Microbiol. Immunol.

, 40(1), 5-11, 1996

Simple Method
for Detection
of Clostridium
botulinum
Type A to F Neurotoxin
Genes
by Ploymerase
Chain Reaction
Kouichi Takeshi1 , Yukako Fujinaga2, Kaoru Inoue2, Hiroshi Nakajima2, Keiji Oguma*,2,
Tetsuya Ueno3, Hiroyuki Sunagawa1, and Tohru Ohyama1
1Hokkaido

Instituteof Public Health, Sapporo, Hokkaido 060, Japan, 2Departmentof Bacteriology, Okayama UniversityMed-

ical School, Okayama,

Gunma 374, Japan
Received

Okayama

July 14, 1995; in revised

700, Japan,

form,

September

and 3Research

25, 1995.

Laboratory,

Accepted

Toa Pharmaceutical

October

Company

Ltd., Tatebayashi,

2, 1995

Abstract: A polymerase chain reaction (PCR)-based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins. In this procedure, the PCR products were
easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected. The specific PCR products were obtained from toxigenic C. botulinum types
A to F, a type E toxin- producing C. butyricum strain, and a type F toxin-producing C. baratii strain, but no
PCR product was detected in nontoxigenic strains of C. botulinum and other clostridial species. The neurotoxin genes were also detected in food products of a seasoned dry salmon and a fermented fish (Izushi)
which had caused type E outbreaks of botulism. Therefore, it is concluded that this PCR- based detection
method can be used for the rapid diagnosis of botulism.
Key words: PCR, Clostridium

botulinum,

Toxin gene

Clostridium botulinum neurotoxins are one of the

most poisonous toxins in the world, and they sometimes cause the food-borne botulism with high lethal
rates. Therefore,a rapid diagnosisand appropriatetreatment are required. At present, the diagnosisof botulism
is confirmedby clinical symptoms, detection and identificationof botulinumtoxinsby mousebioassay,and isolation and identification of C. botulinum organisms.
However, mouse bioassay and colony isolation procedures take several days to complete and it is recommended not to use the animals for diagnosis bacause
of ethical problems. Recently, the DNA sequences of
botulinumtypeA to G toxin geneshave beendetermined
(1-4, 9-11, 13, 14, 16-18), and the polymerase chain
reaction (PCR) detecting botulinum type A to G genes
has beenestablished(5-7, 15). However,the lengthsand
the sites of amplified gene fragments were different
dependingon the studies,and no one used the restriction
enzymedigestion profiles of the amplifiedproducts for
confirmingthe type of toxin genes.
In this manuscript, establishment of the PCR procedure specificallyamplifyingapproximately300 by of the

light

chain

toxin

component

genes,

cific

of

which

have

endonuclease

of

toxin

each

type

only

one

is described.

genes

in

two

of

In

cases

botulinum

cleavage

of

A to

site

addition,

for

the

food-borne

a spe-

detection

botulism

is

described.

Materials

and

Bacterial
ulinum

types

and

C.

(Table

1).

DNA
overnight

11

in

pH

trifugation

30

C and

neurotoxigenic
each

ml

then

of

frozen

medium

at 10,000 ~

2%

glucose,
C.

The
g

for

0.1%
cells
10

were
min

used

cooked

meat
U.S.A.)

C until

strains

LYG

were

Mich.,

at -20

Laboratories),

at 30

in

Detroit,

were
(1%

yeast

use.

cultured

lactalbumin
extract

cysteine

(Difco

hydrochlo-

collected
at 4 C,

bot-

nontoxibutyricum

strains

cultured

Bacterial

250

of
C.

clostridial

were

C.

strain

neurotoxigenic

other

strains

0.5%
7.2)

16

Laboratories,

(Difco

Laboratories),

of
one

and

preparation.

hydrolysate

ride,

E,

and
These

at

total

G strains,

and

(Difco

5 days

to

baratii,

medium
for

strains.

type

genic

Methods

by

suspended

cenin

Abbreviations: EDTA, ethylenediaminetetraacetic acid; MLD,

minimum lethal dose; PCR, polymerase chain reaction; SDS,
sodium dodecyl sulfate; TE buffer, Tris HCl-EDTA buffer; Tris,
tris (hydroxymethyl) aminomethane.

