Patent Publication Number: US-6210876-B1

Title: Nucleic acid primers and probes for detecting Chlamydia pneumoniae

Description:
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 chemistry and the instruments available from Applied Biosystems, Inc, (Foster City, Calif. ); DuPont, (Wilmington, Del.); or Milligen, (Bedford, Mass.). Similarly, and when desirable, the sequences can be labeled using methodologies well known in the art such as described in U.S. patent applications Nos. 5,464,746; 5,424,414; and 4,948,882 all of which are herein incorporated by reference. 
     The term “label ” as used herein means a molecule or moiety having a property or characteristic which is capable of detection. A label can be directly detectable, as with, for example, radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles and the like; or a label may be indirectly detectable, as with, for example, specific binding members. It will be understood that directly detectable labels may require additional components such as, for example, substrates, triggering reagents, light, and the like to enable detection of the label. When indirectly detectable labels are used, they are typically used in combination with a “conjugate”. A conjugate is typically a specific binding member which has been attached or coupled to a directly detectable label. Coupling chemistries for synthesizing a conjugate are well known in the art and can include, for example, any chemical means and/or physical means that does not destroy the specific binding property of the specific binding member or the detectable property of the label. As used herein, “specific binding member” means a member of a binding pair, i.e., two different molecules where one of the molecules through, for example, chemical or physical means specifically binds to the other molecule. In addition to antigen and antibody specific binding pairs, other specific binding pairs include, but are not intended to be limited to, avidin and biotin; haptens and antibodies specific for haptens; complementary nucleotide sequences; enzyme cofactors or substrates and enzymes; and the like. 
     Generally, the sequences provided herein can be employed to detect the presence of  C. pneumoniae  in a test sample by contacting a test sample with at least one of the sequences provided herein under hybridizing conditions and detecting hybridization between the  C. pneumoniae  target sequence and at least one of the sequences designated herein as SEQ ID NOs. 2-7 and 9-14. Several well known methods for detecting hybridization can be employed according to the present invention and may include, for example, the use of gels and stains or detecting a label associated with one or more of the sequences provided herein after performing, for example, a dot blot or amplification reaction. 
     The term “test sample” as used herein, means anything suspected of containing the target sequence. The test sample can be derived from any biological source, such as for example, blood, bronchial alveolar lavage, saliva, throat swabs, ocular lens fluid, cerebral spinal fluid, sweat, sputa, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissues such as heart tissue and the like, or fermentation broths, cell cultures, chemical reaction mixtures and the like. The test sample can be used (i) directly as obtained from the source or (ii) following a pre-treatment to modify the character of the sample. Thus, the test sample can be pre-treated prior to use by, for example, preparing plasma from blood, disrupting cells, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, and the like. 
     A “target sequence” as used herein means a nucleic acid sequence that is detected, amplified, both amplified and detected or otherwise is complementary to one of the sequences herein provided. Thus, a target sequence will be approximately 80% complementary with the sequences provided herein. Additionally, while the term target sequence is sometimes referred to as single stranded, those skilled in the art will recognize that the target sequence may actually be double stranded. 
     “Hybridization” or “hybridizing” conditions are defined generally as conditions which promote annealing between complementary nucleic acid sequences or annealing and extension of one or more nucleic acid sequences. It is well known in the art that such annealing is dependent in a rather predictable manner on several parameters, including temperature, ionic strength, sequence length and G:C content of the sequences. For example, lowering the temperature in the environment of complementary nucleic acid sequences promotes annealing. For any given set of sequences, melt temperature, or Tm, can be estimated by any of several known methods. Typically, diagnostic applications utilize hybridization temperatures which are slightly below the melt temperature. Ionic strength or “salt” concentration also impacts the melt temperature, since small cations tend to stabilize the formation of duplexes by negating the negative charge on the phosphodiester backbone. Typical salt concentrations depend on the nature and valency of the cation but are readily understood by those skilled in the art. Similarly, high G:C content and increased sequence length are also known to stabilize duplex formation because G:C pairings involve 3 hydrogen bonds where A:T pairs have just two, and because longer sequences have more hydrogen bonds holding the sequences together. Thus, a high G:C content and longer sequence lengths impact the hybridization conditions by elevating the melt temperature. 
