Nucleic Acid Probes and Broad-Range Primers from Regions in Dna Directed Rna Polymerase Subunit B Genes, and Methods in Which They are Used
First Claim
1. A diagnostic method for detecting and identifying bacterial species causing infections from a clinical sample, said method comprising:
- a) amplifying DNA isolated from said clinical sample using a mixture of DNA primers that comprises sequences which hybridize with the sequences that originate from conserved regions of rpoB genes encoding DNA directed RNA polymerase [EC;
2.7.7.6] subunit B of bacterial species causing infections, said sequences comprising SEQ ID NOS;
20 and 21 and/or complementary sequences thereof and/or functional fragments thereof, b) contacting the amplified DNA with a desired combination of oligonucleotide probe sequences that hybridize under normal hybridization conditions with hyper-variable regions situated near said conserved regions of rpoB genes encoding DNA directed RNA polymerase [EC;
2.7.7.6] subunit B of bacterial species causing said infections, said sequences being bacterial species specific under said hybridization conditions, and c) detecting the formation of a possible hybridization complex.
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Abstract
The invention relates to nucleic acid probes and to broad-range primers that are useful in the identification of bacterial species and the diagnosis of bacterial infections. Especially, the invention relates to specific nucleic acid probes that originate from hyper-variable regions situated near the conserved sequences of the gene for RNA polymerase beta subunit, rpoB (DNA directed RNA polymerase subunit B) of infection-causing bacteria. The invention also relates to broad-range primers originating from the conserved regions of rpoB genes. In addition, the invention relates to the use of these nucleic acid probes and broad-range primers in the diagnosis of bacterial infections as well as to diagnostic methods in which these nucleic acid probes and broad-range primers are used.
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Citations
16 Claims
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1. A diagnostic method for detecting and identifying bacterial species causing infections from a clinical sample, said method comprising:
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a) amplifying DNA isolated from said clinical sample using a mixture of DNA primers that comprises sequences which hybridize with the sequences that originate from conserved regions of rpoB genes encoding DNA directed RNA polymerase [EC;
2.7.7.6] subunit B of bacterial species causing infections, said sequences comprising SEQ ID NOS;
20 and 21 and/or complementary sequences thereof and/or functional fragments thereof,b) contacting the amplified DNA with a desired combination of oligonucleotide probe sequences that hybridize under normal hybridization conditions with hyper-variable regions situated near said conserved regions of rpoB genes encoding DNA directed RNA polymerase [EC;
2.7.7.6] subunit B of bacterial species causing said infections, said sequences being bacterial species specific under said hybridization conditions, andc) detecting the formation of a possible hybridization complex. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A DNA primer mixture comprising sequences that hybridize with sequences of the conserved regions of rpoB genes encoding DNA directed RNA polymerase [EC:
- 2.7.7.6] subunit B of bacterial species that cause infections, said mixture comprising SEQ ID NOS;
20 and 21 and/or complementary sequences thereof or functional fragments thereof.
- 2.7.7.6] subunit B of bacterial species that cause infections, said mixture comprising SEQ ID NOS;
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12. An oligonucleotide sequence useful in the diagnosis of infection causing bacterial species, wherein said oligonucleotide sequence hybridizes under normal hybridization conditions with a sequence of a hyper-variable region that is bacterial species-specific and is situated near the conserved regions of rpoB genes encoding DNA directed RNA polymerase [EC:
- 2.7.7.6] subunit B of bacterial species causing said infections, said oligonucleotide sequence being bacterial species-specific and said oligonucleotide sequence comprising one of the SEQ ID NOS;
1 to 19 or complementary sequences thereof or functional fragments thereof.
- 2.7.7.6] subunit B of bacterial species causing said infections, said oligonucleotide sequence being bacterial species-specific and said oligonucleotide sequence comprising one of the SEQ ID NOS;
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13. The combination of oligonucleotide probe sequences useful in the diagnosis of infection causing bacterial species comprising any combination of the SEQ ID NOS:
- 1 to 19 or complementary sequences thereof or functional fragments thereof.
- View Dependent Claims (14, 15)
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16. A diagnostic kit for use in the diagnosis of infection-causing bacteria, especially those causing respiratory tract infections, comprising
a) a DNA primer mixture comprising sequences that hybridize with sequences of the conserved regions of rpoB genes encoding DNA directed RNA polymerase [EC: - 2.7.7.6] subunit B of bacterial species causing infections, especially bacterial species that cause respiratory tract infections, said mixture comprising SEQ ID NOS;
20 and 21 or complementary sequences thereof or functional fragments thereof,b) a combination of bacterial species-specific oligonucleotide probe sequences, optionally attached on a solid support, comprising any combination of the SEQ ID NOS;
1 to 19 or complementary sequences thereof or functional fragments thereof,c) positive and optionally negative control probe sequences, and optionally d) reagents required in the amplification, hybridization, purification, washing, and/or detection steps.
- 2.7.7.6] subunit B of bacterial species causing infections, especially bacterial species that cause respiratory tract infections, said mixture comprising SEQ ID NOS;
Specification