Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-06-06
2001-04-03
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06210876
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to
Chlamydia pneumoniae
and, in particular, it relates to oligonucleotides for detecting
Chlamydia pneumoniae
in a test sample.
BACKGROUND OF THE INVENTION
Three species within the genus Chlamydia are clinically important because of their ability to infect and cause disease in a human host.
Chlamydia trachomatis
has been reported as the most common sexually transmitted disease in industrial societies and causes genital infections in both men and women.
Chlamydia psittaci
is responsible for a variety of respiratory tract infections. The most recently characterized and clinically important member of the Chlamydia genus is
Chlamydia pneumoniae
(
C. pneumoniae
) which also is responsible for respiratory tract infections and has been associated with coronary artery disease.
Perhaps because of its fairly recent characterization, the predominant methods for detecting
C. pneumoniae
in a test sample include isolation of the organism in culture, and serology testing. Isolation may include growing the organism in tissue culture cells to produce inclusion bodies which are then detected by fluorescently staining the inclusion bodies using a labeled species-specific-antibody. Serological testing requires two samples from an individual suspected of being infected with
C. pneumoniae.
Two samples are necessary because a significant number of individuals have antibodies to
C. pneumoniae
and a rise in antibody titer to
C. pneumoniae
or a change in antibody class (e.g. IgM to IgG) is measured as an indication of a recent
C. pneumoniae
infection. Because a rise in antibody titer or a change in antibody class is measured, acute and convalescent serum samples are taken. Unfortunately, these samples are often times taken weeks or even months apart. Hence, detecting a
C. pneumoniae
infection can be a time consuming process. Accordingly, there is a need for methods and reagents capable of detecting
C. pneumoniae
in a specific and timely manner.
SUMMARY OF THE INVENTION
The present invention provides nucleic acid sequences that can be used to specifically detect
C. pneumoniae
by using these sequences as oligonucleotide probes and/or primers. Such primers or probes are designated SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO7, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14. Those skilled in the art will recognize that homologs of these sequences and combinations of these sequences can also be employed to detect
C. pneumoniae
in a test sample. Preferably, the sequences are employed in amplification reactions and can be provided in kits along with other reagents for performing an amplification reaction.
Methods provided by the present invention include hybridization assays as well as amplification based assays. Thus, according to one method, a method of detecting the presence of
C. pneumoniae
in a test sample may comprise the steps of (a) contacting the test sample with one or more of the sequences listed above, or their homologs; and (b) detecting hybridization between the above sequences and a
C. pneumoniae
target sequence as an indication of the presence of
C. pneumoniae
in the test sample.
According to another embodiment, a method for detecting the presence of
C. pneumoniae
in a test sample may comprise the steps of (a) forming a reaction mixture comprising nucleic acid amplification reagents, a test sample containing a
C. pneumoniae
target sequence, and at least one primer and one probe oligonucleotide selected from the group consisting of SEQ ID NOs. 2 and 5; SEQ ID NOs. 3 and 4; SEQ ID NOs. 2, 3 and 4; SEQ ID NOs. 2, 3 and 5; SEQ ID NOs. 2, 3, 4 and 5; SEQ ID NOs. 9 and 11; SEQ ID NOs. 10 and 12; SEQ ID NOs. 9, 10 and 11; SEQ ID NOs. 9, 10 and 12; or SEQ ID NOs. 9, 10, 11 and 12; (b) subjecting the mixture to hybridization conditions to generate at least one nucleic acid sequence complementary to the target sequence; (c) hybridizing the probe to the nucleic acid sequence complementary to the target sequence, so as to form a complex comprising the probe and the complementary nucleic acid sequence; and (d) detecting the so-formed complex as an indication of the presence of
C. pneumoniae
in the sample.
According to another embodiment, the invention provides kits which comprise a set of oligonucleotide primers and probes, selected from the SEQ ID NOs. listed above, and amplification reagents.
DETAILED DESCRIPTION OF THE INVENTION
As previously mentioned, the present invention provides nucleic acid sequences, methods for using these sequences and kits containing these sequences, all of which can be employed to specifically detect
C. pneumoniae.
The sequences provided are designated herein as SEQ ID NOs. 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14 and homologs thereof. These sequences are derived from a
C. pneumoniae
gene encoding a cysteine rich outer-membrane-protein (OMP) disclosed in Watson, M. W., et. al,
Journal of Clinical Microbiology,
29(6) p. 1188-1193 (1991) and a
C. pneumoniae
gene encoding a 76 Kilodalton protein (76 kD protein) disclosed in Perez-Melgosa, M., et. al.,
Infection and Immunity,
62(3) p. 880-886 (1994).
With respect to the sequences herein provided, the term “homologs” means those sequences sharing about 80% homology with SEQ ID NOs. 2-7 and 9-14, and more preferably those sequences that share about 90% homology with SEQ ID NOs. 2-7 and 9-14. Thus, sequences that contain about 80% homology with the sequences provided herein and specifically hybridize with
C. pneumoniae
are intended to be within the scope of the present invention. For example, extensions of the present sequences, sequences that are shorter than the present sequences but contain a subset of the present sequences, and those sequences that deviate from the present sequences by minor base substitutions are contemplated as within the scope of the present invention.
Those skilled in the art will recognize various modifications that can be made to the sequences designated SEQ ID NOs. 2-7 and 9-14 without departing from their ability to specifically detect
C. pneumoniae
and share about 80% homology with these sequences. For example, 3′ or 5′ extensions of the present sequences with bases that are complementary to succeeding or preceding bases in either the OMP gene or 76 kD protein gene are considered to be homologs of the present sequences when they share about 80% homology with the present sequences and specifically detect
C. pneumoniae.
Additionally, 3′ or 5′ extensions of present sequences with bases that are not complementary to succeeding or preceding bases in the OMP gene or 76 kD protein gene that share about 80% homology with the present sequences and specifically detect
C. pneumoniae
are contemplated as within the scope of the present invention. Further, base substitutions can be made to SEQ ID NOs. 2-7 and 9-14, but these modified sequences will nevertheless maintain the ability to specifically hybridize with
C. pneumoniae
and share about 80% homology with SEQ ID NOs. 2-7 and 9-14. They are therefore contemplated as within the scope of the present invention. Moreover, sequences that contain about 80% of the sequences designated SEQ ID NOs. 2-7 and 9-14 but have bases deleted from the 3′ or 5′ end, are considered to be within the scope of the term homolog.
The sequences disclosed herein, as well as their homologs, may comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or nucleic acid analogs such as uncharged nucleic acid analogs including but not limited to peptide nucleic acids (PNAs) which are disclosed in International Patent Application WO 92/20702 or morpholino analogs which are described in U.S. Pat. No. 5,185,444, 5,034,506, and 5,142,047 all of which are herein incorporated by reference. Such sequences can routinely be synthesized using a variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistr
Abbott Laboratories
Jones W. Gary
Souaya Jehanne
Yasger Paul D.
LandOfFree
Nucleic acid primers and probes for detecting Chlamydia... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acid primers and probes for detecting Chlamydia..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid primers and probes for detecting Chlamydia... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2503699