Claims
- 1. A method of detecting a sexually transmitted disease caused by Neisseria gonorrhoeae or Chlamydia trachomatis comprising contacting a rRNA or DNA molecule containing sample from a cell sample suspected of containing said rRNA or DNA with a peptide nucleic acid hybridization probe of formula 1, under conditions such that hybridization between said peptide nucleic acid probe and any complementary sample rRNA or DNA sequences coding for said rRNA of Neisseria gonorrhea or Chlamydia trachomatis in said sample, can occur, and detecting binding between said peptide nucleic acid probe and said rRNA or DNA in said sample as indication of presence of said Neisseria gonorrhea or Chlamydia trachomatis in said sample, wherein said peptide nucleic acid probe is specific for one of Neisseria gonorrhea or Chlamydia trachomatis; and ##STR5## wherein f is an integer from 6-30,
- each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine, and uracil, and non-naturally occurring nucleobases, in a sequence enabling the probe to hybridize to 16S or 23S rRNA or DNA sequences from the area coding for said rRNA of Neisseria gonorrhea or Chlamydia trachomatis, each X may be identical or different and selected from a group of hydrophilic linker units, independently selected among --NH(CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 C(O)--, --NH(CHOH).sub.n C(O)--, --(O)C(CH.sub.2).sub.n C(O)--, --(O)C(CH.sub.2 OCH.sub.2)C(O)-- and --NH(CH.sub.2).sub.n C(O)--, wherein n is 0 or integer from 1 to 6, preferably and integer from 1 to 3,
- and wherein
- d is 0 or an integer from 1 to 10,
- e is 0 or an integer from 1 to 3,
- g is 0 or an integer from 1 to 3,
- h is 0 or an integer from 1 to 10,
- R.sup.1 is OH, NH.sub.2 (X).sub.q --L,
- R.sup.2 is H, CH.sub.3 C(O), (X).sub.q --L,
- R.sup.3 is H, the side of a naturally occurring amino acids with the exclusion of Gly, the side chain of a non-naturally occurring alpha amino acid, R'--(X).sub.q or R'--(X).sub.q --l, wherein R' is the side chain of Thr, Ser, Tyr, Cys, His, Asp, Glu, Lys, Trp Orn, or HomoCys, and R.sub.4 is H, the side chain of a naturally occurring amino acid with the exclusion of Gly, the side chain of a non-naturally occurring alpha amino acid, R'--(X).sub.q or R'--(X).sub.q --l, wherein R' is the side chain of Thr, Ser, Tyr, Cys, His, Asp, Glu, Lys, Trp, Orn, or HomoCys, and wherein
- each L may be identical or different and designate a label, and q is 0 or an integer from 1 to 10.
- 2. The method according to claim 1, wherein said probe comprises a sufficient number of nucleobases complementary to the target sequence to form stable hybrids between the PNA probe and the target nucleic acid and a sufficient number of nucleobase mismatches to minimize the hybrid formation between the PNA probe and nucleic acid from closely and distantly related species.
- 3. The method according to claim 2, wherein said probe comprises from 8 to 20 N-(2-aminoethylglycine units.
- 4. The method according to claim 1, wherein
- f is an integer from 8 to 20,
- each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, and non-naturally occurring nucleobases, in a sequence enabling the probe to hybridize to 16S or 23S rRNA or DNA sequences from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis,
- each X may be identical or different and selected from a group of hydrophilic linker units,
- and when
- d is 0 or an integer from 1 to 10,
- e is 0,
- g is 0 or an integer from 1 to 3,
- h is 0 or an integer of 1 to 10, then
- R.sup.1 is OH, NH.sub.2, (X).sub.q or (X).sub.q --L,
- R.sup.2 is H or CH.sub.3 C(O),
- R.sup.3 is H, the side chain of a naturally occurring amino acid with the exclusion of Gly, the side chain of a non-naturally occurring alpha amino acid, R'--(X).sub.q or R'--(X).sub.q --L, wherein R' is the side chain of Thr, Ser, Tyr, Cys, His, Asp, Glu, Lys, Trp, Orn or HomoCys, and
- R.sup.4 is H, the side chain of a naturally occurring amino acid with the exclusion of Gly or the side chain of a non-naturally occurring alpha amino acid,
- or when
- d is 0 or an integer from 1 to 10,
- e is 0 or an integer from 1 to 3,
- g is 0,
- h is 0 or an integer from 1 to 10, then
- R.sup.1 is OH or NH.sub.2,
- R.sup.2 is H, CH.sub.3 C(O), (X).sub.q or (X).sub.q --L,
- R.sup.3 is H, the side chain of a naturally occurring amino acid with the exclusion of Gly or the side chain of a non-naturally occurring alpha amino acid, and
- R.sup.4 is H, the side chain of a naturally occurring amino acid with the exclusion of Gly, the side chain of a non-naturally occurring alpha amino acid, R'--(X).sub.q or R'--(X).sub.q --L, wherein R' is the side chain of Thr, Ser, Tyr, Cys, His, Asp, Glu, Lys, Trp, Orn or HomoCys, and
- each L may be identical or different and designate a label, and
- q is 0 or an integer from 1 to 20.
