m Vol. 44, No.

44




 
 !"#$!#%&' () *+ ,./ 14 0 9 1 3 234)

5 6 7 1
> 6 ?2

8 9 :1
@ABCD3

; * < =1
E 9 F G1, H

Discrimination of Psychoactive Fungi (Commonly Called Magic Mushrooms) Based

on the DNA Sequence of the Internal Transcribed Spacer Region
Takuro M6GJN6B61, Osamu S=>GDI61, Nobuo K6L6=6G61, Kazumasa YD@DN6B62,
Yukiko M6@>CD3 and Yukihiro GD961, H
(1National Institute of Health Sciences: 1181, Kamiyoga, Setagaya-ku, Tokyo 1588501, Japan;
2Faculty of Education, Shiga University: 251, Hiratsu, Otsu 5200862, Japan; 3Kanto-Shinetsu
Regional Narcotic Control O$ce, Ministry of Health, Labor and Welfare: 2414, Nakameguro,
Meguro-ku, Tokyo 1530061, Japan; H Corresponding author)
Magic mushrooms (MMs) are psychoactive fungi containing the hallucinogenic compounds,
psilocin (1) and psilocybin (2). Since June 6, 2002, these fungi have been regulated by the
Narcotics and Psychotropics Control Law in Japan. Because there are many kinds of MMs and
they are sold even as dry powders in local markets, it is very di$cult to identify the original
species of the MMs by morphological observation. Therefore, we investigated the internal
transcribed spacer (ITS) region in the ribosomal RNA gene of MMs obtained in Japanese markets
to classify them by a genetic approach. Based on the size and nucleotide sequence of the ITS
region amplied by PCR, tested MMs were classied into 6 groups. Furthermore, a comparison of
the DNA sequences of the MMs with those of authentic samples or with those found in the
databases (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 Panaeolus
cyanescens among the MMs, but was absent in the Amanita species.
(Received September 3, 2002)
Key words: !"#$!#%&' ( magic mushroom;
IJ%K psilocin; 

 internal transcribed spacer region; %LMNOP Psilocybe genus; QRSNOP Panaeolus
genus; TKUNOP Amanita genus

  !"#$!#%&' (V WX

1, 2 YV LSD 200 , 1V RK 50

`YjhV \^t2), 3)k

M.M.) YV Z[/, 
IJ%KV 1;
IJ%LKV 2;

ab E\]^_-etY%LM

Fig. 1) \]^_`V ab\cd_efg

NOP ( Psilocybe) QRSNOP ( Panaeolus) P

hibaehjk l0V M.M. \m_aen

_-h_hV Yib

opqhrstuaevw\xytV z{|}YV ./

` 50 +Web~4)V 2 11

14 0 6 1~ 1, 2 \]^_-\Z[*

tu1)k
H

1 [m{ 1588501 9
g 1181
2  5200862 . 251
3 z{|}<z{Z[d 1530061
n 2414

Fig. 1.

Structures of hallucinogenic compounds

ITS ? A0BC2^_` 

February 2003

45

 


  M.M. 

tems zy] ,'  - SR-2W XTaitec zy]




,  KUBOTA-6900 XX}]ty] ,' 

! " #$ %&

'( )*+,-

"* ./* M.M. 01
DNA 23,

A Gw G1321A G1316A; Hewlet Pack-

4

! 567289

rRNA    : ; < = > = (internal transcribed

spacer, ITS) ?@A0 BC 
,DE

 LC ; HP1100 K;fe XL G1312

ard zy] , LC/MS ; JMS LC-Mate X< 3

YX}]y] ,' 
ITS
  rRNA large subunit 

4.

 5  FGH9>IJKLM,N M.M.

01
>IJKLM OP,Q 


[ \ 8 (50 mg) , H = | o *   DNeasy
Plant Mini Kit XQiagen zy] ,' genomic DNA


1. 

