316
Anthrax (Bacillus anthracis), Molecular Biology of
RQ
RQ
R
R
R
Q
Q
Q
RQ
1.
2.
3.
4.
Fig. 5
5
0
nuclease real-time PCR assay.
1. A probe complementary to the target
sequence is labeled with a fluorescent
reporter (R) and a quencher (Q). At the
beginning of the reaction, the close
proximity of these molecules allows the
quencher to act on the reporter and little
fluorescent signal can be detected. 2. As
the target sequence is ampliFed, the
probe binds to it. 3. The Taq DNA
polymerase in the reaction uses its 5
0
to
3
0
nuclease activity to degrade the
probe. 4. As the probe is degraded, the
reporter and quencher separate and the
quencher no longer acts on the reporter,
leaving it free to fluoresce. This signal is
used as a measure of the amount of
probe that has been degraded, which is
directly related to the amount of target
sequence in the reaction.
analysis
would
not
be
useful
for
the
identiFcation
of
B. anthracis
.
However,
this conclusion was based on a limited
number of 16S rRNA sequences. During
the
investigation
of
the
outbreak,
the
16S rRNA sequences from a number of
B. anthracis, B. cereus
,and
B. thuringiensis
strains were determined, and it was found
that, indeed, it was possible to distinguish
between the species. All bacillus strains
in this study, including 86
B. anthracis
strains, possessed 16S rRNA types that
were unique to their species. In Genbank,
there are, however, 3 16S rRNA sequences
from
B. anthracis
that fall into a type that
was exclusively found in
B. cereus
in this
study. The explanation for this discrepancy
is unclear at this point, and it may be
due
to
a
methodological
or
an
actual
biological difference.
Real-time PCR has also proven effec-
tive for the rapid identiFcation of anthrax.
The method that has been used is a 5
0
nuclease assay, which exhibits increased
sensitivity compared to a traditional PCR
method (±ig. 5). In addition to primers, 5
0
nuclease assays use a fluorescently labeled
probe that is complementary to the target
sequence. This probe is labeled with a re-
porter fluorophore as well as a quencher
for this reporter. When the probe is added
to the reaction, the quencher and reporter
are in close proximity and little fluorescent
signal is detectable. However, as the tar-
get sequence is ampliFed and the probe
binds to it, the 5
0
to 3
0
exonuclease activ-
ity of the Taq polymerase in the reaction
degrades the probe, releasing the reporter
fluorophore from its quencher and leading
to an increase in the fluorescent signal. In
the case of
B. anthracis
, three primer and
probe sets were used; one each to detect
pXO1, pXO2, and the
B. anthracis
chromo-
some. All fully virulent
B. anthracis
strains
tested were positive for all three targets,
yielding 100% sensitivity for virulent bac-
teria. ±urther, none of the non-
Bacillus.
anthracis
strains, including
B. cereus
and
B. thuringiensis
, tested positive for all three
targets, giving 100% speciFcity, as well.
The lower limit of detection with this assay
is estimated to be 1 pg of DNA or approx-
imately 5 to 10 spores.
B. anthracis
strains
lacking one or the other virulence plasmid
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