Methods for making oligonucleotide probes for the detection and/or quantitation of non-viral organisms

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 a variable region of rRNA, or the encoding DNA, present in said two or more target species, said oligonucleotide comprising a target-complementary sequence obtained by:

• a) aligning rRNA, or the encoding DNA, sequences of said two or more target species with rRNA, or the encoding DNA, sequences of said one or more nontarget species, so as to reveal inter-species hypervariability present in said variable region;
• and
• b) designing said target-complementary sequence of said oligonucleotide by substantially maximizing complementarity of said oligonucleotide to said variable region present in said two or more target species while substantially minimizing the complementarity of said oligonucleotide to said variable region present in said one or more non-target species, such that a duplex formed between said oligonucleotide and each of said two or more target species has a higher T.sub.m than a duplex formed between said oligonucleotide and said one or more non-target species,
• wherein said variable region is located in an rRNA sequence, or a DNA sequence encoding for said rRNA sequence, corresponding to a target region selected from the group consisting of:
• bases 185-225 of E. coli 16S rRNA or the encoding DNA;
• bases 630-675 of E. coli 16S rRNA or the encoding DNA;
• bases 825-860 of E. coli 16S rRNA or the encoding DNA;
• bases 975-1020 of E. coli 16S rRNA or the encoding DNA;
• bases 980-1010 of E. coli 16S rRNA or the encoding DNA;
• bases 1025-1060 of E. coli 16S rRNA or the encoding DNA;
• bases 270-390 of E. coli 23S rRNA or the encoding DNA;
• bases 535-560 of E. coli 23S rRNA or the encoding DNA;
• bases 1150-1200 of E. coli 23S rRNA or the encoding DNA;
• bases 1440-1600 of E. coli 23S rRNA or the encoding DNA;
• bases 1710-1750 of E. coli 23S rRNA or the encoding DNA;
• and
• bases 2190-2330 of E. coli 23S rRNA or the encoding DNA;
• provided that if said target region corresponds to bases 185-225 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Mycobacterium tuberculosis complex organisms,
• if said target region corresponds to bases 630-675 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Legionella organisms,
• if said target region corresponds to bases 825-860 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are group D Streptococcus organisms selected from the group consisting of Streptococcus faecium, Streptococcus faecalis and Streptococcus avium,
• if said target region corresponds to bases 975-1020 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Legionella organisms,
• if said target region corresponds to bases 980-1010 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Campylobacter organisms, and
• if said target region corresponds to bases 1025-1060 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Mycobacterium organisms.

The 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 oligonucleotide to comprise a nucleotide sequence designed by:

• 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 a DNA sequence encoding for said rRNA sequence, corresponding to a target region selected from the group consisting of:
• bases 65-120 of E. coli 5S rRNA or the encoding DNA;
• bases 60-105 of E. coli 16S rRNA or the encoding DNA;
• bases 125-150 of E. coli 16S rRNA or the encoding DNA;
• bases 175-210 of E. coli 16S rRNA or the encoding DNA;
• bases 185-225 of E. coli 16S rRNA or the encoding DNA;
• bases 190-230 of E. coli 16S rRNA or the encoding DNA;
• bases 450-490 of E. coli 16S rRNA or the encoding DNA;
• bases 455-485 of E. coli 16S rRNA or the encoding DNA;
• bases 600-635 of E. coli 16S rRNA or the encoding DNA;
• bases 820-860 of E. coli 16S rRNA or the encoding DNA;
• bases 830-870 of E. coli 16S rRNA or the encoding DNA;
• bases 980-1015 of E. coli 16S rRNA or the encoding DNA;
• bases 995-1030 of E. coli 16S rRNA or the encoding DNA;
• bases 1255-1290 of E. coli 16S rRNA or the encoding DNA;
• bases 270-390 of E. coli 23S rRNA or the encoding DNA;
• bases 535-560 of E. coli 23S rRNA or the encoding DNA;
• bases 1150-1200 of E. coli 23S rRNA or the encoding DNA;
• bases 1440-1600 of E. coli 23S rRNA or the encoding DNA;
• bases 1710-1750 of E. coli 23S rRNA or the encoding DNA;
• and
• bases 2190-2330 of E. coli 23S rRNA or the encoding DNA;
• provided that if said target region corresponds to bases 65-120 of E. coli 5S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,
• if said target region corresponds to bases 60-150 of E. coli 16S rRNA or the encoding DNA, then said target species is Chlamydia trachomatis,
• if said target region corresponds to bases 125-150 of E. coli 16S rRNA or the encoding DNA, then said target species is Neisseria gonorrhoeae;
• if said target region corresponds to bases 185-225 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycobacterium avium or Mycobaterium intracellulare,
• if said target region corresponds to bases 190-230 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,
• if said target region corresponds to bases 450-490 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,
• if said target region region corresponds to bases 450-490 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,
• if said target region corresponds to bases 455-485 of E. coli 16S rRNA or the encoding DNA, then said target species is Neisseria gonorrhoeae,
• if said target region corresponds to bases 600-636 of E. coli 16S rRNA or the encoding DNA, then said target species is Chlamydia trachomatis,
• if said target region corresponds to bases 820-860 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,
• if said target region corresponds to bases 830-870 of E. coli 16S rRNA or the encoding DNA, then said target species is Chlamydia trachomatis,
• if said target region corresponds to bases 980-1015 of E. coli 16S rRNA or the encoding DNA, then said target species is Neisseria gonorrhoeae,
• if said target region corresponds to bases 995-1030 of E. coli 16S rRNA or the encoding DNA, then said target species is Escherichia coli, and
• if said target region corresponds to bases 1255-1290 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae;
• and
• b) substantially maximizing complementarity of said nucleotide sequence to said variable region present in said target species while substantially minimizing complementarity of said nucleotide sequence 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 T.sub.m than a duplex formed between said oligonucleotide and said non-target species.