,0w xy


79 R 13 S7 13 
T "#28U$%2EV&W

42 GH XY

ZH [\] ,'  ^_` (a bc (K.

Y.) 

V&

 >(W

DNA ,?

rRNA @Y,0W

 4
28de;fge 

1
,'

(Table 1) W( 

5), 6)

(sense-1, antisense-1)

I=

,' PCR ,D-2

ITS ?,A 
PCR  5, 50 mL (0.25 mmol / L dNTP, 1.25
unit Gene Taq NT, each 0.5 mmol/L primers)  q

o Je2DE

 * 94k 4 

hij 60k *l)[\ mn*o X50p qo] +

B C  94k 1  d = L  60k 1  D

, XrstuL]  -.*/n  *v

74k 1  30 >I j* 72k 4 

PCR E dJ=; (1p) 342


2. 

F j QIAquick Gel Extraction Kit XQiagen z y] ,

Genomic DNA 0 w x y   DNeasy Plant

Mini Kit XQiagen z y] , ' 

 PCR { |  Gene

'xy Direct Sequencing 2A0 BC

,DE  0w

DNA (

Taq NT XNIPPON GENE X}] y] , ~   2 8

G2BC+%GH9 ( sense-2

dNTP  {|12  ,'

 antisense-1 I=,'

 34' d

J=; Agarose S XNIPPON GENEX}]y] ,

nested PCR

,D RE, Zero Blunt PCR Cloning Kit XInvitro-

DNA == DNA Ladder, 100 bp XBayou Biolabs

gen zy] ,' pCR-Blunt vector H&j A0

zy] ,'

, 

 dJ=;'( DNA 0w

x y   QIAquick Gel Extraction Kit XQiagen z y]

 rRNA large subunit @Y  BC

,'  5  cycle sequencing 5 BigDye Ter-

 I= sense-3, antisense-2 ,' '

minator Cycle Sequencing FS Ready Reaction Kit

I G,'  >(W A0 J

XPE Applied Biosystems z y]  2 8 DYEnamic ET

KBC Clustal W Je7) ,'DE

Terminator Cycle Sequencing Kit XAmersham Biosci-

' I=  Table 2 *v

ences zy] ,'

 1 76.7*R

  ,'  5  F8 *'st=

Table 2.

   J ; X9  0.45 mm; GL Sci

>=>I= TaKaRa PCR Thermal Cycler

MP X:X}]y] ,'

PCR Primers

Name

ences zy] ,' 

3. 

 A0 BC ABI

PRISM 310 Genetic Analyzer XPE Applied BiosysTable 1.

Sense-1
Sense-2
Sense-3
Antisense-1
Antisense-2

Sequence
5-GGAAGTAAAAGTCGTAACAAGG-3
5-TCCGTAGGTGAACCTGCGG-3
5-TCGATGAAGAACGCAGCG-3
5-GCTGCGTTCTTCATCGATGC-3
5-ATCCTGAGGGAAACTTCGGCA-3

Data of Authentic Psilocybe and Panaeolus sp. Samples

Scientic name

Japanese name

Data of collection

PCR product size (bp)a)

Accession No.

Panaeolus sphinctrinus
Panaeolus subbalteatus
Psilocybe argentipes
Psilocybe subaeruginascens
Psilocybe tampanensisb)

Hikagetake
Senbonsaigyougasa
Hikageshibiretake
Oshibiretake

1999.06.03
1983.07.
1983.07.18
1983.09.23
2000.12.04

281
299
291
319
291

AY152728
AB092794
AB092792
AB092793
AF548391

a)
b)

Sense-2 and antisense-1 primers were used.

Purchased in a market in Amsterdam.

Fig. 2. Sequence alignment of ITS region of several magic mushrooms

1, Panaeolus sphinctrinus; 2, Pa. subbalteatus; 3, Psilocybe argentipes; 4, Ps. subaeruginascens; 5, Ps. tampanensis; 6, MM-1; 7, MM-2-1; 8, MM-2-2; 9, MM-3; 10, MM-4; 11, MM5; 12, MM-6

46

Vol. 44, No. 1

ITS -l56m. e&7

February 2003

5.