     Once sequences are selected for a given diagnostic application, the G:C content and length will be known and can be accounted for in determining precisely what hybridization conditions will encompass. Since ionic strength is typically optimized for enzymatic activity, the only parameter left to vary is the temperature. For improved specificity, the hybridization temperature is selected slightly below the Tm of the primers or probe; typically 2-10° C. below the Tm. Thus, obtaining suitable hybridization conditions for a particular primer, probe or primer and probe set is well within ordinary skill of one practicing this art. 
     The sequences provided herein also can be used as amplification primers or probes according to amplification procedures well known in the art. Such reactions include, but are not intended to be limited to, the polymerase chain reaction (PCR) described in U.S. Pat. Nos. 4,683,195 and 4,683,202, the ligase chain reaction (LCR) described in EP-A-320 308, and gap LCR (GLCR) described in U.S. Pat. No. 5,427,930 all of which are herein incorporated by reference. 
     According to a preferred embodiment, the sequences are employed in the “oligonucleotide hybridization PCR” (variably referred to herein as “OH PCR”) amplification reaction as described in U.S. patent application serial No. 08/514,704, filed Aug. 14, 1995, that is herein incorporated by reference. Briefly, the reagents employed in the preferred method comprise at least one amplification primer and at least one internal hybridization probe, as well as other reagents for performing an amplification reaction. 
     The primer sequence is employed to prime extension of a copy of a target sequence and is labeled with either a capture label or a detection label. The probe sequence is used to hybridize with the sequence generated by the primer sequence, and typically hybridizes with a sequence that does not include the primer sequence. Similarly to the primer sequence, the probe sequence is also labeled with either a capture label or a detection label with the caveat that when the primer is labeled with a capture label the probe is labeled with a detection label and vice versa. Detection labels have the same definition as “labels” previously defined and “capture labels” are typically used to separate extension products, and probes associated with any such products, from other amplification reactants. Specific binding members (as previously defined) are well suited for this purpose. Also, probes used according to this method are preferably blocked at their 3′ ends so that they are not extended under hybridization conditions. Methods for preventing extension of a probe are well known and are a matter of choice for one skilled in the art. Typically, adding a phosphate group to the 3′ end of the probe will suffice for purposes of blocking extension of the probe. 
     “Other reagents for performing an amplification reactions” or “nucleic acid amplification reagents” include reagents which are well known and may include, but are not limited to, an enzyme having polymerase activity, enzyme cofactors such as magnesium; salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs) such as for example deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate. 
     The preferred method generally comprises the steps of (a) forming a reaction mixture comprising nucleic acid amplification reagents, at least one hybridization probe, at least one amplification primer and a test sample suspected of containing a target sequence; (b) subjecting the mixture to hybridization conditions to generate at least one copy of a 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 hybrid comprising the probe and the nucleic acid sequence complementary to the target sequence; and (d) detecting the hybrid as an indication of the presence of  C. pneumoniae  in the sample. It will be understood that step (b) of the above method can be repeated several times prior to step (c) by thermal cycling the reaction mixture as is well known in the art. 
     According to the above method, it is preferable to select primers and probes such that the probe sequence has a lower melt temperature than the primer sequences so that upon placing the reaction mixture under hybridization conditions copies of the target sequence or its complement are produced at temperature above the Tm of the probe. After such copies are synthesized, they are denatured and the mixture is cooled to enable the formation of hybrids between the probes and single stranded copies of the target or its complement. The rate of temperature reduction from the denaturation temperature down to a temperature at which the probes will bind to single stranded copies is preferably quite rapid (for example 8 to 15 minutes) and particularly through the temperature range in which an enzyme having polymerase activity is active for primer extension. Such a rapid cooling favors copy sequence/probe hybridization rather that primer/copy sequence hybridization. 
     Upon formation of the copy sequence/probe hybrids, the differential labels (i.e. capture and detection labels) on the copy sequence and probe sequence can be used to separate and detect such hybrids. Preferably, detection is performed according to the protocols used by the commercially available Abbott LCx® instrumentation (Abbott Laboratories; Abbott Park, Ill.). 
     Thus, keeping the preferred method in mind, the sequences of the present invention are preferably provided in groups of at least two different sequences (i.e. at least one primer sequence and at least one probe sequence complementary to the extension product of the primer). Hence, 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; or homologs of these sequences are preferably provided together. 
     The sequences of the present invention can be provided as part of a kit useful for detecting  C. pneumoniae.  The kits comprise one or more suitable containers containing one or more sequences according to the present invention, an enzyme having polymerase activity, and deoxynucleotide triphosphates. Typically, at least one sequence bears a label, but detection is possible without this. 
    
