Detection of Ribosomal RNA using PNA probes
AssigneeDako A/S
InventorsJens J.o slashed.rgen Hyldig-Nielsen, Michael Anders Godskesen
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Abstract
PNA probes for detection of Neisseria gonorrhoeae and Chlamydia trachomatis. Specific peptide nucleic acid (PNA) probes for detecting a sexual transmitted disease caused by Neisseria gonorrhoeae or Chlamydia trachomatis comprising N-(2-aminoethyl)glycine units in amide linkage with the glycine nitrogen connected to naturally occurring nucleobases or non-naturally occurring nucleobases by a methylene carbonyl linker and said probes capable of hybridizing to 16S or 23S rRNA or rDNA of Neisseria gonorrhoeae or Chlamydia trachomatis are described. PNA is a very stable molecule with very high affinity for nucleic acid allowing a PNA probe to be shorter than conventional nucleic acid probes.
Claims38
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.
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.
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.
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.
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.
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.
Dependencies are inferred from the claim text.