LC
 LC/MS 

47

56y 6 t7 7z (MM1rMM6; Table

 


3) w " MM-3  Lee y85)  Psilocybe

   200 mg 

 

semilanceata 56!9  MM-4  Ps. tam-

 (3 : 1) 10 mL 

panensis *+56!:9

!"
 15 #

!"$ 10 # %&' (

!"

)*+,-. *!

  Ps. tampanensis !;z

MM-5 geh MM-6  e`56pw

LC  /01 TSKgel ODS-80Ts QA 24.6f3150

Psilocybe 1geh Panaeolus 1*+56!2<&

mm; 24565  /017 408 9:;<

=[ zy BLAST search D01 e2

=>?=> 254 nm  1.0 mL/min *

<&=no LOV ( A. muscaria; acces-

 10 mL  >  * A@ AB+

sion no. AB015700) OV ( A. pantherana; acces-

CDE (TFA) 0.1FGH 100F I  * B@

sion no. AF438558) gehOV ( A. gem-

TFA 0.1F GHJKALAB * A@ 

mata; accession no. AF335440) !> 2<&p

* B (75/5) JM6N0O,PNQR' 

 Amanita 17!;z

!SN"#$TMDUV T

=4?@[ MM-1, MM-2  *+geh

MDUV%WX# 9.2  YZ 5 ng/*

 (GenBank, EMBL, DDBJ) A B

10 mL [  (\ LC QRSN

wxyz[ ITS -ley 

] ESI  positive mode MCV^ m/z 205

rRNA large subunit CaD56m. E

_`aMCV ([MbH]) SNcd'"!

!F no MM-1  Ps. cubensis (ac-

e TMDUV&f''[

cession no. AF261619) !:u9 (968/

970; 99.8F)  MM-1  Ps. cubensis !;



1.

   MM-2  Panaeolus sp. (accession no. AF

ITS
 

261525) !2<&p (926/934; 99.1F)

()*+gehi,jk+ ITS -lm.no

Panaeolus 1!yz !y

Fig. 2 p PCR q/ (sense-2 r antisense-1) T

MM-2  Ps. cubensis !! DP g@

Ms 281r319 bp !t e[0uvwxyz

G2) Pa. cyanescens !;z

(Table 1, 3) w {1#2||}~'yz

Table 3 p!g i,jk+ Ps. cubensis ge

[ 3i,jk+ 42 =4 ITS -l

h Pa. cyanescens  2 twH4 9 Ig

i, G M.M. FJ?t KL

Table 3. Classication of Magic Mushroom Obtained in

Japanese Markets by Size and DNA Sequencing
Data of PCR Products and the Name of the
Species Elucidated

Group
MM-1
MM-2
MM-3
MM-4
MM-5
MM-6

PCR
Number of
product
samples (n)
size (bp)
22
15
2
1
1
1

314
307
319
291
288
288

!wMy [ no DP g

( 2 tNPAwOPz!"
Q   RST 2R5 
 M.M.  8 w Pa. cyanescens !"UlJ
VWxyz

Species

3 gt,X'yz=4 Pa.
cyanescens geh Ps. tampanensis zz g 1

Psilocybe cubensis
Panaeolus cyanescens
Psilocybe semilanceata
Psilocybe tampanensis
Amanita sp.-1
Amanita sp.-2

Sense-2 and antisense-1 primers were used.

= 4 2MM-2-1 ! MM-2-2 Ps. tampanensis ! MM-4;

Fig. 2, 35 ) Gt, ITS -l5

6y]u!wMy![ Y\Ul
ZVWG no M.M. w? Y
yz!p g Lee y8

Fig. 3. Phylogenetic tree of DNA sequences of the ITS region

Numbers and branch length represent sequence divergence.