    
     The following examples are provided to further illustrate the present invention and not intended to limit the invention. 
     EXAMPLES 
     The following examples demonstrate use of the DNA oligomer primers and probes provided herein for detecting  C. pneumoniae.  The primers and probes used in the examples are identified as SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, SEQUENCE ID NO 9, SEQUENCE ID NO 10, and SEQUENCE ID NO 11. SEQUENCE ID NOs 2, 3 and 4 are specific for the gene encoding the 60kD cysteine rich outer major protein (OMP) of  C. pneumoniae,  a portion of which is designated herein as SEQ ID NO 1. SEQUENCE ID NO 9, 10 and 11 are specific for the gene encoding the 76kD protein of  C. pneumoniae,  a portion of which is designated herein as SEQ ID NO 8. In the following examples, SEQUENCE ID NOs 2 and 3 are used as  C. pneumoniae  amplification primers specific for the OMP region. SEQ ID NO 4 is used as an internal hybridization probe for the OMP amplification product. SEQ ID NOs 9 and 10 are used as amplification primers specific for the 76kD region of  C. pneumoniae  and SEQ ID NO 11 is used as an internal hybridization probe for the 76kD amplification product. 
     In the following examples, “positive-control  C. pneumonia  sequences” (variably referred to as the “ C. pneumoniae  standard”) were derived from  C. pneumoniae  cell lines TW-183, AR-39 and CWL-029 (obtained from the American Type Culture Collection -ATCC-, Rockville, Md.). The sequences were obtained by mixing equal numbers of cells from all three cell lines and collecting DNA with the QIAgen nucleic acid purification method (QIAgen, Inc., Chatsworth, Calif.). 
     Example 1 
     Preparation of  C. pneumoniae  Primers and Probes 
     A. OMP Primers and Probe 
     Target-specific primers and probes were designed to detect the  C. pneumoniae  OMP target sequence by oligonucleotide hybridization PCR. The primers were SEQUENCE ID NO 2 and SEQUENCE ID NO 3. Primer sequences were synthesized using standard oligonucleotide synthesis methodology and haptenated with adamantane at their 5′ ends using standard cyanoethyl phosphoramidite coupling chemistry as described in U.S. Pat. No. 5,424,414 incorporated herein by reference. 
     The detection probe was designed to hybridize with the amplified  C. pneumoniae  OMP target sequence by oligonucleotide hybridization. This probe is SEQUENCE ID NO 4. The probe sequence was synthesized using standard oligonucleotide synthesis methodology and haptenated with 2 carbazoles at the 5′ end using standard cyanoethyl phosphoramidite coupling chemistry as described in U.S. Pat. No. 5,464,746 (herein incorporated by reference), and blocked with phosphate at the 3′ end. Reactivity was assessed against the  C. pneumoniae  standard. 
     B. 76kD Primers and Probe 
     Target-specific primers and probes were designed to detect the  C. pneumoniae  76kD target sequence by oligonucleotide hybridization PCR. The primers were SEQUENCE ID NO 9 and SEQUENCE ID NO 10. Primer sequences were synthesized using standard oligonucleotide synthesis methodology and haptenated with adamantane at their 5′ ends using standard cyanoethyl phosphoramidite coupling chemistry U.S. Pat. No. 5,424,414. 
     The detection probe was designed to hybridize with the amplified  C. pneumoniae  76kD target sequence by oligonucleotide hybridization. This probe is SEQUENCE ID NO 11. The probe sequence was synthesized using standard oligonucleotide synthesis methodology and haptenated with 2 carbazoles at the 5′ end using standard cyanoethyl phosphoramidite coupling chemistry (as above) and blocked with phosphate at the 3′ end. Reactivity was assessed against the  C. pneumoniae  standard. 
