Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-05-16
1997-04-15
Zitomer, Stephanie W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 243, 536 2432, C12Q 168, C12P 1934, C07H 2104
Patent
active
056208472
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates generally to methods and reagents for identifying and detecting bacteria in cerebrospinal fluid (CSF).
BACKGROUND OF THE INVENTION
In order to treat successfully a disease caused by a bacterium, the rapid and accurate detection and identification of the disease-causing bacterium is required. The detection and identification have traditionally been accomplished by pure culture isolation and identification procedures that make use of knowledge of specimen source, growth requirements, visible (colony) growth features, microscopic morphology, staining reactions, and biochemical characteristics.
A number of different bacterial species can cause meningitis when present in the CSF. The species most frequently causing meningitis include: Escherichia coli and other enteric bacteria, Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, and Listeria monocytogenes.
Conventional methods of detection and identification of bacteria in cerebrospinal fluid include the Gram stain, latex agglutination and other antibody-based tests, and culture. The Gram stain and antibody-based tests are rapid (<1 hour), but of low sensitivity (requiring at least 10.sup.4 colony forming units [CFU]bacteria per ml). Culture methods, while sensitive to approximately 2 CFU per ml, require overnight incubation.
A number of scientific publications relating to this invention exist. For example, the polymerase chain reaction has been used to detect individual species of bacteria causing meningitis: Kuritza and Oehler, May, 1991, Abstracts of the General Meeting of the ASM page 84; Deneer and Boychuk, 1991, Applied Environmental Microbiology 57: 606-609; and Kristiansen et al., 1991, Lancet 337: 1568-1569.
In addition, some of the nucleotide sequence data used herein is available in Genbank. The method of reverse dot-blot detection has been described by Saiki et al., 1989. The use of uracil-N-glycosylase has been described by Longo et al., 1990, Gene 93: 125-128.
A method of detecting bacteria in Cerebrospinal Fluid ("CSF") which is both sensitive and rapid would represent a great improvement over current methods of detection. The present invention meets these needs.
SUMMARY OF THE INVENTION
The present invention pertains to methods and reagents for the rapid detection and identification of bacteria in CSF. The detection and identification is based upon the hybridization of nucleotide probes to nucleotide sequences present in a defined species or group of species, but not in others.
In a preferred method, a target region from genomic DNA or complementary DNA transcribed from 16S rRNA is amplified and the resultant amplified DNA is treated with a panel of probes. Each probe in the panel can hybridize to the DNA of a different species or group of species of bacteria found in CSF. The probe which successfully hybridizes to the amplified DNA is determined and the bacterium is classified as a particular species or group of species.
The invention also pertains to specific probes and their complements for identifying bacteria found in CSF. It also pertains to unique oligonucleotide sequences, and mutants, fragments and subsequences thereof, from which such specific probes were derived.
As indicated, also contemplated herein is a panel of probes which will allow the detection and identification of bacteria commonly found in CSF. The panel includes probes for the bacteria causing meningitis listed above as well as bacterial species which are commonly considered contaminants of human clinical samples such as blood or cerebrospinal fluid. Such contaminant species are also capable of causing meningitis; however, these organisms do so at a lower frequency than the agents listed in the "Background of the Invention" and include: Bacillus species, Corynebacterium species, Propionibacterium acnes and other Propionibacterium species, and Staphylococcus epidermidis and other coagulase-negative Staphylococci (Bergey's Manual of Systematic Bacteriology, ed. J. G. Holt
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Greisen Kay S.
Leong Diane U.
Fredman Jeffrey
Hoffman-La Roche Inc.
Johnston George W.
Rocha-Tramaloni Patricia S.
Seminow Raina
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