Methods for making oligonucleotide probes for the detection and/or quantitation of non-viral organisms
First Claim
1. A method for making a probe for use in a hybridization assay which comprises constructing a oligonucleotide that is sufficiently complementary to hybridize to a region of rRNA, or the encoding DNA, selected to distinguish one or more non-viral target species from at least one non-viral non-target species, said oligonucleotide comprising a target-complementary sequence obtained by:
- a) aligning rRNA sequences, or the encoding DNA sequences, of said target species and said non-target species to identify a variable region; and
b) designing said target-complementary sequence of said oligonucleotide by substantially maximizing the complementarity of said oligonucleotide to said variable region present in said target species while substantially minimizing the complementarity of said oligonucleotide to said variable region present in said non-target species, such that a duplex formed between said oligonucleotide and said target species has a higher Tm than a duplex formed between said oligonucleotide and said non-target species, wherein said variable region is located in an rRNA sequence or DNA sequence corresponding to a target region selected from the group consisting of;
bases 60-100 of E. coli 16S rRNA or the encoding DNA; and
bases 405-480 of E. coli 16S rRNA or the encoding DNA, and wherein the percent similarity in the rRNA sequences of said non-target species and said target species is between 90 and 99%.
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Abstract
A method for preparing probes, as well as several probes for use in qualitative or quantitative hybridization assays are disclosed. The method comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a region of rRNA selected to be unique to a non-viral organism or group of non-viral organisms sought to be detected, said region of rRNA being selected by comparing one or more variable region rRNA sequences of said non-viral organism or group of non-viral organisms with one or more variable region rRNA sequences from one or more non-viral organisms sought to be distinguished. Hybridization assay probes for Mycobacterium avium, Mycobacterium intracellulare, the Mycobacterium tuberculosis-complex bacteria, Mycoplasma pneumoniae, Legionella, Salmonella, Chlamydia trachomatis, Campylobacter, Proteus mirabilis, Enterococcus, Enterobacter cloacae, E. coli, Pseudomonas group I, Neisseria gonorrhoeae, bacteria, and fungi also are disclosed.
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Citations
56 Claims
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1. A method for making a probe for use in a hybridization assay which comprises constructing a oligonucleotide that is sufficiently complementary to hybridize to a region of rRNA, or the encoding DNA, selected to distinguish one or more non-viral target species from at least one non-viral non-target species, said oligonucleotide comprising a target-complementary sequence obtained by:
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a) aligning rRNA sequences, or the encoding DNA sequences, of said target species and said non-target species to identify a variable region; and
b) designing said target-complementary sequence of said oligonucleotide by substantially maximizing the complementarity of said oligonucleotide to said variable region present in said target species while substantially minimizing the complementarity of said oligonucleotide to said variable region present in said non-target species, such that a duplex formed between said oligonucleotide and said target species has a higher Tm than a duplex formed between said oligonucleotide and said non-target species, wherein said variable region is located in an rRNA sequence or DNA sequence corresponding to a target region selected from the group consisting of;
bases 60-100 of E. coli 16S rRNA or the encoding DNA; and
bases 405-480 of E. coli 16S rRNA or the encoding DNA, and wherein the percent similarity in the rRNA sequences of said non-target species and said target species is between 90 and 99%. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method for making a probe for use in a hybridization assay to distinguish two or more non-viral target species of a target group of non-viral organisms from one or more non-viral non-target species which comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a variable region of rRNA, or the encoding DNA, present in said target species, said oligonucleotide comprising a target-complementary sequence obtained by:
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a) aligning rRNA sequences, or the encoding DNA sequences, of said target species and said non-target species, so as to reveal inter-species hyper-variability present in said variable region; and
b) designing said target-complementary sequence of said oligonucleotide by substantially maximizing the complementarity of said oligonucleotide to said variable region present in said target species while substantially minimizing the complementarity of said oligonucleotide to said variable region present in said non-target species, such that a duplex formed between said oligonucleotide and each of said target species has a higher Tm than a duplex formed between said oligonucleotide and said non-target species, wherein said variable region is located in an rRNA sequence or DNA sequence corresponding to a target region selected from the group consisting of;
bases 60-100 of E. coli 16S rRNA or the encoding DNA;
bases 405-480 of E. coli 16S rRNA or the encoding DNA; and
bases 980-1050 of E. coli 16S rRNA or the encoding DNA. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for making an oligonucleotide probe able to distinguish between a non-viral target species and a non-viral non-target species belonging to the same genus, which comprises producing said probe to comprise a nucleotide sequence designed by:
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a) aligning a variable region present in said target species and said non-target species, wherein said variable region is located in an rRNA sequence, or the encoding DNA sequence, corresponding to a target region selected from the group consisting of;
bases 60-100 of E. coli 16S rRNA or the encoding DNA;
bases 120-150 of E. coli 16S rRNA or the encoding DNA;
bases 170-230 of E. coli 16S rRNA or the encoding DNA;
bases 405-480 of E. coli 16S rRNA or the encoding DNA;
bases 600-670 of E. coli 16S rRNA or the encoding DNA;
bases 980-1050 of E. coli 16S rRNA or the encoding DNA; and
b) substantially maximizing the complementarity of said nucleotide sequence to said variable region present in said target species while substantially minimizing the complementarity of said nucleotide sequence to said variable region present in said non-target species, such that a duplex formed between said probe and said target species has a higher Tm than a duplex formed between said probe and said non-target species. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
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38. A method for making an oligonucleotide probe able to distinguish two or more non-viral target species belonging to a first genus from one or more non-viral non-target species belonging to a second genus, which comprises producing said probe to comprise a nucleotide sequence designed by:
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a) aligning a variable region present in each of said target species and in said non-target species, wherein said variable region is located in an rRNA sequence, or the encoding DNA sequence, corresponding to a target region selected from the group consisting of;
bases 60-100 of E. coli 16S rRNA or the encoding DNA;
bases 120-150 of E. coli 16S rRNA or the encoding DNA;
bases 170-230 of E. coli 16S rRNA or the encoding DNA;
bases 405-480 of E. coli 16S rRNA or the encoding DNA;
bases 600-670 of E. coli 16S rRNA or the encoding DNA;
bases 820-860 of E. coli 16S rRNA or the encoding DNA;
bases 980-1050 of E. coli 16S rRNA or the encoding DNA; and
b) substantially maximizing the complementarity of said nucleotide sequence to said variable region present in each of said target species while substantially minimizing the complementarity of said nucleotide sequence to said variable region present in said non-target species, such that a duplex formed between said probe and each of said target species has a higher Tm than a duplex formed between said probe and said non-target species, wherein the percent similarity between the rRNA of said first genus and said second genus is between 90% and 99%. - View Dependent Claims (39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)
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Specification