# US 5,703,217 A · Nucleotide fragment of the 23S ribosomal RNA of mycobacteria, derived probes and primers, reagent and detection method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,703,217 A - Document ID: US5703217A - Status: Granted - Kind: A - Application number: 08/403,762 - Filed: 1995-03-23 - Granted: 1997-12-30 - Earliest claimed: 1993-07-23 - Assignees: Bio Merieux - Inventors: Claude Mabilat; Richard Christen - Web page: https://patentgrep.com/patent/US5703217A ## CPC - C07K 14/35: from Mycobacteriaceae (F) - C12Q 1/689: for bacteria - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract Bacteria of the genus Mycobacterium can be detected and/or identified by methods using probes including fragments of a variable region of a 23S ribosomal RNA of a species of the genus Mycobacterium or probes including DNA fragments that are obtained by reverse transcription of the RNA or that form the RNA by transcription. Primers for the reverse transcription of a variable region of a 23S ribosomal RNA sequence of mycobacteria include nucleotide sequences of a 23S RNA of a species of the genus Mycobacterium. ## Claims ### Claim 1 A single-stranded nucleotide fragment of no more than one hundred modified or unmodified nucleotides, wherein said fragment comprises a nucleotide sequence selected from the group consisting of the following nucleotide sequences of a 23S ribosomal RNA of a species of the genus Mycobacterium: beginning at nucleotide No. 137 and ending at nucleotide No. 179, beginning at nucleotide No. 206 and ending at nucleotide No. 248, beginning at nucleotide No. 269 and ending at nucleotide No. 289 of a 23S ribosomal RNA of species other than M. tuberculosis and M. fortuitum, beginning at nucleotide No. 289 and ending at nucleotide No. 290 of a 23S ribosomal RNA of species other than M. tuberculosis and M. fortuitum, beginning at nucleotide No. 341 and ending at nucleotide No. 372, beginning at nucleotide No. 645 and ending at nucleotide No. 661 of a 23S ribosomal RNA of species other than M. tuberculosis and M. fortuitum, beginning at nucleotide No. 1201 and ending at nucleotide No. 1242, beginning at nucleotide No. 1409 and ending at nucleotide No. 1420 of a 23S ribosomal RNA of species other than M. tuberculosis, M. marinum, M. scrofulaceum, M. avium TMC 724, M. leprae, M. kansasii, M. asiaticum, M. ulcerans ATCC 19423, M. malmoense ATCC 29571, M. gordonae, M. lactae ATCC 25854, M. aurum ATCC 23366, M. vaccae ATCC 15483 and M. neoaurum ATCC 25795, beginning at nucleotide No. 1493 and ending at nucleotide No. 1515, beginning at nucleotide No. 1703 and ending at nucleotide No. 1761, beginning at nucleotide No. 1854 and ending at nucleotide No. 1876 of a 23S ribosomal RNA of species other than M. tuberculosis, M. kansasii and M. fortuitum, beginning at nucleotide No. 2126 and ending at nucleotide No. 2161 of a 23S ribosomal RNA of species other than M. avium, M. tuberculosis, M. kansasii and M. fortuitum, their corresponding DNA sequences, and sequences fully complementary to said RNA and DNA sequences, the numbers of the nucleotides corresponding to their position relative to the nucleotide sequence of Escherichia coli 23S ribosomal RNA, wherein each said fragment selectively hybridizes to RNA or DNA of only one or two species of the genus Mycobacterium. ### Claim 2 (depends on 1) The fragment as claimed in claim 1, wherein said fragment comprises at least one sequence selected from the group consisting of: SEQ ID NO 1 to SEQ ID NO 28, SEQ ID NO 30 to SEQ ID NO 37, SEQ ID NO 39 to SEQ ID NO 41, SEQ ID NO 43 to SEQ ID NO 49, SEQ ID NO 51 to SEQ ID NO 67, SEQ ID NO 69 to SEQ ID NO 75, SEQ ID NO 77 to SEQ ID NO 93, SEQ ID NO 95, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 100 to SEQ ID NO 136, SEQ ID NO 139 to SEQ ID NO 144, SEQ ID NO 146 to SEQ ID NO 147, SEQ ID NO 150 to SEQ ID NO 152, SEQ ID NO 154 to SEQ ID NO 158, SEQ ID NO 160, SEQ ID NO 161, SEQ ID NO 163 to SEQ ID NO 174 and SEQ ID NO 178, and their fully complementary sequences. ### Claim 3 (depends on 1) A single-stranded nucleotide fragment of DNA, which is a reverse transcription product of a RNA fragment as claimed in claim 1, or its fully complementary fragment. ### Claim 4 (depends on 1) A single-stranded nucleotide fragment of genomic DNA, whose transcription product is