Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Utility Patent
1995-05-18
2001-01-02
Riley, Jezia (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C514S04400A, C536S023100, C536S024310, C536S025320
Utility Patent
active
06169169
ABSTRACT:
The present invention relates to specific peptide nucleic acid (PNA) probes and methods for detecting a sexual transmitted disease caused by Neisseria gonorrhoeae or Chlamydia trachomatis. More particularly, the invention relates to peptide nucleic acid (PNA) probes capable of hybridizing to 16S or 23S rRNA or DNA from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis in test samples which may contain Neisseria gonorrhoeae and/or Chlamydia trachomatis.
N. gonorrhoeae, the pathogen of gonorrhoea, is still today a very frequent infectious disease worldwide. In males, the genital infection manifests itself as a purulent inflammation and swelling of the urethra. These symptoms occur in 90% of cases of infection. If left untreated, the infection can ascend and after several weeks produce symptoms of prostatitis. In women, no or only slight symptoms occur in 50% of cases of infection. The infection primarily affects the cervix, but also the urethra. In 10 to 15% of women, the infection spreads to the fallopian tubes and can also lead, inter alia, to sterility. Since the course of the infections is often asymptomatic, many carriers contribute unknowingly to the spread of the disease.
Another very wide spread sexual transmitted disease is caused by Chlamydia trachomatis. Among the more serious complications of C. trachomatis infections are ecotropic pregnancy and tubal infertility.
Considering the impact that these two organisms have, rapid and specific diagnostic tests are of utmost importance.
Diagnosis based on selective growth of the pathogenic bacteria is still the “golden standard”, but cell culturing is time-consuming and many clinical isolates are difficult to grow in vitro.
Some of the attempts to replace the slow methods based on cell culturing with faster methods have been based on nucleic acid hybridization using target specific probes.
Attempts have also been made to use nucleic acid probes for diagnosis of infections caused by Neisseria gonorrhoeae or Chlamydia trachomatis.
Nucleic acid probes have to fulfil two criteria in order to be used diagnostically. They must be specific, i.e. the probe should only hybridize to the nucleic acid of the pathogen to be detected in order to exclude false positive test results. They must also be sensitive, i.e. the detection of only a few target molecules should also be possible in order to exclude false negative test results during the early or persistive stages of the infection. It is possible to specifically detect organisms using nucleic acid probes which are complementary to ribosomal RNA (rRNA). Methods based on probes directed against rRNA are very sensitive as many target molecules (10
3
-10
4
copies) are present in each cell.
Methods for identification of organisms using rRNA specific nucleic acid probes have been described in different publications, see e.g. EP-B 0 131 052.
Nucleic acid probes for detection of rRNA of Neisseria gonorrhoeae have been described in EP-A 0 272 009, EP-A 0 408 077 and EP-A 0 574 852. Nucleic acid probes for detection rRNA of Chlamydia trachomatis have been described in WO 90/15159.
Prokaryotic organisms have rRNA molecules which include 5S, 16S and 23S rRNA. Particularly probes complementary to the 16S and/or 23S rRNAs have been used for hybridization as it is known that species variable regions exist within these highly conserved sequences which can be used for species specific probes. However, as rRNAs are generally highly conserved between related species, it will often be difficult to define nucleic acid probes sufficiently specific in terms of species to be able to obtain the required specificity and sensitivity. Another consequence of the conserved character of the rRNA is that the differentiation between two organisms is often based on only one or a few mismatches only in the target sequence which puts high constraints on the stringency needed in the hybridization reaction. A slight deviation from these conditions may result in misidentification.
A very high degree of sequence homology between rRNA of different Neisseria strains makes it difficult to define species specific probes. To obtain the necessary specificity, it is an advantage to use probes as small as possible where the sequence difference between the species makes up a significant proportion of the probe. Typically used nucleic acid probes comprising less than 20 nucleotides are often unable to give reliable results as the melting temperature, T
m
, for typically used nucleic acid complexes is too low.
The melting temperature, T
m
, refers to the temperature at which the strands of a nucleic acid hybrid are half dissociated or denatured.
One of the objects of the present invention was, therefore, to provide relative short species specific probes for detecting Neisseria gonorrhoeae or Chlamydia trachomatis without simultaneously compromising the robustness and sensitivity of the assay.
This object is achieved by selecting a peptide nucleic acid (PNA) probe comprising N-(2-aminoethyl)glycine units in amide linkage with the glycine nitrogen connected to purine or pyrimidine bases by a methylene carbonyl linker and capable of hybridizing to 16S or 23S rRNA or DNA from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis.
The present PNA probes form complexes with complementary nucleic acids which complexes are considerably more stable (higher T
m
value) than would be a similar hybrid formed by a typically used nucleic acid probe of corresponding sequence allowing sensitive assays to be made with shorter probes than is the case of typical nucleic acid probes used today.
Hybridization with traditionally used nucleic acid probes is much faster in solution than in solid phase hybridization. Due to the high affinity of PNA for nucleic acid, even solid phase hybridization between PNA probes and nucleic acid can be performed rapidly allowing greater flexibility in the assay format. Hybridization efficiency is only slightly influenced by pH and salt concentration in the hybridization solution allowing PNAs to hybridize under conditions not favourable for ordinary DNA probes.
Furthermore, PNAs have a higher thermal instability of mismatching bases whereby PNAs exhibit a greater specificity for their complementary nucleic acids than traditionally used nucleic acid probes of corresponding sequence would do (WO 92/20703).
The structure of PNA is not degraded by nucleases or proteases making the PNA molecule very stable in biological solutions.
The present invention relates to PNA probes for detecting Neisseria gonorrhoeae or Chlamydia trachomatis comprising from 6 to 30 N-(2-aminoethyl)glycine units, particularly from 8 to 20 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 or connected to a labelling group and said probe capable of hybridizing to 16S or 23S rRNA or DNA from the area coding for said rRNA of Neisseria gonorrhoeae or Chlamydia trachomatis.
In addition to the backbone and bases, the PNA probes according to the invention may optionally have pendant groups, usually at the termini, to stabilize the end, to label the probe or to increase the solubility.
Furthermore, the invention relates to a method for the detection of sexual transmitted diseases caused by Neisseria gonorrhoeae or Chlamydia trachomatis using at least one PNA probe under hybridization conditions followed by detection of the hybrid formation, which method is characterized in that one or more of the PNA probes according to the present invention is used for specific detection.
A further object of the present invention is test kits for use in the detection of diseases caused by Neisseria gonorrhoeae and/or Chlamydia trachomatis, comprising at least one PNA probe according to the invention and at least one detection reagent.
Peptide nucleic acids (PNAs) are described in WO 92/20702 as compounds comprising a polyamide backbone bearing a plurality of ligands such as naturally oc
Godskesen Michael Anders
Hyldig-Nielsen Jens Jørgen
Dako A/S
Graham & James LLP
Riley Jezia
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