     Example 2 
     Amplification and Detection of  C.pneumoniae    
     A.  C. pneumoniae  OMP Detection. 
     The  C. pneumoniae  standard sample was PCR amplified and detected using the OMP primers (SEQ ID NO 2 and 3) and OMP detection probe (SEQ ID NO 4) described in Example 1.A. The primers were used at a concentrations of 0.2 μM each. Taq polymerase was used at a concentration of 2.5 units. PCR extension was performed using 10×PCR buffer (Perkin Elmer, Foster City, Calif.) which consists of 100 mM Tris-HCl, pH 8.3, 500 mM KCl, at a final concentration of 1×. The final concentration of MgCl 2  was 2 mM and the final concentration of the nucleotides was 0.2 mM each, in a total reaction volume of 0.2 ml. 
     The reaction mixture was amplified in a Perkin-Elmer 480 Thermal Cycler under the following cycling conditions: 97° C. for 30 seconds/59° C. for 30 seconds/72° C. for 30 seconds for 40 cycles. 
     Following amplification, a 100 μl aliquot from the above reaction mixture was added to a separate tube containing 10 μl of the detection probe at a concentration of 40 nM (therefore final detection probe concentration was 3.6 nM). After an initial denaturation step at 97° C. for 5 minutes, probe oligo hybridization was accomplished by lowering the temperature to 15° C. for 10 minutes. 
     Following probe hybridization, reaction products were detected on the Abbott LCx® system (available from Abbott Laboratories, Abbott Park, Ill.). A suspension of anti-carbazole antibody coated microparticles and an anti-adamantane antibody/alkaline phosphatase conjugate (all of which are commercially available from Abbott Laboratories, Abbott Park, Ill.) were used in conjunction with the LCx® to capture and detect the reaction products. The LCx® showed a positive reaction rate of 1144.1 c/s/s using the OMP primer/probe set to detect  C. pneumoniae.    
     B.  C. pneumoniae  76kD detection. 
     The C. pneumoniae standard sample was PCR amplified and detected using the 76kD primers (SEQ ID NO 9 and 10) and 76kD detection probe (SEQ ID NO 11) described in Example 1.B. Concentrations of reagents used in this example were the same as those used in Example 2.A. above. 
     The reaction mixture was amplified, followed by probe oligo hybridization as in 2.A. above. 
     Following probe hybridization, reaction products were detected on the Abbott LCx® system, as above in Example 2.A. The LCx® showed a positive reaction rate of 994.0 c/s/s using the 76kD primer/probe set to detect  C. pneumoniae.    
     Example 3 
     Specificity of  C. pneumoniae  Detection 
     DNA from two other members of the genus Chlamydia,  C. psittaci  and  C. trachomatis,  was purchased from ABI (Advanced Biotechnologies, Inc., Columbia, Md.), diluted to levels representing 7.1×10 4  and 1.26×10 5  elementary bodies, respectively, and assayed side by side with the  C. pneumoniae  standard from Example 2, as described below. 
     A. Specific Detection of  C. pneumoniae  Using the OMP Primers and Probe 
     The OMP primers (SEQ ID NO 2 and SEQ ID NO 3) and OMP detection probe (SEQ ID NO 4) described in Example 1 were used to amplify and detect 3 samples from the genus Chlamydia (TABLE 1) by the method described in 2.A. above. The data from this experiment is presented in TABLE 1 and shows specific amplification and detection of  C. pneumoniae  only, with the 2 other Chlamydia genus samples being non-reactive. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Sample 
                 LCx ® rate (c/s/s) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 
                   C. psittaci 
                 
