EP0133671A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Detection of bacteria by nucleic acid hybridization, labeled probes and test kit therefore
Assignee: Bayer Corp
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Filing: 1984-07-27
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Granted: —
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Expires: 2004-07-27
Abstract
Abstract A nucleic acid hybridization method for detecting bacteria in a test sample, such as a mammalian body fluid, employing a polynucleotide probe having a base sequence which is homologous with at least one base sequence in the nucleic acid content of substantially all of the respective bacteria suspected to be present in the sample. The homologous base sequence in the probe preferably comprises at least a 10 base portion of one of the strands of a gene which codes for the synthesis of a nucleic acid or protein comprised in the mechanism of protein synthesis, e.g., tRNAs, rRNAs, and elongation factors. Resulting hybridization between the probe and single stranded nucleic acids released from bacteria in the sample indicates bacterial presence and can be detected by employing labeled probes or otherwise detecting duplex formation. The method provides a unique, direct and rapid means for detecting the presence of bacteria without the need for conventional microbiological culturing. In particular, the method can be applied conveniently as a screening test for bacterial presence in fluids which are normally sterile, such as urine or blood.
Inventors
- 👤 Robert J. Carrico
- 👤 Leslie H. Deriemer
- 👤 Josephine C. Grosch
- 👤 Gary A. Wilson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 07:22
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6888; C12Q1/689; C12Q1/6813
Claims Analysis
Total Claims: 11
Claim 1
Independent Claim
99 words
A method for detecting bacteria in a test sample, comprising the steps of subjecting the test sample to conditions to release and denature nucleic acids from bacteria present in th sample, contacting the resulting single stranded nucleic acids wi a polynucleotide probe under conditions favorable to hybridizatio between said probe and bacterial base sequences, and determining the presence of hybridized probe, characterized in that the probe comprises a base sequence wh is homologous with at least one base sequence in the nucleic acid content of substantially all of the respective bacteria suspected .to be present in said test sample.
Claim 2
Independent Claim
99 words
A method for detecting bacteria in a test sample, comprising the steps of subjecting the test sample to conditions to release and denature nucleic acids from bacteria present in th sample, contacting the resulting single stranded nucleic acids wi a polynucleotide probe under conditions favorable to hybridizatio between said probe and bacterial base sequences, and determining the presence of hybridized probe, characterized in that the probe comprises a base sequence wh is homologous with at least one base sequence in the nucleic acid content of substantially all of the respective bacteria suspected .to be present in said test sample.
Claim 3
Dependent Claim
61 words
..The method of Claim 1 wherein said homologous base sequence comprises at least a portion of one of the strands of a gene which codes for the synthesis of a nucleic acid or protein comprised in the mechanism of protein synthesis, and preferably is selected from transfer RNAs, ribosomal RNAs, ribosomal protein aminoacyl-tRNA synthetases, initiation factors, elongation factor and releasing factors.
Claim 4
Dependent Claim
137 words
The method of Claim 1 characterized in that it compris the steps of: (a) immobilizing the single stranded nucleic acids resultin from treatment. of the test sample on a solid support, (b) contacting the solid support with a labeled polynucleotide probe having a base sequence of at least about 10 bases which is homologous with a base sequence comprising at least a portion of one of the strands of a gene which codes for the synth of a nucleic acid or protein comprised in the mechanism of protei synthesis, such contact being accomplished under conditions favor to hybridization between the labeled probe and said base sequence of said gene, (c) separating unhybridized labeled probe from said solid support, and (d) determining hybridized labeled probe associated with said support by measuring the label comprised in said labeled probe.
Claim 5
Dependent Claim
118 words
A method of Claim 1 characterized in that it comprises the steps of: (a) contacting the single stranded nucleic acids resulting from treatment of the sample with an immobilized DNA probe having a base sequence of at least about 10 bases which is homologous with a base sequence comprising at least a portion of a ribosomal RNA, such contact being accomplished under conditions favorable to hybridization between the probe and said base sequence of ribosomal RN,' and (b) de: -ting hybridized probe by adding an antibody, or a fragment thereof, capable of binding to DNA'RNA duplexes formed between the DNA probe and said ribosomal RNA, and determining the antibody or fragment thereof which becomes bound to such duplexes.
Claim 6
Dependent Claim
56 words
A labeled polynucleotide probe suitable for the method of Claim 1 characterized in that it comprises a label incorporated with a polynucleotide probe having a base sequence which is homologous with at least one base sequence in the nucleic acid content of substantially all of the respective bacteria suspected to be present in said test sample.
Claim 7
Dependent Claim
19 words
The labeled probe of Claim 5 wherein said homologous base sequence is at least about 10 bases in length.
Claim 8
Dependent Claim
62 words
The labeled probe of Claim 5 wherein said homologous base sequence comprises at least a portion of one of the strands of a gene which codes for the synthesis of a nucleic acid or protein comprised in the mechanism of protein synthesis, and preferably is selected from transfer RNAs, ribosomal RNAs, ribosomal proteins, aminoacyl-tRNA synthetases, initiation factors, elongation factors, and releasing factors.
Claim 9
Dependent Claim
86 words
8 . A test kit suitable for the method of Claim 1 characterized in that it comprises: (1) a solid support capable of immobilizing single stranded nucleic acid resulting from treatment of said test sample to release and denature nucleic acids from bacteria present in said sample, and (2) a labeled polynucleotide probe having a base sequence which is homologous with at least one base sequence in the nucleic acid content of substantailly all of the respective bacteria suspected to be present in said test sample:
Claim 10
Dependent Claim
62 words
The test kit of Claim 8 wherein said homologous base sequence comprises at least a portion of one of the strands of a gene which codes for the synthesis of a nucleic acid or protein comprised in the mechanism of protein synthesis, and preferably is selected from transfer RNAs, ribosomal RNAs, ribosomal proteins, aminoacyl-tRNA synthetases, initiation factors, elongation factors, and releasing factors.
Claim 11
Dependent Claim
77 words
A test kit suitable for the method of Claim 1 characterized in that it comprises: (1) an immobilized DNA probe having a base sequence which is homologous with at least one RNA base sequence in the nucleic acid content of substantially all of the respective bacteria suspected to be present in said test sample, and (2) an antibody, or fragment thereof, capable of binding to DNA'RNA duplexes formed between the DNA probe and said RNA base sequence.
References Cited (Backward Citations)
No citation records indexed.
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