 Vol. 44, No. 1

48
Table 4.

Psilocin Content of Magic Mushrooms

Psilocin content (/dry weight)

Group (species)
max
MM-1
MM-2
MM-3
MM-4
MM-5
MM-6
a)
b)
c)

a)

( Psilocybe cubensis)
( Panaeolus cyanescens)
( Psilocybe semilanceata)
( Psilocybe tampanensis)
( Amanita sp.-1)
( Amanita sp.-2)

min
b)

1.36
2.35
0.055

trace
trace
0.034

meanSD
0.22 0.29
1.19 0.62
0.0450.01
trace
n.d.c)
n.d.

20 samples were used for the determination of psilocin content.

Less than 0.00025.
Not detected.

5)   Ps. semilanceata    Pa. subbalteatus 

STG& 5UV ; M.M.  3 ' 6 

ITS

 


& b STUV:XYZS

   ! "#

T i'8
^_`30ac\G 

!$% (Fig. 2)&

M.M. q
$<8& b

MM-5, 6  Amanita '()*$+,-./01

 i M.M. =>?@.F
MpqG8<

( A. muscaria) -./01 ( A. pantherina) 23(

8UV;& M.M. 

-./01 ( A. gemmata) 4 56789:;&

<8H;4)& G%Igq

<;()* !=>?@.8ABC


| ;

M  !

$ d,l->D-.E (3; Fig. 1) FG<8H

O"q$&

;I JKL
MN O
$8
PQ&
R
 ; ITS
STUV
WX


1)

Y Z  [ \ G & 5  U V Psilocybe ' 5  Panaeolus ' 3  Amanita ' 2 5]^_`3

0a
 <bcdefg hOU

2)

V: (Fig. 3)&

g iNj
M kl3m_nopqGg
 rst(3u8vw
xyzg{|}
;<8~O& Ps. cubensis, u Pa. cyanescens, u Amanita sp., u&

3)
4)

2.


=>?@.F TUV Table 4

5)

b8& !FG Pa. cyanescens 

$I xy Ps. cubensis, Ps. semilanceata, Ps. tampanensis 8 Amanita ' 2 Nsg4%&

 Mussho# ; > M.M.

M

6)

 T =>?@.F5] Pa. cyanescens (0.43), Ps. semilanceata (0.23), Ps. cubensis
(0.09), Ps. tampanensis (0.02) 8
G2)& <

  
   N j  g 4 %  Ps. semilanceata (MM-3) ! UV$&

W
 M.M. qf


M rRNA W ITS

7)


# 169 (2002) $% |%$%|%
&qO#'O# (\ 14 ) 5
7*.
Mussho#, F., Madea, B., Beike, J., Hallucinogenic mushrooms on the German marketsimple instruction for
examination and identication. Forensic Science International, 113, 389395 (2000).
Nakahara, Y. Yakubutsu Ranyou No Kagaku,
Tokyo, Kenseisha, 1999. (ISBN 4876393826)
Stamets, P. Psilocybin Mushrooms of the World, Berkeley, CA, USA, Ten Speed Press, 1996. (ISBN 089815-839-7)
Lee, J. C., Cole, M., Linacre, A., Identication of members of the genera Panaeolus and Psilocybe by a DNA
testA preliminary test for hallucinogenic fungi.
Forensic Science International, 112, 123133 (2000).
White, T. J., Bruns, T. D., Lee, S. B., J. Taylor, W., PCR
Protocols: Amplication and Direct Sequencing of
Fungal Ribosomal Genes for Phylogenetics, Innis, M.
A., Gelfand, D. H., Sninsky, J. J., White T. J., ed., San
Diego, CA, USA, Academic Press, 1990, p. 315322.
(ISBN 0-12372-181-4)
Thompson, J. D., Higgins, D. G., Gibson, T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting,
position-specic gap penalties and weight matrix
choice. Nucl. Acids Res., 22, 4,6734,680 (1994).