                 26.2 
               
               
                   
                 
                   C. trachomatis 
                 
                 23.9 
               
               
                   
                   C. pneumoniae  (Positive Control) 
                 1144.1 
               
               
                   
                   
               
            
           
         
       
     
     B. Specific Detection of  C. pneumoniae  Using the 76kD Primers and Probe 
     The 76kD primers (SEQ ID NO 9 and SEQ ID NO 10) and 76kD detection probe (SEQ ID NO 11) described in Example 1 were used to amplify and detect 3 samples from the genus Chlamydia (TABLE 2) by the method described in 2.B. above. The data from this experiment is presented in TABLE 2 and shows specific amplification and detection of  C. pneumoniae  only, with the 2 other Chlamydia genus samples being non-reactive. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Sample 
                 LCx ® rate (c/s/s) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 
                   C. psittaci 
                 
                 47.1 
               
               
                   
                 
                   C. trachomatis 
                 
                 34.5 
               
               
                   
                   C. pneumoniae  (Positive Control) 
                 994.0 
               
               
                   
                   
               
            
           
         
       
     
     Example 4 
     Sensitivity of  C. pneumoniae  Detection 
     A panel of  C. pneumoniae  cells which had been quantified using immunofluorescence to determine the number of Inclusion Forming Units (IFU) in each sample, were lysed and tested by the current methodology. Salmon sperm DNA was used as a negative control and the  C. pneumoniae  standard DNA as a positive control. 
     A. Sensitivity of the  C. pneumoniae  OMP Primers and Probe 
     The OMP primers (SEQ ID NO 2 and SEQ ID NO 3) and OMP detection probe (SEQ ID NO 4), described in Example 1, were used to amplify and detect a quantified panel of  C. pneumoniae  cells (TABLE 3) by a unit dose modification of the method used in Examples 2 and 3, namely: the primers, at a concenetration of 0.3 μM each, detection probe, at a concentration of 8 nM, as well as the other reagents were added to a single amplification vessel. Taq polymerase was used at a concentration of 2.5 units. PCR extension was performed in 10×PCR buffer (Perkin Elmer, Foster City, Calif.) which consists of 100 mM Tris-HCl, pH 8.3, 500 mM KCl, at a final concentration of 1×. The final concentration of MgCl 2  was 2 mM and the final concentration of the nucleotides was 0.2 mM each, in a total reaction volume of 0.2 ml. 
     The reaction mixture was amplified in a Perkin-Elmer 480 Thermal Cycler under the following cycling conditions: 97° C. for 30 seconds/59° C. for 30 seconds/72° C. for 30 seconds for 40 cycles. After maintaining the reaction mixture at 97° C. for 5 minutes, probe oligo hybridization was accomplished by lowering the temperature to 15° C. for 10 minutes. 
     Following probe hybridization, reaction products were detected on the Abbott LCx® system. The data from this experiment is presented in TABLE 3 and shows detection of  C. pneumoniae  at concentrations as low as 0.06 IFU/reaction. 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                 
                   C. pneumaniae 
                 
                 LCx ® rate 
               
               
                 Sample # 
                 (IFU/reaction) 
                 (c/s/s) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 50000.00 
                 2305 
               
               
                 2 
                 12500.00 
                 2320 
               
               
                 3 
                 15625.00 
                 2341 
               
               
                 4 
                 3906.25 
                 2215 
               
               
                 5 
                 976.56 
                 2361 
               
               
                 6 
                 244.14 
                 2262 
               
               
                 7 
                 61.04 
                 2329 
               
               
                 8 
                 15.26 
                 2262 
               
               
                 9 
                 3.81 
                 2302 
               
               
                 10 
                 0.95 
                 2241 
               
               
                 11 
                 0.06 
                 1804 
               
               
                 12 
                 0.05 
                 29 
               
               
                   
               
            
           
         
       
     
     Additional testing was performed in triplicate at concentrations below 1 IFU/reaction. The results, shown in TABLE 4, indicate consistent detection of  C. pneumoniae  at concentrations of 0.38 IFU/reaction. 
     
