushroom edibility. Interestingly, the nuclear ITS sequence-based identification revealed that some mushroom samples ingested are genetically similar to C. globosum. Hence, the mushroom samples acquired from 2014 to 2015 were re-examined using the molecular data, peptide toxin analysis and morphological data. We analyzed the combined two molecular loci dataset to infer phylogenetic relationships by using maximum likelihood and maximum parsimony methods. Our results showed that mushroom samples from eight clinically reported cases were separated into two clades including C. molybdites and C. globosum clades. Chlorophyllum molybdites differs from C. globosum in having a larger pileus and cheilocystidia. Moreover, based on our findings C. globosum contained alpha-amanitin and beta-amanitin with an average level of 0.0059 and 0.0013 mg/g dry weight, respectively. There was no any report on toxic substances occurring in this species. Having ingested the poisonous C. molybdites, all patients showed a short latent period of 1 to 2 hours after the meal, nausea, vomiting, severe diarrhea and abdominal pain. The clinical symptoms described in the cases were similar to those of gastrointestinal syndrome revealed by Bresinsky and Besl [21]. In case of C. globosum, latent period is longer than above case. There was no death reported in our resulted from the ingestion of C. globosum. Although amanitins were detected in C. globosum, but there was in low concentration. According to Duffy [22] the amanitins are potently toxic to humans with a lethal dose of alpha-form ca. 0.1 mg/kg of body weight. In conclusion, we suggest that DNA-based identification is particularly suitable for detection and diagnosis of gastrointestinal irritant mushrooms in the genus Chlorophyllum. This method can separate the species of Chlorophyllum which contain amatoxins. Discovery of C. globosum and C. molybdites were new for the clinical records of mushroom poisoning in Thailand. ACKNOWLEDGEMENT This work was financially supported by the Department of Medical Sciences, Ministry of Public Health (Thailand).

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