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Shokuhin Eiseigaku Zasshi 2003, 44(1), p.44.pdf

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m‹¼ Vol. 44, No. 1 44      ! #$!#%&' () *+ ,./ 14 0 9 1 3 234) 5 6 7 ῍῍1 6 ῎ ?῍2 8 9 :῍1 @ABCD῍3 ; * =῍1 E 9 F G῍1, H Discrimination of Psychoactive Fungi (Commonly Called “Magic Mushrooms”) Based on the DNA Sequence of the Internal Transcribed Spacer Region Takuro M6GJN6B6῍1, Osamu S= GDI6῍1, Nobuo K6L6=6G6῍1, Kazumasa YD@DN6B6῍2, Yukiko M6@ CD῍3 and Yukihiro GD96῍1, H (῍1National Institute of Health Sciences: 1ῌ18ῌ1, Kamiyoga, Setagaya-ku, Tokyo 15
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             14  9  3      1     1   1     2   3   1,  Discrimination of Psychoactive Fungi (Commonly Called “Magic Mushrooms”) Basedon the DNA Sequence of the Internal Transcribed Spacer Region Takuro M 6GJN6B6  1 , Osamu S =>GDI6  1 , Nobuo K 6L6=6G6  1 , Kazumasa Y D@DN6B6  2 ,Yukiko M 6@>CD  3 and Yukihiro G D96  1,  (  1 National Institute of Health Sciences: 1  18  1, Kamiyoga, Setagaya-ku, Tokyo 158  8501, Japan;  2 FacultyofEducation, Shiga University: 2  5  1, Hiratsu, Otsu 520  0862, Japan;   3 Kanto-Shin’etsuRegional Narcotic Control O $ ce, Ministry of Health, Labor and Welfare: 2  4  14, Nakameguro,Meguro-ku, Tokyo 153  0061, Japan;   Corresponding author)‘Magic mushrooms’ (MMs) are psychoactive fungi containing the hallucinogenic compounds,psilocin ( 1 ) and psilocybin ( 2 ). Since June 6, 2002, these fungi have been regulated by theNarcotics and Psychotropics Control Law in Japan. Because there are many kinds of MMs andthey are sold even as dry powders in local markets, it is very di $ cult to identify the srcinalspecies of the MMs by morphological observation. Therefore, we investigated the internaltranscribed spacer (ITS) region in the ribosomal RNA gene of MMs obtained in Japanese marketsto classify them by a genetic approach. Based on the size and nucleotide sequence of the ITSregion amplified by PCR, tested MMs were classified into 6 groups. Furthermore, a comparison ofthe DNA sequences of the MMs with those of authentic samples or with those found in thedatabases (GenBank, EMBL and DDBJ) made it possible to identify the species of tested MMs.Analysis by LC revealed that psilocin ( 1 ) was contained at the highest level in  Panaeoluscyanescens  among the MMs, but was absent in the  Amanita  species.(Received September 3, 2002) Key words:   magicmushroom;   psilocin;   internal transcribed spacer region;    Psilocybe  genus;    Panaeolus genus;    Amanita  genus          M.M.            1 ;     2 ;Fig. 1            M.M.          14  6  1, 2  1)     1    158  8501   1  18  1  2    520  0862   2  5  1  3      153  0061   2  4  14 1, 2    LSD  200  1    50     2), 3)   (  Psilocybe )  (  Panaeolus )     50  4)    11 Fig. 1.  Structures of hallucinogenic compounds   Vol. 44, No. 144          M.M.                           M.M.  DNA                 rRNA    (internal transcribedspacer, ITS)                  M.M.        1.             13   13           42                     (K.Y.)       4    1    (Table 1)          60              50                  2.    Genomic DNA         DNeasy PlantMini Kit   Qiagen         PCR  GeneTaq NT   NIPPON GENE           dNTP               Agarose S   NIPPON GENE         DNA  DNA Ladder, 100 bp   Bayou Biolabs          DNA      QIAquick Gel Extraction Kit   Qiagen         cycle sequencing   BigDye Ter-minator Cycle Sequencing FS Ready Reaction Kit  PE Applied Biosystems     DYEnamic ETTerminator Cycle Sequencing Kit   Amersham Biosci-ences         1                          0.45  m m; GL Sci-ences        3.     TaKaRa  PCR Thermal CyclerMP                   ABIPRISM 310 Genetic Analyzer   PE Applied Biosys-tems          SR-2 W   Taitec       KUBOTA-6900               LC    HP1100       G1312A      G1321A      G1316A; Hewlet Pack-ard       LC/MS    JMS LC-Mate               4. ITS  rRNA large subunit    (50 mg)        DNeasyPlant Mini Kit   Qiagen       genomic DNA           DNA         rRNA      (sense-1, antisense-1) 5), 6)    PCR  ITS      PCR      50  m L (0.25 mmol/L dNTP, 1.25unit Gene Taq NT, each 0.5  m mol/L primers)             94  4       94   1       60   1       74  1    30        72  4   PCR       (1  )         QIAquick Gel Extraction Kit   Qiagen         Direct Sequencing        DNA              sense-2  antisense-1    nested PCR       Zero Blunt PCR Cloning Kit   Invitro-gen       pCR  -Blunt vector               rRNA large subunit         sense-3, antisense-2                         Clustal W  7)           Table 2   Table 1.  