Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-12
2001-04-17
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S024330
Reexamination Certificate
active
06218125
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for determining the presence or absence of
Chlamydia trachomatis
in patients. The method involves using nucleic acid primers to amplify specifically the
Chlamydia trachomatis
cryptic plasmid, preferably using one of the techniques of Strand Displacement Amplification (SDA), thermophilic Strand Displacement Amplification (tSDA) or fluorescent real time thermal Strand Displacement Amplification.
BACKGROUND OF THE INVENTION
Chlamydia trachomatis
is the causative agent of trachoma (which is the greatest single cause of blindness), inclusion conjunctivitis, infant pneumonitis, urethritis and lymphogranuloma venereum. Diagnosis and detection of this organism is often on the basis of the pathologic or clinical findings and may be confirmed by isolation and staining techniques.
C. trachomatis
includes a cryptic plasmid which is approximately 7.5 kb in size and is present in multiple copies in the organism. The presence of multiple copies makes this plasmid a good target for diagnostic purposes for assays using nucleic acid amplification techniques.
The following terms are defined herein as follows:
An amplification primer is a primer for amplification of a target sequence by extension of the primer after hybridization to the target sequence. Amplification primers are typically about 10-75 nucleotides in length, preferably about 15-50 nucleotides in length. The total length of an amplification primer for SDA is typically about 25-50 nucleotides. The 3′ end of an SDA amplification primer (the target binding sequence) hybridizes at the 5′ end of the target sequence. The target binding sequence is about 10-25 nucleotides in length and confers hybridization specificity on the amplification primer. The SDA amplification primer further comprises a recognition site for a restriction endonuclease 5′ to the target binding sequence. The recognition site is for a restriction endonuclease which will nick one strand of a DNA duplex when the recognition site is hemimodified, as described by G. Walker, et al. (1992.
PNAS
89:392-396 and 1992
Nucl. Acids Res.
20:1691-1696). The nucleotides 5′ to the restriction endonuclease recognition site (the “tail”) function as a polymerase repriming site when the remainder of the amplification primer is nicked and displaced during SDA. The repriming function of the tail nucleotides sustains the SDA reaction and allows synthesis of multiple amplicons from a single target molecule. The tail is typically about 10-25 nucleotides in length. Its length and sequence are generally not critical and can be routinely selected and modified. As the target binding sequence is the portion of a primer which determines its target-specificity, for amplification methods which do not require specialized sequences at the ends of the target the amplification primer generally consists essentially of only the target binding sequence. For amplification methods which require specialized sequences appended to the target other than the nickable restriction endonuclease recognition site and the tail of SDA (e.g., an RNA polymerase promoter for 3SR, NASBA or transcription based amplification), the required specialized sequence may be linked to the target binding sequence using routine methods for preparation of oligonucleotides without altering the hybridization specificity of the primer.
A bumper primer or external primer is a primer used to displace primer extension products in isothermal amplification reactions. The bumper primer anneals to a target sequence upstream of the amplification primer such that extension of the bumper primer displaces the downstream amplification primer and its extension product.
The terms target or target sequence refer to nucleic acid sequences to be amplified. These include the original nucleic acid sequence to be amplified, the complementary second strand of the original nucleic acid sequence to be amplified and either strand of a copy of the original sequence which is produced by the amplification reaction. These copies serve as amplifiable targets by virtue of the fact that they contain copies of the sequence to which the amplification primers hybridize.
Copies of the target sequence which are generated during the amplification reaction are referred to as amplification products, amplimers or amplicons.
The term extension product refers to the copy of a target sequence produced by hybridization of a primer and extension of the primer by polymerase using the target sequence as a template.
The term species-specific refers to detection, amplification or oligonucleotide hybridization in a species of organism or a group of related species without substantial detection, amplification or oligonucleotide hybridization in other species of the same genus or species of a different genus.
The term assay probe refers to any oligonucleotide used to facilitate detection or identification of a nucleic acid. For example, in the present invention, assay probes are used for detection or identification of
C. trachomatis
cryptic plasmid nucleic acids. Detector probes, detector primers, capture probes and signal primers as described below are examples of assay probes.
SUMMARY OF THE INVENTION
The present invention provides oligonucleotides useful as amplification primers and assay probes for specific detection and identification of
Chlamydia trachomatis.
The specific probes amplify the
C. trachomatis
cryptic plasmid with little or no detectable amplification of either human DNA or DNA of other microorganisms. Two regions of the
C. trachomatis
cryptic plasmid were selected to develop nucleic acid primers which would specifically amplify this plasmid without showing crossreactivity with human DNA or other microorganism DNA.
The oligonucleotides of the invention may be used after culture as a means for confirming the identity of the cultured organism. Alternatively, they may be used prior to culture or in place of culture for detection and identification of
C. trachomatis
cryptic plasmid nucleic acid using known amplification methods. In either case, the inventive oligonucleotides and assay methods provide a means for rapidly discriminating between
C. trachomatis
and other microorganisms, allowing the practitioner to identify rapidly this microorganism without resorting to the more traditional procedures customarily relied upon. Such rapid identification of the specific etiological agent involved in an infection provides information which can be used to determine appropriate therapy within a short period of time.
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Spears, Patricia A. et al., Analytical Biochemistry; 247, pp. 130-137 (1997).
Walker, G.T. et al., Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system;PNAS,89, pp. 392-396 (1992).
Walker, G.T. et al., Strand Displacement amplification—an isothermal, in vitro DNA amplification technique;Nucl. Acids Res.,20, pp. 1691-1696 (1992).
Berger Dolores M.
Foxall Paul A.
Becton Dickinson and Company
Highet David W.
Low Christopher S. F.
Srivastava Devesh
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