Jpn. J. Infect. Dis., 57, 29-32, 2004
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Short Communication
Preparation of a Positive Control DNA for
Molecular Diagnosis of Bacillus anthracis
Satoshi Inoue*, Akira Noguchi, Kiyoshi Tanabayashi and Akio
Yamada
Department of Veterinary Science, National Institute of Infectious
Diseases, Tokyo 162-8640, Japan
(Received October 16, 2003. Accepted December 25, 2003)
*Corresponding author: Mailing address: Laboratory of Transmission
Control, Department of Veterinary Science, National Institute
of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640,
Japan. Tel: +81-3-5285-1111 ext. 2620, Fax: +81-3-5285-1179, E-mail:
sinoue@nih.go.jp
SUMMARY: Bacillus anthracis is considered to be
one of the most potent biological weapons because of its highly
pathogenic nature and efficiency of transmission. Routinely, a
presumptive diagnosis of anthrax is achieved if the bands with
predicted sizes are detected after the PCR targeted to the pag
and cap genes residing on pXO1 and pXO2 plasmids, respectively.
A positive control DNA prepared from the standard strains of B.
anthracis (PAI and PAII) is usually included in the PCR tests.
The handling of living B. anthracis, however, requires
physical containment. The inclusion of DNA from B. anthracis
as a positive control in the PCR test also has a potential risk
of cross contamination that may confuse the results. In order
to circumvent such problems, we attempted to construct a recombinant
plasmid harboring the fragments of the pag and cap
genes that could be distinguished from authentic sequences by
the presence the restriction-enzyme site, the EcoRV site
for the pag gene and the BamHI site for the cap
gene, respectively, which were newly introduced. The strategy
reported here provides a safe and reproducible positive-control
DNA template. It also allows the detection of possible cross contamination,
indicating that this strategy would be useful and convenient for
the molecular identification of not only B. anthracis
but also other highly pathogenic microbes.
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