a RNA fragment as claimed in claim 1, or its fully complementary fragment. ### Claim 5 (depends on 1) A probe for the identification of one or two species of bacteria of the genus Mycobacterium, said probe comprising, as its sole nucleotide sequence, a probe sequence of no more than one hundred modified or unmodified nucleotides, said probe sequence comprising at least eight contiguous modified or unmodified nucleotides of the nucleotide sequence of the fragment as claimed in claim 1, or a homologous sequence of at least eight nucleotides that hybridizes to said nucleotide sequence of said fragment, wherein said probe selectively hybridizes to RNA or DNA of only one or two species of the genus Mycobacterium. ### Claim 6 (depends on 5) The probe as claimed in claim 5, which comprises a sequence of eight to fifty modified or unmodified nucleotides of the nucleotide sequence of said fragment. ### Claim 7 A probe for the identification of one or two species of bacteria of the genus Mycobacterium, said probe comprising, as its sole nucleotide sequence, a first sequence of no more than one hundred modified or unmodified nucleotides, said first sequence comprising a second sequence selected from the group consisting of the following sequences: beginning at nucleotide No. 10 and ending at nucleotide No. 24 of at least one of sequences SEQ ID NO 1 to 14, beginning at nucleotide No. 26 and ending at nucleotide No. 45 of at least one of sequences SEQ ID NO 1 to 14, beginning at nucleotide No. 1 and ending at nucleotide No. 20 of at least one of sequences SEQ ID NO 15 to 28, beginning at nucleotide No. 26 and ending at nucleotide No. 43 of at least one of sequences SEQ ID NO 15 to 28, beginning at nucleotide No. 4 and ending at nucleotide No. 22 of at least one of sequences SEQ ID NO 29 to 41, beginning at nucleotide No. 12 and ending at nucleotide No. 29 of at least one of sequences SEQ ID NO 42 to 53, beginning at nucleotide No. 36 and ending at nucleotide No. 56 of at least one of sequences SEQ ID NO 42 to 53, beginning at nucleotide No. 13 and ending at nucleotide No. 32 of at least one of sequences SEQ ID NO 54 to 67, beginning at nucleotide No. 5 and ending at nucleotide No. 23 of at least one of sequences SEQ ID NO 68 to 79, beginning at nucleotide No. 15 and ending at nucleotide No. 33 of at least one of sequences SEQ ID NO 80 to 93, beginning at nucleotide No. 1 and ending at nucleotide No. 21 of at least one of sequences SEQ ID NO 94 to 106 and SEQ ID NO 166 to 178, beginning at nucleotide No. 44 and ending at nucleotide No. 76 of at least one of sequences SEQ ID NO 94 to 106 and SEQ ID NO 166 to 178, beginning at nucleotide No. 67 and ending at nucleotide No. 85 of at least one of sequences SEQ ID NO 94 to 106 and SEQ ID NO 166 to 178, beginning at nucleotide No. 6 and ending at nucleotide No. 26 of at least one of sequences SEQ ID NO 107 to 120, beginning at nucleotide No. 1 and ending at nucleotide No. 18 of at least one of sequences SEQ ID NO 121 to 136, beginning at nucleotide No. 39 and ending at nucleotide No. 55 of at least one of sequences SEQ ID NO 121 to 136, beginning at nucleotide No. 11 and ending at nucleotide No. 26 of at least one of sequences SEQ ID NO 139 to 144, SEQ ID NO 146, SEQ ID NO 147 and SEQ ID NO 149 to 152, beginning at nucleotide No. 5 and ending at nucleotide No. 22 of at least one of sequences SEQ ID NO 153 to 165, their corresponding DNA sequences, and sequences fully complementary to said RNA and DNA sequences, wherein said probe selectively hybridizes to RNA or DNA of only one or two species of the genus Mycobacterium. ### Claim 8 (depends on 7) The probe as claimed in claim 7, for the identification of M. xenopi, which comprises a RNA sequence beginning at nucleotide No. 59 and ending at nucleotide No. 76 of the sequence SEQ ID NO 173, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences. ### Claim 9 (depends on 7) The probe as claimed in claim 7, for the identification of M. fortuitum, which comprises a RNA sequence beginning at nucleotide No. 6 and ending at nucleotide No. 22 of the sequence SEQ ID NO 117, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences. ### Claim 10 (depends on 7) The probe as claimed in claim 7, for the identification of M. tuberculosis, which comprises a RNA sequence beginning at nucleotide