Jpn. J. Infect. Dis., 55, 2002

Laboratory and Epidemiology Communications

Amplification of Irrelevant Sequence from Bacillus subtilis
Using a Primer Set Designed for Detection ofthe pag Gene of Bacillus anthracis
Osamu Fujita, Satoshi Inoue, Masashi Tatsumi, Tsuneo Kamiyama, Shoichi Akaishi1,
Tomoko Ootani1, Tsuneaki Kawai1, Takashi Hirochi1, Yumiko Sakamoto1,
Kazumichi Tamura2, Haruo Watanabe2 and Akio Yamada*
Department of Veterinary Science andDepartment of Bacteriology I, National Institute of Infectious Diseases,
1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640 and
1
Sapporo City Institute of Public Health,
Kikusui 9-1-5-22, Shiroishi-ku, Sapporo, Hokkaido 003-8505

2

Communicated by Kazuo Kato
(Accepted July 26, 2002)

that the sample may contain bacteria harboring a pXO1
pathogenic plasmid. We have thus attempted to determine
whether or not the bacteria were indeed B. anthracis. First,
microplate hybridization (kindly provided by Dr. Matsunaga
of Wakunaga Pharmaceutical Co., Ltd., Hiroshima) wasemployed for detection of the Ba813 chromosomal gene thatwas thought to be anthrax-specific (2). The results revealed
no significant signals, suggesting that the bacteria did nothave sequences homologous to Ba813. Second, real-time PCR
was performed using a LightCycler-Bacillus anthracis
Detection Kit (Roche Diagnostics, Tokyo). No anthrax-specificsignals were detected when the sample was tested for thepresence of pag and cap genes. We then sequenced the amplicon
after cloning it into the pCR2.1 plasmid. The nucleotide
sequence showed substantial homology with B. subtilis ATPdependent deoxyribonuclease, but no homology with any
known B. anthracis genes was determined. An increase in
the annealing temperature had no effect on the production of

Anthrax is one of the major bacterial zoonoses caused byspore-forming Gram-positive rods, Bacillus anthracis. Afterthe bioterrorist attack in the USA in September 2001, theprobability of anthrax attacks has increased worldwide, andthe number of hoaxes involving “white powder” has increasedin Japan as well. Since rapid and precise diagnosis is the mostpowerful measure to counter bioterrorism, it is of particularly
importance to have a diagnostic tool to detect anthrax.
Polymerase chain reaction (PCR) is regarded as one of suchdiagnostic method for the detection of anthrax, althoughdefinitive diagnosis can only be made when other methods
support the PCR results. The PCR method recommended by
the World Health Organization (WHO) is widely accepted inJapan as the standard approach. A presumptive diagnosis ofanthrax is made if bands with predicted sizes are detected
after PCR with primer sets targeted to pag and cap genes
residing on pXO1 and pXO2 plasmids, respectively (1).
On March 27, 2002, “white powder” was found disseminatedinside a public restroom located in Sapporo City. The powder
was subjected to microbiologic tests and PCR at the Sapporo
City Institute of Public Health directly and after cultivationin TSB medium. Although no bacterium was found when thesample was directly examined, Gram-positive, spore-forming
bacilli were detected after cultivation in TSB medium (Table 1).
Since the bacteria were motile and hemolytic, the possibility
that isolated bacteria were B. anthracis was ruled out; however,the PCR with primers designed for detection of the pag gene
resulted in amplification of a 596 bp fragment (Fig. 1). The
presence of the PCR amplicon of this size usually indicates

Table 1. Characteristics of bacteria isolated after cultivationin TSB media
Smear

Gram stain
Spore
CapsulePCR

PagCapIsolation*Corresponding author: Tel: +81-3-5285-1111 ext. 2601, Fax:
+81-3-5285-1179, E-mail: yamada@nih.go.jp

Blood Agar
NGKG agar
BCA**Bacillus cereus Agar

99

Gram +, rods+–+–
hemolytic
lecithinase+, Medusa’s headlecithinase+, Medusa’s head-Jpn. J. Infect. Dis., 55, 2002tests indicated that the bacteria isolated belonged to the B.
subtilis group (data not shown). These findings indicate that
the PCR protocol recommended by WHO is not sufficiently
specific, unless the internal nucleotide sequence is determined,
since there remains the possibility that a bacterium exists,
the genome of which contains sequence(s) amplifiable by theprimers. Moreover, the size of the amplified fragments isindistinguishable from those amplified from B. anthracis.
This article appeared in the Infectious Agents Surveillance
Report, vol. 23, no. 7, 2002 in Japanese.
REFERENCES
1. World Health Organization (1998): Guidelines for thesurveillance and control of anthrax in human and animals.
3rd ed. World Health Organization, Geneva.
2. Patra G., Sylvester, P., Ramisse, V., Therasse, J. andGuesdon, J.-L. (1996): Isolation of a specific chromosomic
DNA sequence of Bacillus anthracis and its possible usein diagnosis. FEMS Immunol. Med. Microbiol., 15, 223231.

Fig. 1. Effect of annealing temperature on amplification of fragmentsfrom the Sapporo sample.
M: 100 bp Ladder
S: Sapporo Sample
P: Positive control (B. anthracis Pasteur II)

these bands when the sample was subjected to PCR using
the same pag primer set (Fig. 1). The results of biochemical100