- 5. The method according to claim 1, wherein
- f is an integer from 8 to 20,
- each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, and non-naturally occurring nucleobases, in a sequence enabling the probe to hybridize to 16S or 23S rRNA or DNA sequences from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis,
- each X may be identical or different and selected from a group of hydrophilic linker units,
- and when
- d is 1,
- e is 0,
- g is 0 or an integer from 1 to 3,
- h is 0 or an integer from 1 to 10, then
- R.sup.1 is OH, NH.sub.2, (X).sub.q or (X).sub.q --L,
- R.sup.2 is H or CH.sub.3 C(O),
- R.sup.3 is H or the side chain of a naturally occurring amino acid with the exclusion of Gly, and
- R.sup.4 is H or the side chain of Thr, Ser, Asn, Asp, Glu, Gln, Lys, Arg or His, or when
- d is 1,
- e is 0,
- g is 0 or an integer from 1 to 3,
- h is an integer of 1 to 10, then
- R.sup.1 is OH or NH.sub.2,
- R.sup.2 is H or CH.sub.3 C(O),
- R.sup.3 is R'--(X).sub.q or R'--(X).sub.q --L, wherein R' is the side chain of Thr, Ser, Tyr, Cys, His, Asp, Glu, Lys or Trp, and
- R.sup.4 is H or the side chain of Thr, Ser, Asn, Asp, Glu, Gln, Lys, Arg or His,
- or when
- d is 0 or an integer from 1 to 10,
- e is 0 or an integer from 1 to 3,
- g and h are 0, then
- R.sup.1 is OH or NH.sub.2,
- R.sup.2 is (X).sub.q or (X).sub.q --L, and
- R.sup.4 is H or the side chain of a naturally occurring amino acid with the exclusion of Gly,
- or when
- d is 0 or an integer from 1 to 10,
- e is 0 or an integer from 1 to 3,
- g and h are 0, then
- R.sup.1 is OH or NH.sub.2,
- R.sup.2 is H or CH.sub.3 C(O), and
- R.sup.4 is R'--(X).sub.q or R'--(X).sub.q --L, wherein R' is the side chain of Thr, Ser, Tyr, Cys, His, Asp, Glu, Lys or Trp, and
- each L may be identical or different and designate a label, and
- q is 0 or an integer from 1 to 20.
- 6. The method according to claim 1, wherein
- f is an integer from 8 to 20,
- each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, and non-naturally occurring nucleobases, in a sequence enabling the probe to hybridize to 16S or 23S rRNA or DNA sequences from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis,
- each X may be identical or different and selected from a group of hydrophilic linker units,
- d and h are 1,
- e is 0,
- g is 0 or an integer from 1 to 3,
- R.sup.1 is NH.sub.2,
- R.sup.2 is H,
- R.sup.3 is R'--(X).sub.q --L, where R' is the side chain of Lys, and
- R.sup.4 is H or the side chain of Thr, Ser, Asn, Asp, Glu, Gin, Lys, Arg or His, and
- each L designates a label, and
- q is 0 or an integer from 1 to 20.
- 7. The method according to claim 1, wherein
- f is an integer from 8 to 20,
- each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, and non-naturally occurring nucleobases, in a sequence enabling the probe to hybridize to 16S or 23S rRNA or DNA sequences from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis,
- each X may be identical or different and selected from a group of hydrophilic linker units,
- d is 1,
- e is 0,
- g is an integer from 1 to 3,
- h is an integer 1 to 10,
- R.sup.1 is NH.sub.2,
- R.sup.2 is H,
- R.sup.3 is H or the side chain of a naturally occurring amino acid with the exclusion of Gly, and
- R.sup.4 is H or the side chain of Thr, Ser, Asn, Asp, Glu, Gin, Lys, Arg or His.