*Address correspondence to Keiji Oguma, Department of

Bacteriology,Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, Okayama 700, Japan.
5

K. TAKESHI

Table

20

ml

col

1. Source

of

LYG

4000

medium

(Nakarai

penicillin
of

Tesque,

at

centrifugation,

the

sodium

sulfate

were

dodecyl
extracted

Marmur

were

to

lysed

(SDS).

purified

60

The
by

sequences
shown

Type-specific

genes

were

reported
in Table

Applied

Mo.,

U.S.A.),

and

2.5

min.

with

procedure

380A,

2.

designed

Elmer

After

to

3%

of

by

These

(1,

primers

City,

purification

4,

were

on
8,

for
the
11,

Calif.,

14,

18),
on

and
(Applied

PCR

and

annealing

microliters

electrophoresis

an

solution
by

of

312

Kyoto,

electrophoresis.

reaction

mixture

The
containing

PCR

was
25

performed
ng

of

template

in

and

in

2.0%

gel

was

U.S.A.),

at

agarose

out

for

C for

25

1 min
extension.

subjected
an

to

mini-size

(Cosmobio
an

the

Perkin-Elmer

with

Mupid

then

(Perkin-

was

gel

with

dNTP,

using

carried

72

nm
8.3),

denaturation,

products

of

and

was
C for

each

by

PJ2000,

1 min

PCR

stained

with

enzyme

products
then

nm

polymerase

100

Corp.,

ethidium

bromide

photographed

at

a Transilluminator

a wave-

(Funakoshi

Japan).

Restriction
PCR

the

(1 g/ml)

length
Ltd.,

Biosys-

and

of

system
The

DNA

at 94

C,
(pH

of

Calif.,

procedure

1 min

at 95

Tris-HCl

200 m

(Model

PCR
of

10 min
mM

gelatin,

cycler

The

C for

10

Norwalk,

consisting

Tokyo).

Incorp.).

a 100111

Corp.).

for

KCl,

AmpliTaqTm

thermal

as

Mate,

purified

of
Corp.,

electrophoresis

gene

(PCR

U.S.A.),

cartridge

toxin

synthesized

Synthesizer

Ten

detecting

preheated
mM

100 g/ml

units

GeneAmp

cycles

been
50

Cetus

Cetus

DNAs
reported

sets

based

DNA

Foster

oligonucleotide

primer

previously

Biosystems

Model

MgCl2,

mm

90

had

primer,

1.5

chromosomal

the

of

which

each

102 g/ml

Louis,

for

U/ml

of

at 55

primers.

to F toxin

tems

cells

103

(12).

PCR
A

and

DNA

gly-

and

Tokyo)

St.

30

polyethylene

Japan),

Ltd.,

Chemial,

incubated

8%

Kyoto,

Seiyaku

(Sigma

then

of cultures

containing

G (Banyu

lysozyme

and

and origin

ET AL

were

resuspended

analysis
concentrated
in

10 l

of

PCR

by
of

ethanol
TE

products.

The

precipitation
buffer

(10

mM

DETECTION
Table 2. Sequences

Tris-HC1,

1 mM

concentrated

for

1 hr

the

treated

a 4%

agarose

sequences

were

subjected

mg/ml

salmon

specific
also

(1 ~

3).

SSC-0.1%

food

at 37

been
sterile

Inc.,

mM

and

of PCR.

botulinum
(or

of

amplified
ethidium
with

out

M Na,

citrate

at 65
plus

6 ~

SSC-1%
100 g/ml
of

SSC-0.1%

min

gel

and

the

Mass.,

and

C for

0.15

SDS-5 ~

Den-

salmon

SDS

sperm
with

2 ~

at room

tem-

and

for

6.0)

was

of

mixed

each

sera

purchased

Japan).

