Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-12
2002-09-10
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S023700, C536S024320, C536S024330
Reexamination Certificate
active
06448016
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the detection of
Bacillus anthracis.
More specifically, the invention relates to anthrax-specific polymorphic signature sequences identified on, and isolated from the anthrax chromosome DNA for use as
B. anthracis
-specific markers. Primer sets and hybridization probes designed from these DNA fragment sequences, as well as amplification of the fragments, can be used in a variety of platforms for anthrax detection.
BACKGROUND OF THE INVENTION
Anthrax—primarily a disease of herbivorous animals but of rare occurrence in humans—is caused by Bacillus anthracis. Cutaneous anthrax is acquired via injured skin or membranes, entry sites where the spore germinate into vegetative cells. Proliferation of vegetative cells results in gelatinous edema. Alternatively, inhalation of the spores results in high fever and chest pain. Both types can be fatal unless the invasive aspect of the infection can be intercepted.
Bacillus anthracis
is a biological warfare (BW) agent. Ten grams of anthrax spore can kill as many people as a ton of the chemical warfare agent, sarin. Due to the highly lethal nature of anthrax and BW agents in general, there is great need for the development of sensitive and rapid BW agent detection. Current detection technology for biological warfare agents have traditionally relied on time-consuming laboratory analysis or onset of illness among people exposed to the BW agent.
In theory, the use of specific antibodies or distinguishing DNA probes are the two approaches to modernizing detection technology in this field. However, antibody-based detection of threat agents suffers from drawbacks. For example, interference from other environmental contaminants precludes detection, or detection limits of current levels fail to meet the detection thresholds set by governmental testing protocols. Alternatively, the threat agent, such as with anthrax spore, may be poorly immunogenic.
Since a sample suspected of containing a BW agent like
B. anthracis
could contain such a small yet lethal amount of spores, and an overwhelming amount of other interfering materials, the ability to amplify the agent's genomic material affords a choice of target sites for developing signature probes for specific detection of that agent. Development of highly discriminating techniques are crucial to achieving the stated goals of rapid and sensitive BW detection.
Current PCR-based detection methods of
B. anthracis
rely on the use of primers amplifying tripartite exotoxin genes and/or the polyglutamic capsule genes (Jackson et al, Proc. Natl. Acad. Sci, 95: 1224-9 (1998)). Both sets of genes comprise virulence factors and are located on the two indigenous plasmids of anthrax bacteria, pXO1 (174 kbp; toxin) and pXO2 (95 kbp; capsule). Under normal conditions, the two plasmids in
B. anthracis
do not move across the related bacilli of the “
B. cereus
group” (which is comprised of
B. anthracis, B. cereus, B. thuringiensis
and
B. mycoides
(although
B. mycoides
does not produce toxin and therefore may be grouped differently from the other three members)). However, under certain conditions, these plasmids are known to be transferred from
B. anthracis
to
B. cereus
and
B. thuringiensis
(Ruhfel et al, J. Bact., 157: 708-11 (1984)). Yet
B. cereus
and
B. thuringiensis
containing one or both of these plasmids do not cause anthrax. Therefore, detection of anthrax based solely on virulence factors can give rise to a false-positive determination.
Two chromosomal DNA fragment sequences from
B. anthracis
have been previously identified and used in identifying the presence of
B. anthracis
bacteria. One, designated Ba813, is a 277 bp long DNA fragment (Patra et al.,
FEMS Microbiol.
15: 223-231 (1996)) and the other, vrrA, is a region of sequence variability containing variable repeats (caa tat caa caa) (Anderson et al.,
J. Bacteriol.
178: 377-384 (1996)).
Additionally, Yamada et al (U.S. Pat. No. 6,087,104) identified unique regions of the DNA gyrase sub-unit B (gyrB) gene for each of the closely related bacteria of the
B. cereus
group, and designed oligonucleotide primers corresponding to those unique regions for amplification-based detection methods. However, amplification of DNA segments unique to each of the
B. cereus
group members occurred only when the correct target strain DNA by itself was present in the PCR protocol.
However, since the development of more rapid and more sensitive BW detection methodologies is of such importance to the military as well as public health sectors of the U.S. government, there is great need to continue the process of identifying, cloning, and sequencing of polymorphic DNA markers from chromosomal DNA of threat agents. With this purpose in mind, comprehensive libraries of BW agent-specific signature sequences can be built, and from there useful diagnostic primers and probes can be designed for highly discriminating detection methods. The present invention as herein described fulfills these objectives.
SUMMARY OF THE INVENTION
Accordingly, it is an embodiment of the invention to provide purified and isolated DNA sequences from the chromosomal DNA of
B. anthracis,
as shown in
FIGS. 1
a
-
1
f
and corresponding to SEQ ID NOS: 1-7.
Another embodiment of the invention is the use of these sequences in diagnostic assays to accurately analyze samples for environmental contamination by
B. anthracis
spores and for the early diagnosis of anthrax in human and non-human animals. In this embodiment resides the capability not only to distinguish over closely related Bacillus species, thereby affording more sensitive and reliable detection for anthrax, but also to detect a very minuscule concentration of anthrax spores in the presence of an overwhelming amount of unrelated non-specific environmental DNA.
In yet another embodiment, the invention provides for primer pairs (SEQ ID NOS: 8-15) designed from the aforementioned purified and isolated DNA sequences of the first embodiment. The primer pairs are useful in carrying out PCR amplification-based detection of
B. anthracis.
Alternatively, the invention further provides hybridization probes designed from the novel isolated DNA sequences of the first embodiment, such probes being useful in a number of assay platforms for
B. anthracis.
Kits useful in the practice of the invention are also provided, containing at least one container comprising at least one pair of primers or at least one hybridization probe specifically designed to selectively amplify or bind, respectively, chromosomal DNA of
B. anthracis.
These and other embodiments of the invention will be better appreciated by the following detailed disclosure.
REFERENCES:
patent: 6087104 (2000-07-01), Yamada et al.
Chehab et al. Proceedings of the National Academy of Sciences. (1989) 86: 9178-9182.*
Rastogi. 1999. Proceedings of the ERDCC Science Conference Chemistry, Biology, Defense Research. pp. 515-524.*
Patra, G., et al., “Isolation of a Specific Chromosmic DNA Sequence ofBacillus Anthracisand Its Possible Use in Diagnosis”, FEMS Immunology and Mical Microbiology 15 (1996) pp. 223-231.
Williams, JG, et al, “DNA Polymorphisms Amplified by Arbitrary Primers are usefuls as Genetic Markers”, (Abstract Only), Nucleic Acids Research, vol. 18, Issue 22, pp. 6531-6535.
Jackson, P., et al, “PCR Analysis of Tissue Samples from the 1979 Sverdlovsk Anthrax Victims: The Presence of MultipleBacillus AnthracisStrains in Different Victims” Microbiology, vol. 95, pp. 1224-1229, Feb. 1998.
Andersen, G., et al, “Identification of a Region of Genetic Variability AmongBacillus AnthracisStrains and Related Species” Journal of Bacteriology, Jan. 1996, vol. 178, No. 2, pp. 377-384.
Cheng Tu-Chen
Rastogi Vipin K.
Biffoni Ulysses John
Myers Carla J.
The United States of America as represented by the Secretary of
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