No. 1 and ending at nucleotide No. 21 of the sequence SEQ ID NO 94, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences. ### Claim 11 A probe comprising, as its sole nucleotide sequence, a probe sequence of no more than one hundred modified or unmodified nucleotides, said probe sequence comprising a nucleotide sequence selected from the group consisting of the following sequences of a 23S RNA of a species of the genus Mycobacterium: beginning at nucleotide No. 137 and ending at nucleotide No. 179, beginning at nucleotide No. 206 and ending at nucleotide No. 248, beginning at nucleotide No. 269 and ending at nucleotide No. 289, beginning at nucleotide No. 289 and ending at nucleotide No. 290, beginning at nucleotide No. 341 and ending at nucleotide No. 372, beginning at nucleotide No. 645 and ending at nucleotide No. 661, beginning at nucleotide No. 1201 and ending at nucleotide No. 1242, beginning at nucleotide No. 1409 and ending at nucleotide No. 1420, beginning at nucleotide No. 1493 and ending at nucleotide No. 1515, beginning at nucleotide No. 1703 and ending at nucleotide No. 1761, beginning at nucleotide No. 1854 and ending at nucleotide No. 1876, beginning at nucleotide No. 2126 and ending at nucleotide No. 2161 of species other than M. avium, their corresponding DNA sequences, and sequences fully complementary to said RNA and DNA sequences, the numbers of the nucleotides corresponding to their position relative to the nucleotide sequence of Escherichia coli 23S ribosomal RNA, wherein said probe selective hybridizes to RNA or DNA of only one or two species of the genus Mycobacterium. ### Claim 12 A primer for the specific reverse transcription of a variable region of a 23S ribosomal RNA sequence of mycobacteria into a complementary DNA sequence, said primer consisting of a sequence of no more than one hundred modified or unmodified nucleotides, said sequence comprising a nucleotide sequence of at least eight contiguous nucleotides of at least one of the following nucleotide sequences of a 23S RNA of a species of the genus Mycobacterium: beginning at nucleotide No. 137 and ending at nucleotide No. 179, beginning at nucleotide No. 206 and ending at nucleotide No. 248, beginning at nucleotide No. 269 and ending at nucleotide No. 289, beginning at nucleotide No. 289 and ending at nucleotide No. 290, beginning at nucleotide No. 341 and ending at nucleotide No. 372, beginning at nucleotide No. 645 and ending at nucleotide No. 661, beginning at nucleotide No. 1201 and ending at nucleotide No 1242, beginning at nucleotide No. 1409 and ending at nucleotide No 1420, beginning at nucleotide No. 1493 and ending at nucleotide No 1515, beginning at nucleotide No. 1703 and ending at nucleotide No 1761, beginning at nucleotide No. 1854 and ending at nucleotide No 1876, beginning at nucleotide No. 2126 and ending at nucleotide No 2161 of species other than M. avium, their corresponding DNA sequences, and sequences fully complementary to said RNA and DNA sequences, the numbers of the nucleotides corresponding to their position relative to the nucleotide sequence of Escherichia coli 23S ribosomal RNA, wherein said primer selectively hybridizes to RNA or DNA of only one or two species of the genus Mycobacterium. ### Claim 13 (depends on 3) A primer for specific enzymatic amplification of the DNA sequence as claimed in claim 3, said primer consisting of a sequence of no more than one hundred modified or unmodified nucleotides, said sequence comprising a nucleotide sequence of at least eight contiguous modified or unmodified nucleotides of at least one of the following nucleotide sequences of a 23S RNA of a species of the genus Mycobacterium: beginning at nucleotide No. 137 and ending at nucleotide No. 179, beginning at nucleotide No. 206 and ending at nucleotide No. 248, beginning at nucleotide No. 269 and ending at nucleotide No. 289, beginning at nucleotide No. 289 and ending at nucleotide No. 290, beginning at nucleotide No. 341 and ending at nucleotide No. 372, beginning at nucleotide No. 645 and ending at nucleotide No. 661, beginning at nucleotide No. 1201 and ending at nucleotide No. 1242, beginning at nucleotide No. 1409 and ending at nucleotide No. 1420, beginning at nucleotide No. 1493 and ending at nucleotide No. 1515, beginning at nucleotide No. 1703 and