After

mixture

was

The

20

10

template

was
the

of

Chiba

incubation

at 37

two
Type

test.

Toxin

(MLD)

U/ml

per

of

1 hr,

ml

antitoxin

Institute

C for

(Chiba,
0.5

injected

into

for

6 days.

Fragments

by

observed

steps,
into

amount.

dose
10

Serum

intraperitoneally
were

10-fold

injected

toxin

lethal

volume

was
buffer

centrifugation,
in

neutralization

minimum
equal

mice

of
min

products

After

detect
by

from

injected

ml
10

M phosphate

diluted

dilution
to

with

had

g,

as

food

0.2

gelatin.

determined
of

were

used

of

sterile

serially

intraperitoneally
toxin

40
for

(10,000 ~

were

grams

of

0.2%

ml

heated

and

in

were

type

1990,

centrifuged

Ten
ml

were

0.5

and

flat-

of

PCR.

containing

then

1989

(25 l)

10

supernatants

fermented

oubreaks

homogenized

then

bioassay.
in

in

were

and

the

mixtures

supernatants

preparations

carried

C,
The

solutions

mice
of

30

cooled,

The

(pH

mem-

was

each

C,

homogenized

solution-100

hybridization

Template
type

0317011

serial

carried

and

EDTA-

0.2 ~

Westborough,

Denhardt's

washes

pi)

Japan

buffer.

Mouse

(10

nylon

95

salmon

caused

Hokkaido,
at -

TE

dry
had

ml

of

two

the

mice.

given.

Sensitivity

wavelength

in

agarose

-pore

kept

DNA

toxin

which
in

min).
of

the

products

in a 2%

DNA,

stringency

were

botulism

on

products
on

seasoned

(Izushi),

of

products.

PCR

PCR

0.45-m

is 0.015

38

PCR

the

BY PCR

hybrydization

products,

fish

10 l

based

The

SDS-5 ~

at

SDS,

diluting

for

was

SSC

solution-20

C.

the

electrophoresis

prepared

Separations,

sperm

then

RIMD

of
restriction

Japan)

of

GENES

used in Southern

at

7.0)-0.5%

perature

to

to electrophoresis

overnight

DNA,

with

Thereafter,

analysis

Prehybridization

hardt's

probes

microliter

Kyoto,

10 l.

a MagnaGraph

SSC

pH

PCR

Ltd.,

subjected

(Table

to

NaC1,

by

was

were

(Micron

hr in 6 ~

treated

of

probes

gene

U.S.A.).

One

was

TOXIN

used in the PCR

gel.

type

on

8.0).

Syuzo

hybridization

toxin

from

pH

products

volume

Oligonucleotide

was

oligonucleotide

solution

each

out

Table 3. The internal

EDTA,

a total

Southern

brane

primers

(Takara
in

blotted

of the oligonucleotide

PCR

endonuclease

OF BOTULINUM

260

bromide
food

and

Iwanai),
nm

in

10-fold
by

DNA

PCR,

E strains,
were

the

staining
products.

obtained

A-190

adjusted

to

spectrophotometer.

steps,
and

solutions

each
their

after
Ten

and
1.0

Eat

preparation

Amplification

were

of

each

The

detected

products
igenic

of

of

PCR

clostridial

electrophoresis.
grams

After

diluted

products

Results

the

the

were

of

Toxin

Genes

performed

strains

strains
type

was

by

on

using

demonstrated
from
primer

the

primer

only

which

the

sets

were

DNAs

sets.

when

DNAs
identical.

PCR

from

were

different
The

the

type

PCR
of

tox-

extracted
The

products

and

K. TAKESHI
1

Fig.