- 8. The method according to claim 1, wherein
- f is an integer from 8 to 20,
- each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, and non-naturally occurring nucleobases, in a sequence enabling the probe to hybridize to 16S or 23S rRNA or DNA sequences from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis,
- each X may be identical or different and selected from a group of hydrophilic linker units,
- d, g and h are 0,
- e is 0 or an integer from 1 to 3,
- R.sup.1 is NH.sub.2, and
- R.sup.2 is L or CH.sub.3 C(O), and
- L designates a label.
- 9. The method according to claim 1, wherein the label L is biotin, 5-(and 6)-carboxyfluorescein, fluorescein isothiocyanate or dinitro benzoic acid.
- 10. The method according to claim 1, wherein
- f is an integer from 13 to 20,
- each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, and non-naturally occurring nucleobases, in a sequence enabling the probe to hybridize to 16S or 23S rRNA or DNA sequences from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis,
- d, e, g and h are 0,
- R.sup.1 is NH.sub.2,
- R.sup.2 is (X).sub.q --L or CH.sub.3 C(O),
- wherein each X is --NH(CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 C(O)--, L is 5-(and 6)-carboxyfluorescein, fluorescein isothiocyanate, biotin or dinitro benzoic acid, n is an integer from 1 to 6, preferably from 1 to 3, and q is an integer from 1 to 3.
- 11. The method according to claim 1 characterized in that the probe is selected among probes of the general formula (Ia) which are probes of the general formula (I) wherein d, e, g and h are 0 ##STR6## wherein each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, and non-naturally occurring nucleobases, in a sequence enabling the probe to hybridize to 16S or 23S rRNA or DNA sequences from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis, and
- f is 20; R.sup.2 is (X).sub.q --L, wherein X is --NH(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C(O)--; q is 1 or 2 and L is biotin;
- f is 18; R.sup.2 is (X).sub.q --L, wherein X is --NH(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C(O)--; q is 1 or 2 and L is biotin;
- f is 18; R.sup.2 is (X).sub.q --L, wherein X is --NH(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C(O)--; q is 1 or 2 and L is 5-(and 6)-carboxyfluorescein or fluorescein isothiocyanate;
- f is 17; R.sup.2 is (X).sub.q --L, wherein X is --NH(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C(O)--; q is 1 or 2 and L is 5-(and 6)-carboxyfluorescein or fluorescein isothiocyanate;
- f is 16; R.sup.2 is (X).sub.q --L, wherein X is --NH(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C(O)--; q is 1 or 2 and L is biotin;
- f is 15; R.sup.2 is (X).sub.q --L, wherein X is --NH(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C(O)--; q is 1 or 2 and L is 5-(and 6)-carboxyfluorescein or fluorescein isothiocyanate;
- f is 15; R.sup.2 is (X).sub.q --L, wherein X is --NH(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C(O)--; q is 1 or 2 and L is dinitro benzoic acid;
- f is 15; R.sup.2 is (X).sub.3, wherein (X).sub.3 is HO(O)CCH.sub.2 C(O)(NH--(CH.sub.2 CH.sub.2 O).sub.2 CH2C(O)).sub.2 --; and
- f is 13; R.sup.2 is (X).sub.q --L, wherein X is --NH(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C(O)--; q is 1 or 2 and L is 5-(and 6)-carboxyfluorescein or fluorescein isothiocyanate.
- 12. The method according to claim 1 for detecting Neisseria gonorrhoeae, wherein each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, in a sequence comprising all or part of one of the Neisseria gonorrhoeae specific sequences 1a to 5a to allow hybridization to target sequences in 16S rRNA of Neisseria gonorrhoeae or to DNA sequences from the area coding for said rRNA.
- 13. The method according to claim 1 for detecting Neisseria gonorrhoeae, wherein each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, in a sequence comprising all or part of one of the Neisseria gonorrhoeae specific sequences 6a to 9a to allow hybridization to target sequences in 23S rRNA of Neisseria gonorrhoeae or to DNA sequences from the area coding for said rRNA.
- 14. The method according to claim 1 for detecting Chlamydia trachomatis, wherein each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, in a sequence comprising all or part of one of the Chlamydia trachomatis specific sequences 10a to 20a to allow hybridization to target sequences in 16S rRNA of Chlamydia trachomatis or to DNA sequences from the area coding for said rRNA.
- 15. The method according to claim 1 for detecting Chlamydia trachomatis, wherein each B is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine and uracil, in a sequence comprising all or part of one of the Chiamydia trachomatis specific sequences 21a to 33a to allow hybridization to target sequences in 23S rRNA of Chlamydia trachomatis or to DNA sequences from the area coding for said rRNA.