       
         
           
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                 
                   C. pneumoniae 
                 
                 LCx ® rate 
               
               
                 Sample # 
                 (IFU/reaction) 
                 (c/s/s) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 0.38 
                 2477 
               
               
                 1 
                 0.38 
                 2277 
               
               
                 1 
                 0.38 
                 2414 
               
               
                 2 
                 0.10 
                 33 
               
               
                 2 
                 0.10 
                 1764 
               
               
                 2 
                 0.10 
                 2414 
               
               
                 3 
                 0.02 
                 34 
               
               
                 3 
                 0.02 
                 31 
               
               
                 3 
                 0.02 
                 29 
               
               
                 Negative Control 
                   
                 77 
               
               
                 Negative Control 
                   
                 70 
               
               
                 Negative Control 
                   
                 89 
               
               
                 Positive Control 
                   
                 1876 
               
               
                 Positive Control 
                   
                 1987 
               
               
                 Positive Control 
                   
                 1919 
               
               
                   
               
            
           
         
       
     
     B. Sensitivity of the  C. pneumoniae  76kD Primers and Probe 
     The 76kD primers (SEQ ID NO 9 and SEQ ID NO 10) and 76kD detection probe (SEQ ID NO 11), described in Example 1, were used to amplify and detect a quantified panel of  C. pneumoniae  cells (TABLE 5) by the unit dose method described in Example 4.A. above. The primers were used at a concentration of 0.3 μM and the detection probe was used at a concetration of 8 nM. The other reaction mixture components were the same as in 4.A. above with the exception of MgCl 2  which was used at a final concentration of 1 mM. 
     The reaction mixture was amplified, followed by probe oligo hybridization as in 4.A. above. 
     Following probe hybridization, reaction products were detected on the Abbott LCx® system. The data from this experiment is presented in TABLE 5 and shows detection of  C. pneumoniae  at concentrations as low as 0.05 IFU/reaction. 
     
       
         
           
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                 
                   C. pneumoniae 
                 
                 LCx ® rate 
               
               
                 Sample # 
                 (IFU/reaction) 
                 (c/s/s) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 50000.00 
                 1657 
               
               
                 2 
                 12500.00 
                 1776 
               
               
                 3 
                 15625.00 
                 1686 
               
               
                 4 
                 3906.25 
                 1624 
               
               
                 5 
                 976.56 
                 1685 
               
               
                 6 
                 244.14 
                 1646 
               
               
                 7 
                 61.04 
                 4688 
               
               
                 8 
                 15.26 
                 1622 
               
               
                 9 
                 3.81 
                 1628 
               
               
                 10  
                 0.95 
                 1522 
               
               
                 11  
                 0.06 
                 21 
               
               
                 12  
                 0.05 
                 984 
               
               
                 Negative Control 
                   
                 41 
               
               
                 Positive Control 
                   
                 576 
               
               
                   
               
            
           
         
       
     
     Additional testing was performed in triplicate at concentrations below 1 IFU/reaction. The results shown in TABLE 6 indicate consistent detection of  C. pneumoniae  at concentrations of 0.38 IFU/reaction. 
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                 
                   C. pneumoniae 
                 
                 LCx ® rate 
               
               
                 Sample # 
                 (IFU) 
                 (c/s/s) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 0.38 
                 1488 
               
               
                 1 
                 0.38 
                 1410 
               
               
                 1 
                 0.38 
                 1378 
               
               
                 2 
                 0.10 
                 26 
               
               
                 2 
                 0.10 
                 25 
               
               
                 2 
                 0.10 
                 560 
               
               
                 3 
                 0.02 
                 27 
               
               
                 3 
                 0.02 
                 21 
               
               
                 3 
                 0.02 
                 31 
               
               
                 Negative Control 
                   
                 26 
               
               
                 Negative Control 
                   
                 30 
               
               
                 Negative Control 
                   
                 34 
               
               
                 Positive Control 
                   
                 1531 
               
               
                 Positive Control 
                   
                 1572 
               
               
                 Positive Control 
                   
                 47 
               
               
                   
               
            
           
         
       
     
     Example 5 
     Sensitivity and Specificity of  C. pneumoniae  OMP and 76kD Primers and Probes 
     The OMP primers (SEQ ID NO 2 and SEQ ID NO 3) and OMP detection probe (SEQ ID NO 4) or the 76kD primers (SEQ ID NO 9 and SEQ ID NO 10) and 76KD detection probe (SEQ ID NO 11), as described in Example 1, were used to amplify and detect previously quantified genomic DNA from both  C. pneumoniae  and  Mycoplasma pneumoniae  ( M. pneumoniae ), using the respective methods in Example 4 above. The data from this experiment is presented in TABLE 7 and shows detection of  C. pneumoniae  by both OMP and 76kD primer/probe sets at genomic DNA of 15.6 pg/ml, with no cross-detection of  M. pneumoniae  genomic DNA. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                   
                 Genomic 
                 OMP LCx ® 
                 76kD 
               