1.

out

(BRL,

5,

with

CS-11/CS-22;

C-CB19

D-South

DNA

lane

12,

BL

3112

1873,
bands

F-Langeland
DNA

those

11,
DNA

with

E-Sapporo,
to

electrophoresis.
Md.,

the

DS-11/DS-22;
C.

with

F-Denmark
respective

FS-11/FS-22;
DNAs

6,

also
toxin

produced
type

DNA
with

C-468

9,

BL
with

DNA
lane

E-Iwanai

5262

DNA

10

11

12

13

Fig. 2. Digestion patterns of PCR products with restriction

enzymes. Lane 1, BioMarkerTMLow (Funakoshi Ltd., Kyoto,
Japan); lane 2, 283 by product amplified from A-190; lane 3,
digestion of the lane 2 preparation with Hpa I; lane 4, 315 by
product amplified from B-Lamanna; lane 5, digestion of the lane
4 preparationwith Ssp I; lane 6, 290 by product amplified from CStockholm; lane 7, digestion of the lane 6 preparation with Rsa I;
lane 8, 497 by product amplified from D-South African; lane 9,
digestion of the lane 8 preparation with Rsa I; lane 10, 266 by
product amplified from E-Iwanai; lane 11, digestionof the lane 10
preparation with Aha III; lane 12, 332 by product amplified from
F-Langeland; lane 13, digestion of the lane 12 preparation with
Aha III.

ES-11/ES-22;

lane
from

1 kbp

CS-11/CS-22;
lane

DNA

by

1,

CS-11/CS-22;

butyricum

(E)-Sapporo

FS-11/FS-22.
and

of

with

was

meth-

DNA

lane

DNA

10,

in

2, A-190

with

CS-11/CS-22;

detected

B-Lamanna

DNA

(c)-A02

PCR

Lane
lane

13

Clostrid-

was

U.S.A.);
3,

PCR.

described

product

lane

12

different

as

after

lane
lane

pairs

PCR

with

ES-11/ES-22;

ES-11/ES-22;

from

primer

11

by

each

with
7,

10

fragments

C-Stockholm

lane

African

with

with

DNA

extracted

of

Bethesda,

4,

gene

AS-11/AS-22;
lane

lane

of

staining

with

BS-11/BS-22;

DNA

type

(10 l)

Ladder

amplified

(25 l)

each

bromide

DNA

8,

DNAs

and

One-tenth

ethidium

of toxin

with

cultures

ods.

Amplification

carried
ium

ET AL

13,

A-901,
the
DNAs

C.

baratii

B-17,
same
(data

Dsized
not

shown).

on the agarose gels of type A, B, C, D, E, and F C. botulinum toxigenic strains were 283, 315, 290, 497, 266,
and 332 bp, respectively(Fig. 1). The DNAs from neurotoxigenicC. butyricumstrain BL 5262, and C. baratii
BL 3112 also demonstrated the same sized products as
those of the type E and F strains, respectively. On the
contrary, with the DNAs extracted from the toxigenic
strainsof C. argentinens(or C. botulinumtype G), G-89
and G-2740, nontoxigenic strains of (C)-A02, (E)-Sapporo, and 11 strains of other clostridia listed in Table 1,
no PCR product was obtained even though the primer
sets for all types were employed.
Identificationof the PCR Products
Identificationof the PCR productswere confirmedby
digestion of the amplified fragments by restriction
enzymes (Fig. 2). Digestion patterns were in agreement with the sizes estimated from the sequence data;
type A (283 bp) showed two restrictionfragmentsof 246
and 37 by by Hpa I, type B (315 bp) showed 167 and
148 by by Ssp I, type C (290 bp) showed 155 and 135 by
by Rsa I, type D (497 bp) showed 332 and 165 by by Rsa
I, type E (266 bp) showed 184 and 82 by by Aha III, and
type F (332 bp) showed 220 and 112 by by Aha III.
The PCR products were also confirmed by Southern