- 16. A method according to claim 1, characterized in that the hybridization complex is captured on a solid phase before measuring the extent of hybridization.
- 17. A method according to claim 1, characterized in that a PNA probe according to claim 1 is used in capturing the hybridization complex.
- 18. A method according to claim 1, characterized in that in measuring the resulting hybridization a signal amplifying system is used.
- 19. Kit for detecting Neisseria gonorrhoeae and/or Chlamydia trachomatis, characterized in that it comprises at least one PNA probe and a detection system with at least one detecting reagent, wherein said PNA probe comprises from 6 to 30 N-(2-aminoethyl)glycine units in peptide linkage with the glycine nitrogen connected to naturally occurring nucleobases or non-naturally occurring nucleobases by a methylene carbonyl linker or connected to a labelling group and said probe capable of hybridizing to target sequences in 16S or 23S rRNA or DNA from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis.
- 20. Kit according to claim 19, characterized in that it furthermore comprises a solid phase capture system.
- 21. Kit according to claim 20, characterized in that the detection system comprises a signal amplifying system.
- 22. An assay for detecting rRNA which may be present in a test sample, comprising:
- a. fixing said test sample and thereafter contacting said fixed test sample in situ with a peptide nucleic acid (PNA) probe capable of attaching to said rRNA in said test sample conjugated to an indicator reagent comprising signal generating compound capable of generating a measurable signal; and
- b. detecting said measurable signal as an indication of the presence of rRNA in the test sample.
- 23. The assay of claim 22, wherein quantitation is performed by exciting fluorescence.
- 24. The assay of claim 22, wherein said signal generating compound is fluorescein.
- 25. In a fluorescence in situ hybridization assay for detecting the presence of rRNA which may be present in a test sample comprising the steps of (a) fixing said test sample, (b) hybridizing said rRNA in said test sample with a probe capable of attaching to said rRNA in said test sample conjugated to a signal generating compound capable of generating a measurable signal and (c) detecting the presence of rRNA in said test sample by measuring the detectable signal, wherein the improvement comprises hybridizing said test sample with a peptide nucleic acid (PNA) probe.
- 26. The fluorescence in situ hybridization assay of claim 25, wherein said signal generating compound is fluorescein.
- 27. A method for detecting rRNA in a sample, comprising the steps of:
- (a) contacting a smear of bacterial cells with one or more PNA probes, under conditions suitable for in situ hybridization to occur between the PNA probes(s) and any target rRNA in the sample; and
- (b) detecting any complexes formed between said PNA probes(s) and said bacterial rRNA.
- 28. The method of claim 27 wherein the assay format is fluorescence in situ hybridization.
- 29. The method of claim 28, wherein said PNA probe(s) are labeled with fluorescein.
- 30. A method for detecting an rRNA or rDNA target, comprising the steps of:
- (a) releasing nucleic acid from bacteria present in a test sample;
- (b) contacting one or more detectably labeled PNA probes with rRNA or rDNA target in solution under conditions which promote hybridization between the PNA probe and any target nucleic acid present and
- (c) detecting the PNA-nucleic acid complex formed.
- 31. The method of claim 30, wherein the PNA-nucleic acid complex is immobilized to a solid support.
- 32. The method of claim 31, wherein the PNA-nucleic acid complex is immobilized to a solid support using a capture probe.
- 33. The method of claim 31, wherein the PNA-nucleic acid complex is immobilized to a solid support using a capture antibody specific for PNA-nucleic acid complexes.
- 34. The method of claim 31, wherein said PNA probes(s) are immobilized to a solid support.
- 35. The method of claim 32 wherein, the capture probe is a species specific PNA probe not used in the hybridization reaction for detecting the target nucleic acid.
- 36. The method of claim 30, wherein said PNA-nucleic acid complex is detected using a primary antibody which reacts specifically with PNA-nucleic acid complexes.
- 37. The method of claim 36, wherein said primary antibody comprises a label.
- 38. The method of claim 36, wherein said primary antibody is unlabeled, and the detection system comprises a labeled secondary antibody which specifically binds to said primary antibody.
Priority Claims (1)
Number |
Date |
Country |
Kind |
0572/94 |
May 1994 |
DKX |
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CROSS-REFERENCE
This is a division of Ser. No. 08/443,930 filed on May 18, 1995.
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Divisions (1)
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Number |
Date |
Country |
Parent |
443930 |
May 1995 |
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