               
                   
                   
                 DNA 
                 rate 
                 LCx ® rate 
               
               
                   
                 Sample 
                 (pg/ml) 
                 (c/s/s) 
                 (c/s/s) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 
                   C. pneumoniae 
                 
                 5000 
                 2417 
                 1864 
               
               
                   
                   
                 1250 
                 2438 
                 1882 
               
               
                   
                   
                 312 
                 2543 
                 1827 
               
               
                   
                   
                 78 
                 2420 
                 1772 
               
               
                   
                   
                 15.6 
                 2481 
                 1653 
               
               
                   
                 
                   M. pneumoniae 
                 
                 5000 
                 37 
                 20 
               
               
                   
                   
                 1250 
                 38 
                 22 
               
               
                   
                   
                 312 
                 46 
                 18 
               
               
                   
                   
                 78 
                 34 
                 26 
               
               
                   
                   
                 15.6 
                 41 
                 30 
               
               
                   
                 Buffer 
                 0 
                 38 
                 21 
               
               
                   
                   
               
            
           
         
       
     
     Example 6 
     Comparison of  C. pneumoniae  Detection by OH-PCR and Culture 
     A. OH-PCR and Culture Detection of  C. pneumoniae  in nasopharyngeal swab samples. 
     Test results from twenty-five nasopharyngeal swab samples obtained from patients that were tested for  C. pneumoniae  by traditional culture methodology were compared to results obtained using OMP primers (SEQ ID NO 2 and SEQ ID NO 3) and OMP detection probe (SEQ ID NO 4) or the 76kD primers (SEQ ID NO 9 and SEQ ID NO 10) and 76kD detection probe (SEQ ID NO 11) as described in Example 1. Sample DNA was isolated using the QIAgen nucleic acid purification method and amplified and detected by the respective OMP or 76kD methods as in Example 4 above. Results are shown in Table 8.  C. pneumoniae  was used as a positive control and salmon sperm DNA was used as a negative control. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 8 
               
               
                   
                   
               
               
                   
                   
                   
                 OMP LCx ® 
                 76kD LCx ® 
               
               
                   
                   
                   
                 rate 
                 rate 
               
               
                   
                 Sample # 
                 Culture 
                 (c/s/s) 
                 (c/s/s) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                  1 
                 − 
                 23 
                 32 
               
               
                   
                  2 
                 − 
                 952 
                 644 
               
               
                   
                  3 
                 − 
                 18 
                 24 
               
               
                   
                  4 
                 − 
                 37 
                 24 
               
               
                   
                  5 
                 − 
                 20 
                 26 
               
               
                   
                  6 
                 − 
                 1499 
                 2180 
               
               
                   
                  7 
                 − 
                 23 
                 25 
               
               
                   
                  8 
                 − 
                 24 
                 19 
               
               
                   
                  9 
                 − 
                 23 
                 24 
               
               
                   
                 10 
                 − 
                 23 
                 25 
               
               
                   
                 11 
                 − 
                 29 
                 22 
               
               
                   
                 12 
                 + 
                 1538 
                 2188 
               
               
                   
                 13 
                 − 
                 14 
                 24 
               
               
                   
                 14 
                 − 
                 25 
                 25 
               
               
                   
                 15 
                 + 
                 1510 
                 2264 
               
               
                   
                 16 
                 + 
                 1670 
                 2190 
               
               
                   
                 17 
                 + 
                 1532 
                 2140 
               
               
                   
                 18 
                 + 
                 1455 
                 2107 
               
               
                   
                 19 
                 − 
                 22 
                 28 
               
               
                   
                 20 
                 + 
                 1609 
                 2258 
               
               
                   
                 21 
                 + 
                 1580 
                 2237 
               
               
                   
                 22 
                 + 
                 1525 
                 2226 
               
               
                   