hybridization test using type-specific internal oligonucleotide probes (Fig. 3). These results clearly demonstrated that our type-specific primers were capable of
amplifying the sequences from corresponding toxin
types with high specificity.
Sensitivityof PCR
PCR was performed with serially diluted type A (A190) and E (E-Iwanai) DNAs. The lowest amount of
templateDNAs of type A and E whichproducedobservable products on agarose gels was 2.5 pg (Fig. 4). The
DNAs of other types also showed similar sensitivityon
the PCR with 25 cycles.
Amplificationof ToxinGene Fragments byPCR in Food
Products
PCR was performedwith the two food products(seasoned dry salmon and fermented flatfish) which had
caused the outbreaks of type E botulism in 1989 and
1990. As controls, commercially sold fermented fish
products (Izushi)which showed no toxigenicityin mice
were also checked. Both the food products demonstrated the band of 266 by by PCRwith the typeE primerset,
that was digested into 184 and 82 by by Aha III (Fig.5).
No gene fragmentswere amplifiedwith the other primer
sets. Also, no toxin gene fragments were amplified
from commercially sold Izushi by any of the primer
sets.
Toxin amounts and toxin types in these two food
products were also determined by bioassay with mice.

DETECTION
a)

OF BOTULINUM

TOXIN

GENES

BY PCR

c)

b)

d)

g)

f)

e)

Fig. 3. Southern hybridization with type-specific oligonucleotide probes. PCR products on ethidium bromide-stained gel is shown in
panel a). Lane M, 1 kbp DNA Ladder (BRL); lane A, 283 by product amplified from A-190; lane B, 315 by product amplified from BLamanna; lane C, 290 by product amplified from C-Stockholm; lane D, 497 by product amplified from D-South African; lane E, 497
by amplified from E-Iwanai; lane F, 332 by product amplified from F-Langeland. These PCR products were reacted with labelled typespecific oligonucleotide probes. Panel b), type A-specific probe; panel c), type B-specific probe; panel d), type C-specific probe; panel
e), type D-specific probe; panel f), type E-specific probe; panel g), type F-specific probe.
1

10

11

12

13

14

15
1

Fig. 4. Sensitivity of PCR. PCRs were performed on serial 10fold diluted template DNAs from A-190 and E-Iwanai. Lane 1,
BioMarkerTm
Low; lane 2-8, A-190 DNAs from 25 ng (lane 2) to
250 fg (lane 8); lane 9-15, E-Iwanai DNAs from 250 ng (lane 9)
to 250 fg (lane 15).
The 1-g preparations of a seasoned dry salmon and fermented flatfish products demonstrated
20 and 40
MLD/ml without the activation by trypsin treatment,
respectively. The toxin amounts in the foods were similar to those assayed in 1989 and 1990, and they were
neutralized by antiserum against type E toxin.
Discussion
PCR was performed on the DNAs obtained from toxigenic botulinum type A to F strains, and neurotoxi-

10

11

12

13

Fig. 5. Amplification of toxin gene fragments by PCR in food

products. Existence of toxin gene fragments were analyzed by
PCR in contaminated food products, seasoned dry salmon (lanes
2-8) and fermented flatfish (lane 9). Lane 1, BioMarkernTM
Low;
lanes 2-5, negative for the PCR products amplified with AS11/AS-22 (lane 2), BS-11/BS-22 (lane 3), CS-11/CS-22 (lane
4), DS-11/DS-22 (lane 5) and FS-11/FS-22 (lane 8); lane 6,266
by product amplified with ES-11/ES-22; lane 7, digestion of the
lane 6 preparation by Aha HI; lane 9,266 by product amplified
from fermented flatfish with ES-11/ES-22; lanes 10-13, negative
for the PCR products amplified from commercially sold fermented flatfish with ES-11/ES-22.