A method for making an oligonucleotide provbe 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 oligonucleotide probe to comprise a nucleotide sequence designed by:

• a) aligning a variable region present in each of said two or more target species and in said one or more non-target species, wherein said variable region is located in an rRNA sequence, or a DNA sequence encoding for said rRNA sequence, corresponding to a target region selected from the group consisting of:
• bases 185-225 of E. coli 16S rRNA or the encoding DNA;
• bases 405-428 of E. coli 16S rRNA or the encoding DNA;
• bases 440-475 of E. coli 16S rRNA or the encoding DNA;
• bases 630-675 of E. coli 16S rRNA or the encoding DNA;
• bases 705-735 of E. coli 16S rRNA or the encoding DNA;
• bases 825-860 of E. coli 16S rRNA or the encoding DNA;
• bases 975-1020 of E. coli 16S rRNA or the encoding DNA;
• bases 980-1010 of E. coli 16S rRNA or the encoding DNA;
• bases 1025-1060 of E. coli 16S rRNA or the encoding DNA;
• bases 1125-1155 of E. coli 16S rRNA or the encoding DNA;
• bases 270-390 of E. coli 23S rRNA or the encoding DNA;
• bases 350-395 of E. coli 23S rRNA or the encoding DNA;
• bases 365-405 of E. coli 23S rRNA or the encoding DNA;
• bases 535-560 of E. coli 23S rRNA or the encoding DNA;
• bases 540-575 of E. coli 23S rRNA or the encoding DNA;
• bases 1150-1200 of E. coli 23S rRNA or the encoding DNA;
• bases 1440-1600 of E. coli 23S rRNA or the encoding DNA;
• bases 1570-1610 of E. coli 23S rRNA or the encoding DNA;
• bases 1585-1620 of E. coli 23S rRNA or the encoding DNA;
• bases 1710-1750 of E. coli 23S rRNA or the encoding DNA;
• and
• bases 2190-2330 of E. coli 23S rRNA or the encoding DNA;
• provided that if said target region corresponds to bases 185-225 of E. coli 16S rRNA or the encoding DNA, then said first genus is Mycobacterium;
• if said target region corresponds to bases 405-428 of E. coli 16S rRNA or the encoding DNA, then said first genus is Campylobacter,
• if said target region corresponds to bases 440-475 of E. coli 16rRNA or the encoding DNA, then said first genus is Campylobacter,
• if said target region corersponds to bases 630-675 of E. coli 16S rRNA or the encoding DNA, then said first genus is Legionella,
• if said target region corresponds to bases 705-735 of E. coli 16S rRNA or the encoding DNA, then said first genus is Campylobacter,
• if said target region corresponds to bases 825-860 of i E. coli 16S rRNA or the encoding DNA, then said first genus is Streptococcus,
• if said target region corresponds to bases 975-1020 of E. coli 16S rRNA or the encoding DNA, then said first genus is Legionella,
• if said target region corresponds to bases 980-1010 of E. coli 16S rRNA or the encoding DNA, then said first genus is Campylobacter,
• if said target region corresponds to bases 1025-1060 of E. coli 16S rRNA or the encoding DNA, then said first genus Mycobacterium,
• if said target region corresponds to bases 1125-1155 of E. coli 16S rRNA or the encoding DNA, then said first genus is Salmonella,
• if said target region corresponds to bases 350-395 of E. coli 23S rRNA or the encoding DNA, then said first genus is Legionella,
• if said target region corresponds to bases 365-405 of E. coli 23S rRNA or the encoding DNA, then said first genus is Pseudomonas,
• if said target region corresponds to bases 540-575 of E. coli 23S rRNA or the encodng DNA, then said first genus is Mycobacterium,
• if said targer region corresponds to bases 1570-1610 of E. coli 23S rRNA or the encoding DNA, then said first genus is Mycobacterium, and
• if said target region corresponds to bases 1585-1620 of E. coli 23S rRNA or the encoding DNA, then said first genus is Legionella;
• and
• b) substantially maximizing complementarity of said nucleotide sequence to said variable region present in each of said two or more target species while substantially minimizing the complementarity of said nucleotide sequence to said variable region present in said one or more non-target species, such that a probe:target duplex formed between said oligonucleotide probe and each of said two or more target species has a higher T.sub.m than a duplex formed between said oligonucletodide probe and said one or more non-target species;
• wherein the percent similarity between the rRNA of said first genus and said second genus is between 90% and 99%.