                 23 
                 − 
                 19 
                 20 
               
               
                   
                 24 
                 + 
                 2348 
                 1393 
               
               
                   
                 25 
                 + 
                 2215 
                 1464 
               
               
                   
                 Neg Control 
                   
                 24 
                 85 
               
               
                   
                 Neg Control 
                   
                 78 
                 30 
               
               
                   
                 Pos Control 
                   
                 1568 
                 2061 
               
               
                   
                 Pos Control 
                   
                 2048 
                 1353 
               
               
                   
                   
               
            
           
         
       
     
     Ten samples were identified as positive for  C. pneumoniae  by culture (#12, 15, 16, 17,18, 20, 21, 22, 24 and 25), all of which were also detected as positive by both OMP and 76kD assay methods. Two additional samples (#2 and 6) were found positive by both the OMP and 76kD  C. pneumoniae  primer/probe sets using OH-PCR on the LCx®. 
     B. Detection of  C. pneumoniae  in Throat Swab and Nasopharyngeal Swab Samples Using the OMP Primer/Probe Set and Culture 
     Eighteen paired throat swab and nasopharyngeal swab samples obtained from patients were tested for  C. pneumoniae  by traditional culture methodology and compared to  C. pneumoniae  detection using OMP primers (SEQ ID NO 2 and SEQ ID NO 3) and OMP detection probe (SEQ ID NO 4) as described in Example 1. Sample DNA was isolated using the QIAgen nucleic acid purification method and amplified and detected by the OMP method as in Example 4.A. above. 
     The results using the OMP  C. pneumoniae  primer/probe set showed concordance with standard culture, with all samples negative by both methods. 
     While the invention has been described in detail and with reference to specific embodiments, it will be apparent to one skilled in the art that various changes and modifications may be made to such embodiments without departing from the spirit and scope of the invention. 
     
       
         
           
             14 
           
           
             
               230 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               genomic DNA (C. pneumoniae) 
             
             
               unknown 
             
             1
TAGAAATTTG CCAGTCCGTT CCAGAATACG CTACTGTAGG ATCTCCTTAC                50
CCTATTGAAA TCCTTGCTAT AGGCAAAAAA GATTGTGTTG ATGTTGTGAT               100
TACACAACAC CTACCTTGCG AAGCTGAATT CGTAAGCAGT GATCCAGAAA               150
CAACTCCTAC AAGTGATGGG AAATTAGTCT GGAAAATCGA TCGCCTGGGT               200
GCAGGAGATA AATGCAAAAT TACTGTATGG                                     230 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             2
CAGTCCGTTC CAGAATACGC TACTG                                           25 
           
           
             
               23 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             3
TGCATTTATC TCCTGCACCC AGG                                             23 
           
           
             
               20 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
              4
CCAGAAACAA CTCCTACAAG                                                 20 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             5
CTTGTAGGAG TTGTTTCTGG                                                 20 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             6
CAGTAGCGTA TTCTGGAACG GACTG                                           25 
           
           
             
               23 base pairs 
               nucleic acid 
               SINGLE 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             7
CCTGGGTGCA GGAGATAAAT GCA                                             23 
           
           
             
               150 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               genomic DNA (C. pneumoniae) 
             
             
               unknown 
             
             8
TACCTCAACA TCACTAGCTG ACATACAGGC TGCTTTGGTG AGCCTCCAGG                50
ATGCTGTCAC TAATATAAAG GATACAGCGG CTACTGATGA GGAAACCGCA               100
ATCGCTGCGG TGTGGGAAAC TAAGAATGCC GATGCAGTTA AAGTTGGCGC               150 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             9
CTAGCTGACA TACAGGCTGC TTTGG                                           25 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             10
CATCGGCATT CTTAGTTTCC CACTC                                           25 
           
           
             
               16 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             11
TTCCTCATCA GTAGCC                                                     16 
           
           
             
               16 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             12
GGCTACTGAT GAGGAA                                                     16 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             13
CCAAAGCAGC CTGTATGTCA GCTAG                                           25 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               synthetic DNA 
             
             
               unknown 
             
             14
GAGTGGGAAA CTAAGAATGC CGATG                                           25