genic C. butyricum and C. baratii strains by using typespecific primer sets. In these preparations,
the specific
PCR products were observable
on agarose gels only in
the

type-matched

combinations

of the DNA

prepara-

K. TAKESHI

10

tions and the primer sets. On the contrary, no PCR

product was obtained with the DNAs from nontoxigenic strainsof C. botulinumand other clostridialspecies
even though all of the primer sets were employed.
In the PCR previouslyperformedfor detectingtypeA
to G toxin genes, the amplified products were sometimes very large (more than 1.3kbp), and none employed
the restrictionenzyme digestion profiles in order to confirm the PCR products. This time, we designed the
primer sets to amplify approximately 300 by products
which could be separated into two fragments by the
specificenzyme digestion. Therefore,the gene products
were easily confirmed by their digestion profiles rather
than by performing the hybridization test with specific
probes. The time requiredfor the PCRwas less than 3 hr.
The sensitivity of our procedure was similar to those
reported previously (7, 15); 2.5 pg of template DNAs
(approximately875 cells) could be detected by staining
their amplified products on agarose gels with ethidium
bromide. Of course, the detection sensitivity was
increased about 100 times by Southern hybridization
with the type-specificprobes (data not shown).
The usefulness of PCR was also confirmed in the
two specimens of food-bornebotulism; one was caused
by a seasoned dry salmon in 1989 and the other by a
fermentedflatfish(Izushi)in 1990. The toxin amountsin
the contaminated foods were only 20 to 40 MLD/g.
However, the toxin gene fragments were amplifiedby
PCR from these foods, and the amplified toxin gene
types were identicalto thoseestimatedby the mouseneutralization test. From these data, it is concluded that the
developed PCR procedureis rapid, specific,and reliable
for identifyingthe C. botulinumtype A to F toxin genes,
and that it can be used for diagnosis of food-bornebotulism in addition to or instead of isolation of organisms
and identification of toxin in mice.
We are grateful

to Kiyomi

Hamada

for her technical

ET AL

Biochim. Biophys. Acta 1216: 487-491.

4) East, A.K., Richardson, P.T., Allaway, D., Collins, M.D.,
Roberts, T.A., and Thompson, D.E. 1992. Sequence of the

5)

6)

7)

8)

9)

10)

11)

12)

13)

assistance.

References

14)

1) Binz, T., Kurazono,H., Wille,M., Frevert,J., Wernars,K.,

and Niemann, H. 1990. The complete sequence of botulinumneurotoxintypeA and comparisonwithotherclostridial neurotoxins.J. Biol, Chem.265: 9153-9158.
2) Binz, T., Kurazono,H., Popoff,M.R.,Eklund,M.W.,Sakaguchi, G., Kozaki, S., Krieglstein,K., Henschen,A., Gill,
D.M., and Niemann,H. 1990. Nucleotidesequenceof the
gene encoding Clostridiumbotulinumneurotoxintype D.
NucleicAcids Res. 18:5556.
3) Campbell, K., Collins, M.D., and East, A.K. 1993.
Nucleotidesequence of the gene coding for Clostridium
botulinum(Clostridiumargentinense)type G neurotoxin:
genealogicalcomparisonwithother clostridialneurotoxins.

15)

16)

gene encoding type F neurotoxin of Clostridium botulinum.

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evaluation in food samples. Appl. Environ. Microbiol. 61:
389-392.
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organisms. J. Clin. Microbiol. 32: 1911-1917.
Ferreira, J.L., Baumstark, B.R., Hamdy, M.K., and Mccay,
S.G. 1993. Polymerase chain reaction for detection of type A
Clostridium botulinum in foods. J. Food Prot. 56: 18-20.
Fujii, N., Kimura, K., Yashiki, T., Indoh, T., Murakami, T.,
Tsuzuki, K., Yokosawa, N., and Oguma, K. 1991. Cloning of
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DETECTION

OF BOTULINUM

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17) Whelan, S.M., Elmore, M.J., Bodsworth, N.J., Atkinson,
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TOXIN

GENES

BY PCR

11

18) Whelan, S.M., Elmore, M.J., Bodsworth, N.J., Brehm, J.K.,

Atkinson, T., and Minton, N.P. 1992. Molecular cloning of
the Clostridium botulinum structural gene encoding the type
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sequence. Appl. Environ. Microbiol. 58: 2345- 2354.