Abstract

A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba813 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus anthracis and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide C1 was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba813 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anthracis.

References

[1]
Gordon
R.E.
Haynes
W.C.
Pang
C.H.-N.
(
1973
)
The genus Bacillus
.
United States Department of Agriculture, Agricultural Handbook No. 427
 .
U.S. Government Printing Office
,
Washington, DC
.
[2]
Carlson
C.R.
Caugant
D.A.
Kølsto
A.-B.
(
1994
)
Genotypic diversity among Bacillus cereus and Bacillus thuringiensis strains
.
Appl. Environ. Microbiol.
 
60
,
1719
1725
.
[3]
Priest
F.G.
Goodfellow
M.
Todd
C.
(
1988
)
A numerical classification of the genus Bacillus
.
J. Gen. Microbiol.
 
134
,
1847
1882
.
[4]
Mikesell
P.
Ivins
B.E.
Ristroph
J.D.
Dreier
T.M.
(
1983
)
Evidence for plasmid-mediated toxin production in Bacillus anthracis
.
Infect. Immun.
 
39
,
371
376
.
[5]
Green
B.D.
Battisti
L.
Koehler
T.M.
Thorne
C.B.
Ivins
B.E.
(
1985
)
Demonstration of a capsule plasmid in Bacillus anthracis
.
Infect. Immun.
 
49
,
291
297
.
[6]
Uchida
I.
Sekizaki
T.
Hashimoto
K.
Terakado
N.
(
1985
)
Association of the encapsulation of Bacillus anthracis with a 60 megadalton plasmid
.
J. Gen. Microbiol.
 
131
,
363
367
.
[7]
Kaneko
T.
Nozaki
R.
Aizawa
K.
(
1978
)
Deoxyribonucleic acid relatedness between Bacillus anthracis and Bacillus cereus and Bacillus thuringiensis
.
Microbiol. Immunol.
 
22
,
639
641
.
[8]
Böhm
R.
Späth
G.
(
1990
)
The taxonomy of Bacillus anthracis according to DNA-DNA hybridization and G+C content
.
Salisbury Medical Bulletin No. 68 Special Supplement
 ,
29
31
.
[9]
Turnbull
P.C.B.
Hutson
R.A.
Ward
M.J.
Jones
M.N.
Quinn
C.P.
Finnie
N.J.
Duggleby
C.J.
Krammer
J.M.
Melling
J.
(
1992
)
Bacillus anthracis but not always anthrax
.
J. Appl. Bacteriol.
 
72
,
21
28
.
[10]
Priest
F.G.
Kaji
D.A.
Rosato
Y.B.
Canhos
V.P.
(
1994
)
Characterization of Bacillus thuringiensis and related bacteria by ribosomal RNA gene restriction fragment length polymorphisms
.
Microbiology
 
140
,
1015
1022
.
[11]
Chevrier
D.
Rasmussen
S.R.
Guesdon
J.-L.
(
1993
)
PCR product quantitation by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells
.
Mol. Cell. Probes
 
7
,
187
197
.
[12]
Sambrook
J.
Fritsch
E.F.
Maniatis
T.
(
1989
)
Molecular Cloning: a Laboratory Manual
 .
Cold Spring Harbor Laboratory Press
,
Cold Spring Harbour, NY
.
[13]
Hohn
B.
Collins
J.
(
1980
)
A small cosmid for efficient cloning of large DNA fragments
.
Gene
 
11
,
291
298
.
[14]
Hohn
B.
Murray
K.
(
1977
)
Packaging recombinant molecules into bacteriophage particles in vitro
.
Proc. Natl. Acad. Sci. USA
 
74
,
3259
3263
.
[15]
Feinberg
A.P.
Vogelstein
B.
(
1983
)
A technique for radiolabelling DNA restriction endonuclease fragments to high specificity activity
.
Anal. Biochem.
 
132
,
6
13
.
[16]
Sanger
F.
Nicklen
S.
Coulson
A.R.
(
1977
)
DNA sequencing with chain-terminating inhibitors
.
Proc. Natl. Acad. Sci. USA
 
74
,
5463
5467
.
[17]
Fouet
A.
Sonensheim
A.L.
(
1990
)
A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis
.
J. Bacteriol.
 
172
,
835
844
.
[18]
Southern
E.M.
(
1975
)
Detection of specific sequences among DNA fragments separated by gel electrophoresis
.
J. Mol. Biol.
 
98
,
503
517
.
[19]
Rasmussen
S.R.
Larsen
M.R.
Rasmussen
S.E.
(
1991
)
Covalent immobilization of DNA onto polystyrene microwells: The molecules are only bound at the 5′ end
.
Anal. Biochem.
 
198
,
138
142
.
[20]
Harrell
L.J.
Andersen
G.L.
Wilson
K.H.
(
1995
)
Genetic variability of Bacillus anthracis and related species
.
J. Clin. Microbiol.
 
33
,
1847
1850
.
[21]
Henderson
I.
Duggleby
C.J.
Turnbull
P.C.
(
1994
)
Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR
.
Int. J. Syst. Bacteriol.
 
44
,
99
105
.
[22]
Chevrier
D.
Popoff
M.Y.
Dion
M.P.
Hermant
D.
Guesdon
J.-L.
(
1995
)
Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate
.
FEMS Med. Microbiol. Immunol.
 
10
,
245
252
.
[23]
Makino
S.-I.
Iinuma-Okada
Y.
Maruyama
T.
Ezaki
T.
Sasakawa
C.
Yoshikawa
M.
(
1993
)
Direct detection of Bacillus anthracis DNA in animals by polymerase chain reaction
.
J. Clin. Microbiol.
 
31
,
547
551
.
[24]
Johns
M.
Harrington
L.
Titball
R.W.
Leslie
D.L.
(
1994
)
Improved methods for the detection of Bacillus anthracis spores by the polymerase chain reaction
.
Lett. Appl. Bacteriol.
 
18
,
236
238
.
[25]
Carl
M.
Hawkins
R.
Coulson
N.
Lowe
J.
Robertson
D.L.
Nelson
W.M.
Titball
R.W.
Woody
J.N.
(
1992
)
Detection of spores of Bacillus anthracis using the polymerase chain reaction
.
J. Infect. Dis.
 
165
,
1145
1148
.
[26]
Reif
T.C.
Johns
M.
Pillai
S.D.
Carl
M.
(
1994
)
Identification of capsule-forming Bacillus anthracis spores with the PCR and a novel dual-probe hybridization format
.
Appl. Environ. Microbiol.
 
60
,
1622
1625
.
[27]
Ramisse
V.
Patra
G.
Vaude-Lauthier
V.
Sylvestre
P.
Thérasse
J.
Guesdon
J.-L.
(
1996
)
Multiplex PCR assay for identification of Bacillus anthracis and differentiation from Bacillus cereus group bacteria
.
Salisbury Medical Bulletin No. 87 Special Supplement
 ,
S1
.