Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-08
2002-10-29
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S022100
Reexamination Certificate
active
06472155
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and compositions for the detection of Bacillus species such as
Bacillus anthracis
and
Bacillus globigii
as well as
Clostridium perfringens
. It relies on nucleic acid sequence differences in spore protein genes carried in the genomic sequence of these organisms.
BACKGROUND OF THE INVENTION
The genus Bacillus is composed of rod-shaped, gram-positive, aerobic or (under some conditions) anaerobic bacteria widely found in soil and water. Most strains of Bacillus are not pathogenic for humans and only infect them incidentally in their role as soil organisms; a notable exception is
Bacillus anthracis
, which causes anthrax in humans and domestic animals.
In addition to its role as a naturally occurring pathogen,
Bacillus anthracis
may also be used as a biological weapon. Because Bacillus organisms are widely distributed in the environment, and because they are very closely related genetically, there is need for a reliable method to distinguish species members in various types of samples.
Considerable efforts have been made to develop sensitive, species specific tests for Bacillus microorganisms with only limited results. Most of the
Bacillus anthracis
PCR detection systems have targeted plasmid sequences, yet mobility of these genetic elements make them an unreliable detection target. Several of the PCR type assays to genomic sequences target non-gene-coding sequences, which often proves not to correlate well with species identity. The weaknesses in each of these systems underscores the need for a more reliable Bacillus identification method.
SUMMARY OF THE INVENTION
The subject invention provides novel methods and materials for specifically identifying certain rod-shaped, gram-positive bacteria. These methods and materials rely on the discovery that certain small acid-soluble protein (sasp) gene targets are present in the bacterial coats of Bacillus and Clostridium organisms. These sasp gene targets may be identified by a technique described here as “heterologous PCR”. More specifically, these methods and materials provide means for specifically identifying certain members of the Bacillus genus, especially
Bacillus anthracis
and
Bacillus globigii
, and members of the Clostridium genus, especially
Clostridium perfringens
. Using heterologous PCR, novel gene targets are identified that yield species-specific regions for detection and identification. Based on this technique, assay techniques for identifying the organisms
Bacillus anthracis, Bacillus globigii
and
Clostridium perfringens
have been developed.
Another aspect of the invention comprises use of a small acid soluble protein (sasp-B) gene sequence in
Bacillus anthracis
that contains a specific region of stable nucleotide insertion absent in even the closest Bacillus relatives. This unique biomarker is a probe-binding region for the specific detection of
Bacillus anthracis
. Additional regions of sequence within the gene were used to develop a
Bacillus anthracis
specific amplification and detection system. The system is capable of distinguishing
Bacillus anthracis
from near neighbors during two phases of the amplified assay: during the amplification reaction, and by probe hybridization to the
Bacillus anthracis
specific biomarker within the amplified product for sequence confirmation.
As another aspect of the invention, the present
Bacillus anthracis
detection system either alone, or multiplexed with one or more additional genomic and/or virulence plasmid markers, is of use as a diagnostic or confirmatory tool in Public Health and clinical laboratories, as well as in the law enforcement sector. The system may be adapted to a variety of amplification and detection platforms in order to accommodate the technical and fiscal capabilities of the laboratory, or used in ‘field’ settings.
As yet another aspect of the invention, building upon the discovery that small acid-soluble spore protein genes maintain species-specific sequence signatures, analogous regions were identified among the sasp of
Bacillus globigii
(which is used as a non-pathogenic ‘surrogate’ for
Bacillus anthracis
in research and development applications, defense, and emergency response modeling, etc.). Taking advantage of this discovery,
Bacillus globigii
specific PCR was designed and reduced to practice.
Another aspect of the invention is to provide a two-step amplification/detection system. A particular sasp gene is selected for amplification, and its identity determined by a species-specific probe. Although the present invention is described in detail in connection with a PCR, it is understood that, based on the present teachings, other amplification and/or identification systems could be devised.
REFERENCES:
patent: 5985557 (1999-11-01), Prudent et al.
patent: WO 95/21912 (1995-08-01), None
Cabrera-Martinez et al. FEMS Microbiology Letters. vol. 77. pp. 127-132. 1991.*
Sun and Setlow. J. Bacteriology. vol. 169. pp. 308-3093. 1987.
Aston David J.
Chew Michelle S.
Jones W. Gary
Mahoney John W.
Taylor Janell E.
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