ending at nucleotide No. 1761, beginning at nucleotide No. 1854 and ending at nucleotide No. 1876, beginning at nucleotide No. 2126 and ending at nucleotide No. 2161 of species other than M. avium, their corresponding DNA sequences, and sequences fully complementary to said RNA and DNA sequences, the numbers of the nucleotides corresponding to their position relative to the nucleotide sequence of Escherichia coli 23S ribosomal RNA, wherein said primer selectively hybridizes to RNA or DNA of only one or two species of the genus Mycobacterium. ### Claim 14 (depends on 5) A reagent for detecting, identifying or detecting and identifying at least one species of mycobacteria in a biological sample, which comprises a first probe according to claim 5 that is a capture probe and a second probe according to claim 5 that is a detection probe, wherein said capture probe and said detection probe have different nucleotide sequences. ### Claim 15 (depends on 14) The reagent as claimed in claim 14, wherein the capture probe is bound directly or indirectly to a solid support. ### Claim 16 (depends on 14) The reagent as claimed in claim 14, wherein the detection probe is labeled by means of a label selected from the group consisting of: radioactive isotopes, enzymes, chromophoric chemical compounds, chromogenic compounds, fluorogenic compounds, luminescent compounds, nucleotide base analogs and biotin. ### Claim 17 (depends on 5) A reagent for detecting or identifying at least two different species of mycobacteria in a biological sample, which comprises a mixture of probes as claimed in claim 5. ### Claim 18 (depends on 7) A method of selective detection, identification or detection and identification of a species of mycobacteria in a biological sample, comprising exposing 23S ribosomal RNA extracted from bacteria contained in said sample to at least one probe as claimed in claim 7, and detecting hybridization of said probe. ### Claim 19 (depends on 18) The method as claimed in claim 18, wherein the probe is selected from the group consisting of: a probe for identification of M. xenopi comprising a RNA sequence beginning at nucleotide No. 59 and ending at nucleotide No. 76 of the sequence SEQ ID NO 173, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences; a probe for identification of M. fortuitum comprising a RNA sequence beginning at nucleotide No. 6 and ending at nucleotide No. 22 of the sequence SEQ ID NO 117, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences; and a probe for identification of M. tuberculosis comprising a RNA sequence beginning at nucleotide No. 1 and ending at nucleotide No. 21 of the sequence SEQ ID NO 94, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences. ### Claim 20 (depends on 7) A method of selective detection, identification or detection and identification of a species of mycobacteria in a biological sample, comprising denaturing genomic DNA or RNA extracted from bacteria contained in said sample, exposing said denatured DNA or RNA to at least one probe as claimed in claim 7, and detecting hybridization of said probe. ### Claim 21 (depends on 20) The method as claimed in claim 20, wherein the probe is selected from the group consisting of: a probe for identification of M. xenopi comprising a RNA sequence beginning at nucleotide No. 59 and ending at nucleotide No. 76 of the sequence SEQ ID NO 173, a corresponding DNA sequences, or a sequence fully complementary to said RNA and DNA sequence; a probe for identification of M. fortuitum comprising a RNA sequence beginning at nucleotide No. 6 and ending at nucleotide No. 22 of the sequence SEQ ID NO 117, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences; and a probe for identification of M. tuberculosis comprising a RNA sequence beginning at nucleotide No. 1 and ending at nucleotide No. 21 of the sequence SEQ ID NO 94, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences. ### Claim 22 (depends on 7) A method of selective detection, identification or detection and identification of a species of mycobacteria in a biological sample, comprising exposing DNA obtained by reverse transcription of 23S ribosomal RNA of bacteria contained in said sample to at least one probe as claimed in claim 7, and detecting hybridization of said probe. ### Claim 23 (depends on 22) The method as