Data of Authentic  Psilocybe  and  Panaeolus  sp. SamplesScientific name Japanese name Data of collection PCR product size (bp) a) Accession No.  Panaeolus sphinctrinus Panaeolus subbalteatus Psilocybe argentipes Psilocybe subaeruginascens Psilocybe tampanensis b) HikagetakeSenbonsaigyougasaHikageshibiretakeOshibiretake  1999.06.031983.07.1983.07.181983.09.232000.12.04281299291319291AY152728AB092794AB092792AB092793AF548391 a) Sense-2 and antisense-1 primers were used. b) Purchased in a market in Amsterdam. Table 2.  PCR PrimersName SequenceSense-1Sense-2Sense-3Antisense-1Antisense-25  -GGAAGTAAAAGTCGTAACAAGG-3  5  -TCCGTAGGTGAACCTGCGG-3  5  -TCGATGAAGAACGCAGCG-3  5  -GCTGCGTTCTTCATCGATGC-3  5  -ATCCTGAGGGAAACTTCGGCA-3  February 2003 ITS       45        F       i     g  .      2  .      S   e   q   u   e   n   c   e   a     l     i   g   n   m   e   n    t   o     f     I     T     S   r   e   g     i   o   n   o     f   s   e   v   e   r   a     l   m   a   g     i   c   m   u   s     h   r   o   o   m   s     1 ,      P   a   n   a   e   o     l   u   s   s    p     h     i   n   c    t   r     i   n   u   s    ;     2 ,      P   a .   s   u     b     b   a     l    t   e   a    t   u   s    ;     3 ,      P   s     i     l   o   c   y     b   e   a   r   g   e   n    t     i    p   e   s    ;     4 ,      P   s .   s   u     b   a   e   r   u   g     i   n   a   s   c   e   n   s    ;    5 ,      P   s .    t   a   m    p   a   n   e   n   s     i   s    ;     6 ,     M     M  -     1   ;    7 ,     M     M  -     2  -     1   ;     8 ,     M     M  -     2  -     2   ;     9 ,     M     M  -     3   ;     1     0 ,     M     M  -     4   ;     1     1 ,     M     M  -    5   ;     1     2 ,     M     M  -     6 46    Vol. 44, No. 1  5. LC  LC/MS                       200 mg          (3 : 1) 10 mL     15        10                   LC     TSKgel ODS-80Ts QA   4.6 f  150mm;                40     254 nm    1.0 mL/min       10  m L            A      (TFA) 0.1   100        B  TFA 0.1         A     B (75/5)                   9.2       5 ng/    10  m L        LC    ESI  positive mode    m /  z  205    ([M  H]  )           1. ITS        ITS     Fig. 2    PCR   (sense-2  antisense-1)  281  319 bp    (Table 1, 3)                   42      ITS     6      (MM1  MM6; Table3)     MM-3  Lee    5)   Psilocybe semilanceata           MM-4   Ps. tam- panensis             Ps. tampanensis     MM-5  MM-6      Psilocybe    Panaeolus            BLAST search       (  A. muscaria ; acces-sion no. AB015700)    (  A. pantherana ; acces-sion no. AF438558)  (  A. gem-mata ; accession no. AF335440)        Amanita             MM-1, MM-2      (GenBank, EMBL, DDBJ)       ITS     rRNA large subunit                    MM-1   Ps. cubensis  (ac-cession no. AF261619)     (968/970; 99.8  )    MM-1   Ps. cubensis         MM-2   Panaeolus  sp. (accession no. AF261525)    (926/934; 99.1  )   Panaeolus      MM-2    Ps. cubensis      2)  Pa. cyanescens     Table 3          Ps. cubensis   Pa. cyanescens  2    9          M.M.                  2                       M.M.  8   Pa. cyanescens                   Pa.cyanescens   Ps. tampanensis  1      MM-2-1  MM-2-2   Ps. tampanensis   MM-4;Fig. 2, 3          ITS                    M.M.         Lee   Table 3.  Classification of Magic Mushroom Obtained inJapanese Markets by Size and DNA SequencingData of PCR Products and the Name of theSpecies ElucidatedGroup Number ofsamples ( n )PCRproductsize   (bp)SpeciesMM-1MM-2MM-3MM-4MM-5MM-622152111314307319291288288  Psilocybe cubensis Panaeolus cyanescens Psilocybe semilanceata Psilocybe tampanensis Amanita  sp.-1  Amanita  sp.-2   Sense-2 and antisense-1 primers were used. Fig. 3.  Phylogenetic tree of DNA sequences of the ITS regionNumbers and branch length represent sequence divergence.February 2003 ITS           47
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