Oligonucleotide probes for the detection of periodontal pathogens
AssigneeMicroProbe, Inc.
InventorsDennis E. Schwartz, Roy H. Kanemoto, Susan M. Watanabe, Kim Dix
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Abstract
This invention relates to compositions of oligonucleotide probes for use in the detection of bacteria associated with medical disorders of the human mouth, wherein said probes consist essentially of a segment of nucleic acid capable of selectively hybridizing under hybridizing conditions, to the 16S or 23S ribosomal RNA [rRNA] of said bacteria. Methods for detection, as well as diagnostic kits for the assay of these bacterium, are also disclosed.
Claims22
A composition of polynucleotide probes for the detection of bacteria associated with a periodontal disease, wherein said probes comprise a selective sequence of 10 to 100 nucleotides or nucleotide analogs capable of selectively hybridizing, under hybridizing conditions, to hypervariable regions of ribosomal RNA of the bacteria with the provision that any additional nucleotides covalently bound to said segment do not hybridize under said conditions to nucleic acids of bacteria found in the human mouth wherein said selective sequence comprises in whole or in part a nucleic acid sequence selected from the group consisting of:
- for Bacteroides gingivalis:
- 5'CAATACTCGTATCGCCCGTTATTC3';
- 5'GTATAAAAGAAGTTTAMAATCCTT3';
- 5'CCTTAGGACAGTCTTCCTTCACGC3';
- 5'CTGTGGAAGCTTGACGGTATATCG3';
- 5'GGTTTTCACCATCAGTCATCTACA3';
- 5'ATTACCCTAGTGCGCCCCTTGCGG3';
- and
- 5'CCGATGCTTATTCTTACGGTACAT3';
- for Actinobacillus (ex. Hameophilus) actinomycetemcomitans:
- 5'ATTTAACGTCAATTTGGCATGCTA3';
- 5'CTTCGGGCACYAGGGCTAAACCCC3';
- 5'ACCCATCTCTGAGTTCTTCTTCGG3';
- 5'GTGGTAAACGCCCCCCTCTCGGTT3';
- and
- 5'TGGCATGCTATTAACACACCAACC3';
- for Bacteroides intermedius Type 1:
- ' GGTCCTTATTCGAAGGGTAAATGC3';
- 5'CACGTGCCCCACTTTACTCCCCAA3';
- 5'TAGCCGCTAACGCCAGGCGCTAAC3';
- 5'CCCTAGGYGCGCTCCTCGCGGTTA3';
- 5'GAGTCAACATCTCTGTATCCTGCG3';
- and
- 5'TTGCCCTAGGTCGCTCCTCGCGGT3';
- for Bacteroides intermedius Type 2:
- 5'AGACGCCCCGAAGGAAGCCTATGT3';
- 5'ATGAGGTACATGCAATGGCGCACA3';
- and
- 5'CGTGCGCCAATTTATTCCCACATA3';
- for Bacteroides forsythus:
- 5'CACAAGGTACATGCAATAAAATAC3';
- 5'CGTATCTCATTTTATTCCCCTGTA3';
- 5'AGCTCTCACTCTCCGTCGTCTACA3';
- 5'AATACACGTATCTCATTTTATTCC3';
- and
- 5'GAAGAAAGCTCTCACTCTCCGTCG3';
- 5'CTGTAGAGCTTACACTATATCGCA3';
- for Eikenella corrodens:
- ' GTACGCTACTAAGCAATCAAGTTG3';
- 5'TTAGGTACCGTCAGCAAAAAGTGG3';
- 5'GCACTTCCCTTTTCTTCCCTAACA3';
- 5'TACCGTGGCAAGCGGGCTCCTTGC3';
- and
- 5'CTTCCGTCTCTGGAAGGTTCCGTAC3';
- for Fusobacterium nucleatum:
- 5'GTCATCGTGCACACAGAATTGCTG3';
- 5'GTTGGTACCGTCATTTTTTTCTTC3';
- 5'AGGTTTCCCCGAAGGCACTGAAAC3';
- 5'TCAGACTCTCGGTCCATTGTCCAA3';
- and
- 5'AAACATCTCTGTCTCATTCCTAAG3';
- for Wolinella recta:
- 5'GTACCGTCATAATTCTTTCCCAAG3';
- 5'GGACCATAACCGGTTTGGTATTTG3';
- 5'GCATTACTGCCTCGACTAGCGAAG3';
- and
- 5'CTTGGGTACCGTCATAATTCTTTCC3';
- for Streptococcus intermedius:
- ' GTACCGTCACAGTATGAACTTTCC3';
- 5'TTCTCACACTCGTTCTTCCTTAAC3';
- and
- 5'TTTCCATTCTCACACTCGTTCTTC3';
- and combinations thereof wherein M represents A or C and Y represents C or T.