claimed in claim 22, wherein the probe is selected from the group consisting of: a probe for identification of M. xenopi comprising a RNA sequence beginning at nucleotide No. 59 and ending at nucleotide No. 76 of the sequence SEQ ID NO 173, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences; a probe for identification of M. fortuitum comprising a RNA sequence beginning at nucleotide No. 6 and ending at nucleotide No. 22 of the sequence SEQ ID NO 117, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences; and a probe for identification of M. tuberculosis comprising a RNA sequence beginning at nucleotide No. 1 and ending at nucleotide No. 21 of the sequence SEQ ID NO 94, a corresponding DNA sequence, or a sequence fully complementary to said RNA and DNA sequences. ### Claim 24 (depends on 1) The fragment as claimed in claim 1, wherein said nucleotide sequence is selected from the group consisting of the following nucleotide sequences of the 23S RNA: beginning at nucleotide No. 137 and ending at nucleotide No. 179, beginning at nucleotide No. 206 and ending at nucleotide No. 248, beginning at nucleotide No. 289 and ending at nucleotide No. 290 of a 23S ribosomal RNA of species other than M. tuberculosis and M. fortuitum, beginning at nucleotide No. 341 and ending at nucleotide No. 372, beginning at nucleotide No. 1201 and ending at nucleotide No. 1242, beginning at nucleotide No. 1409 and ending at nucleotide No. 1420 of a 23S ribosomal RNA of species other than M. tuberculosis, M. marinum, M. scrofulaceum, M. avium TMC 724, M. leprae, M. kansasii, M. asiaticum, M. ulcerans ATCC 19423, M. malmoense ATCC 29571, M. gordonae, M. lactae ATCC 25854, M. aurum ATCC 23366, M. vaccae ATCC 15483 and M. neoaurum ATCC 25795, beginning at nucleotide No. 1493 and ending at nucleotide No. 1515, beginning at nucleotide No. 1703 and ending at nucleotide No. 1761, their corresponding DNA sequences, and sequences fully complementary to said RNA and DNA sequences. ### Claim 25 (depends on 1) The fragment as claimed in claim 1, wherein said nucleotide sequence is selected from the group consisting of the following nucleotide sequences of the 23S RNA: beginning at nucleotide No. 137 and ending at nucleotide No. 179, beginning at nucleotide No. 206 and ending at nucleotide No. 248, beginning at nucleotide No. 269 and ending at nucleotide No. 289 of a 23S ribosomal RNA of species other than M. tuberculosis and M. fortuitum, beginning at nucleotide No. 289 and ending at nucleotide No. 290 of a 23S ribosomal RNA of species other than M. tuberculosis and M. fortuitum, beginning at nucleotide No. 341 and ending at nucleotide No. 372, beginning at nucleotide No. 645 and ending at nucleotide No. 661 of a 23S ribosomal RNA of species other than M. tuberculosis and M. fortuitum, beginning at nucleotide No. 1201 and ending at nucleotide No. 1242, beginning at nucleotide No. 1493 and ending at nucleotide No. 1515, beginning at nucleotide No. 1703 and ending at nucleotide No. 1761, beginning at nucleotide No. 1854 and ending at nucleotide No. 1876 of a 23S ribosomal RNA of species other than M. tuberculosis, M. kansasii and M. fortuitum, their corresponding DNA sequences, and sequences fully complementary to said RNA and DNA sequences. ### Claim 26 (depends on 5) A method of selective detection, identification or detection and identification of a species of mycobacteria in a biological sample, comprising exposing 23S ribosomal RNA extracted from bacteria contained in said sample to at least one probe as claimed in claim 5, and detecting hybridization of said probe. ### Claim 27 (depends on 5) A method of selective detection, identification or detection and identification of a species of mycobacteria in a biological sample, comprising denaturing genomic DNA or RNA extracted from bacteria contained in said sample, exposing said denatured DNA or RNA to at least one probe as claimed in claim 5, and detecting hybridization of said probe. ### Claim 28 (depends on 5) A method of selective detection, identification or detection and identification of a species of mycobacteria in a biological sample, comprising exposing DNA obtained by reverse transcription of 23S ribosomal RNA of bacteria contained in said sample to at least one probe as claimed in claim 5, and detecting hybridization of said probe.