A method of detecting, in a sample obtained from the mouth of a human patient, bacteria associated with periodontal disease, said method comprising the steps of:
- lysing the microbial cells to free ribosomal RNA in said sample;
- contacting said ribosomal RNA, under hybridizing conditions, with polynucleotide probes comprising a selective sequence of 10 to 100 nucleotides or nucleotide analogs capable of selectively hybridizing to the hypervariable regions of the ribosomal RNA of said bacteria;
- and
- detecting hybridization complexes as an indication of the presence of the microbial cell in the sample;
- wherein said selective sequence comprises in whole or in part a nucleic acid segment selected from the group consisting of:
- for Bacteroides gingivalis:
- ' CAATACTCGTATCGCCCGTTATTC3';
- 5'GTATAAAAGAAGTTTAMAATCCTT3';
- 5'CCTTAGGACAGTCTTCCTTCACGC3';
- 5'CTGTGGAAGCTTGACGGTATATCG3';
- 5'GGTTTTCACCATCAGTCATCTACA3';
- 5'ATTACCCTAGTGCGCCCCTTGCGG3';
- and
- 5'CCGATGCTTATTCTTACGGTACAT3';
- for Actinobacillus (ex. Haemophilus) actinomycetemcomitans:
- 5'ATTTAACGTCAATTTGGCATGCTA3';
- 5'CTTCGGGCACYAGGGCTAAACCCC3';
- 5'ACCCATCTCTGAGTTCTTCTTCGG3';
- 5'GTGGTAAACGCCCCCCTCTCGGTT3';
- and
- 5'TGGCATGCTATTAACACACCAACC3';
- for Bacteroides intermedius Type 1;
- 5'GGTCCTTATTCGAAGGGTAAATGC3;
- 5'CACGTGCCCCACTTTACTCCCCAA3;
- 5'TAGCCGCTAACGCCAGGCGCTAAC3;
- 5'CCCTAGGYGCGCTCCTCGCGGTTA3;
- 5'GAGTCAACATCTCTGTATCCTGCG3;
- and
- 5'TTGCCCTAGGTCGCTCCTCGCGGT3';
- for Bacteroides intermedius Type 2:
- 5'AGACGCCCCGAAGGAAGCCTATGT3';
- 5'ATGAGGTACATGCAATGGCGCACA3';
- and
- 5'CGTGCGCCAATTTATTCCCACATA3';
- for Bacteroides forsythus:
- ' CACAAGGTACATGCAATAAAATAC3';
- 5'CGTATCTCATTTTATTCCCCTGTA3';
- 5'AGCTCTCACTCTCCGTCGTCTACA3';
- 5'AATACACGTATCTCATTTTATTCC3';
- 5'GAAGAAAGCTCTCACTCTCCGTCG3';
- and
- 5'CTGTAGAGCTTACACTATATCGCA3';
- for Eikenella corrodens:
- 5'GTACGCTACTAAGCAATCAAGTTG3';
- 5'TTAGGTACCGTCAGCAAAAAGTGG3';
- 5'GCACTTCCCTTTTCTTCCCTAACA3';
- 5'TACCGTGGCAAGCGGGCTCCTTGC3';
- and
- 5'CTTCCGTCTCTGGAAGGTTCCGTAC3';
- for Fusobacterium nucleatum:
- 5'GTCATCGTGCACACAGAATTGCTG3';
- 5'GTTGGTACCGTCATTTTTTTCTTC3';
- 5'AGGTTTCCCCGAAGGCACTGAAAC3';
- 5'TCAGACTCTCGGTCCATTGTCCAA3';
- and
- 5'AAACATCTCTGTCTCATTCCTAAG3';
- for Wolinella recta:
- ' GTACCGTCATAATTCTTTCCCAAG3';
- 5'GGACCATAACCGGTTTGGTATTTG3';
- 5'GCATTACTGCCTCGACTAGCGAAG3';
- and
- 5'CTTGGGTACCGTCATAATTCTTTCC3';
- for Streptococcus intermedius:
- 5'GTACCGTCACAGTATGAACTTTCC3';
- 5'TTCTCACACTCGTTCTTCCTTAAC3';
- and
- 5'TTTCCATTCTCACACTCGTTCTTC3';
- and combinations thereof wherein M represents A or C and Y represents C or T.
A method according to claim 111 wherein the bacteria is Wolinella recta and said probes comprise a nucleic acid segment selected from the group consisting of:
- 5'GTACCGTCATAATTCTTTCCCAAG3';
- 5'GGACCATAACCGGTTTGGTATTTG3';
- 5'GCATTACTGCCTCGACTAGCGAAG3';
- 5'CTTGGGTACCGTCATAATTCTTTCC3';
- and combination thereof.
A diagnostic kit for use in determining the presence of a specific polynucleotide sequence which comprises in a container a synthetic oligonucleotide probe comprising a selective sequence of 10 to 100 nucleotides or nucleotide analogs complementary to the hypervariable region of the ribosomal RNA of bacteria associated with a human oral peridontal disease;
- wherein said selective sequence comprises in whole of in part a nucleic acid segment selected from the group consisting of:
- for Bacteroides gingivalis:
- 5'CAATACTCGTATCGCCCGTTATTC3';
- 5'GTATAAAAGAAGTTTAMAATCCTT3';
- 5'CCTTAGGACAGTCTTCCTTCACGC3';
- 5'CTGTGGAAGCTTGACGGTATATCG3';
- 5'GGTTTTCACCATCAGTCATCTACA3';
- 5'ATTACCCTAGTGCGCCCCTTGCGG3';
- and
- 5'CCGATGCTTATTCTTACGGTACAT3';
- for Actinobacillus (ex. Haemophilus) actinomycetemcomitans:
- 5'ATTTAACGTCAATTTGGCATGCTA3';
- 5'CTTCGGGCACYAGGGCTAAACCCC3';
- 5'ACCCATCTCTGAGTTCTTCTTCGG3';
- 5'GTGGTAAACGCCCCCCTCTCGGTT3';
- and
- 5'TGGCATGCTATTAACACACCAACC3';
- for Bacteroides intermedius Type 1:
- ' GGTCCTTATTCGAAGGGTAAATGC3';
- 5'CACGTGCCCCACTTTACTCCCCAA3';
- 5'TAGCCGCTAACGCCAGGCGCTAAC3';
- 5'CCCTAGGYGCGCTCCTCGCGGTTA3';
- 5'GAGTCAACATCTCTGTATCCTGCG3';
- and
- 5'TTGCCCTAGGTCGCTCCTCGCGGT3';
- for Bacteroides intermedius Type 2:
- 5'AGACGCCCCGAAGGAAGCCTATGT3';
- 5'ATGAGGTACATGCAATGGCGCACA3';
- and
- 5'CGTGCGCCAATTTATTCCCACATA3';
- for Bacteroides forsythus:
- 5'CACAAGGTACATGCAATAAAATAC3';
- 5'CGTATCTCATTTTATTCCCCTGTA3';
- 5'AGCTCTCACTCTCCGTCGTCTACA3';
- 5'AATACACGTATCTCATTTTATTCC3';
- 5'GAAGAAAGCTCTCACTCTCCGTCG3';
- and
- 5'CTGTAGAGCTTACACTATATCGCA3';
- for Eikenella corrodens:
- ' GTACGCTACTAAGCAATCAAGTTG3';
- 5'TTAGGTACCGTCAGCAAAAAGTGG3';
- 5'GCACTTCCCTTTTCTTCCCTAACA3';
- 5'TACCGTGGCAAGCGGGCTCCTTGC3';
- and
- 5'CTTCCGTCTCTGGAAGGTTCCGTAC3';
- for Fusobacterium nucleatum:
- 5'GTCATCGTGCACACAGAATTGCTG3';
- 5'GTTGGTACCGTCATTTTTTTCTTC3';
- 5'AGGTTTCCCCGAAGGCACTGAAAC3';
- 5'TCAGACTCTCGGTCCATTGTCCAA3';
- and
- 5'AAACATCTCTGTCTCATTCCTAAG3';
- for Wolinella recta:
- ' GTACCGTCATAATTCTTTCCCAAG3';
- 5'GGACCATAACCGGTTTGGTATTTG3';
- 5'GCATTACTGCCTCGACTAGCGAAG3';
- and
- 5'CTTGGGTACCGTCATAATTCTTTCC3';
- for Streptococcus intermedius:
- 5'GTACCGTTCACAGTATGAACTTTCC3';
- 5'TTCTCACACTCGTTCTTCCTTAAC3';
- and
- 5'TTTCCATTCTCACACTCGTTCTTC3';
- and combinations thereof.
Dependencies are inferred from the claim text.