Patent Publication Number: US-6218125-B1

Title: Assay for Chlamydia trachomatis by amplification and detection of Chlamydia trachomatis cryptic plasmid

Description:
This application is a divisional of application 08/963,927, filed Nov. 4, 1997, now U.S. Pat. No. 6,096,501. 
    
    
     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. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The various objects, advantages and novel features of the present invention will be readily understood from the following detailed description when read in conjunction with the appended drawings in which: 
     FIG. 1 indicates bumpers, primers and detectors used for System B. 
     FIGS. 2A-B show the phosphorimages resulting from using a matrix of test conditions using various combinations of primers, buffer conditions and temperatures to perform tSDA with a portion of  C. trachomatis  cryptic plasmid (in plasmid pCT 16) as the target. 
     FIGS. 3A and 3B show the phosphorimages of tSDA reactions performed using pCT16 as the target but using two different, but complementary, detectors. The reactions were performed using 3% DMSO, 7% glycerol, 6 mM magnesium, 35 mM potassium phosphate and were amplified at 52° C. The four combinations of primers (1, 2, 3 and 4) were used. The four samples in FIG. 3A were detected with an extension primer which hybridized to the anti-sense strand of the amplified product in FIG.  3 B. The same four samples were detected with the complement of the detector used in FIG.  3 A. The detector used for the experiments shown in FIG. 3B hybridizes to the sense strand of the amplified product. 
     FIG. 4 shows the phophorimage results of performing tSDA using pCT16 as a target and using various combinations of primers and buffer conditions to test sensitivity. Target was present at from 10 2  to 10 6  copies. Each of primer combinations 1, 2, 3 and 4 was tested across each range of target copy number. The experiments were performed under two sets of buffer conditions as explained in Example 2. 
     FIG. 5 shows the results of an assay to check the specificity and crossreactivity of primer combination 2. Target nucleic acid consisted of fourteen serovars of  C. trachomatis  (A, B, Ba, C, D, E, F, G, H, I, J, K L 2  and L 3 ) to test specificity as well as  C. pneumoniae, C. psittaci  and  N. gonorrhoeae  to test crossreactivity. Each serovar was tested at both 10 4  and 10 3  genome copies while the crossreactant tests used 10 7  genome copies of the tested organisms. Lanes with control (pCT 16) and no target were also included. 
     FIG. 6 shows the results of an assay to test the sensitivity of primer combination 2 for  C. trachomatis  cryptic plasmid in the presence of various amounts of human DNA ranging from 300 to 3000 ng. 
     FIG. 7 shows the results of an assay for crossreactivity using primer combination 2. Nine pools consisting of a total of 35 different organisms representing 30 different species were tested to determine if there was any crossreaction. Each species was tested at 10 7  genomic copies. For each pool, the left lane indicates amplification of the pool alone, whereas the right lane indicates the amplification of the pool plus 200 copies of pCT 16. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to oligonucleotides, amplification primers and assay probes which exhibit  Chlamydia trachomatis -specificity in nucleic acid amplification reactions. Also provided are methods for detecting and identifying  C. trachomatis  cryptic plasmid nucleic acids using the oligonucleotides of the invention. The preferred methods are to use SDA, tSDA or homogeneous real time fluorescent tSDA. These methods are known to those skilled in the art from references such as U.S. Pat. No. 5,547,861 and U.S. Pat. No. 5,648,211, U.S. patent application Ser. No. 08/865,675, filed May 30, 1997 and U.S. patent application Ser. No. 08/855,085 filed May 13, 1997, the disclosures of which are hereby specifically incorporated herein by reference. 
     The primers of the present invention were designed based on the sequence of  C. trachomatis  cryptic plasmid which is available from GenBank. The plasmid sequences were aligned and sectioned into manageable regions (300-500 bp). Two regions (B and F) were selected for further study. Regions B and F were sequenced for several serovars of  C. trachomatis  and tSDA systems were designed in these areas. The primers used to amplify and sequence the B and F regions, respectively, are: 
     B Region Amplification and Sequencing Primers 
     CTpB1P 5′-GAAGATCGAGTAGACGTAATAT-3′ (SEQ ID NO: 1) 
     CTpB2P 5′ ATAGCAGATATATCTAGAACCTT-3′ (SEQ ID NO: 2) 
     CTpB3N 5′-TTGGATCGAAATGTAATACCGA-3′ (SEQ ID NO: 3) 
     CTpB4N 5′-ACTTCTGATTTTCAAGGTGGAT-3′ (SEQ ID NO: 4) 
     F Region Amplification and Sequencing Primers 
     CTpFPL 5′-CAAAACTGCGTCTTTGCTGATA-3′ (SEQ ID NO: 5) 
     CTpFPR 5′-GGGTGTGACTGTGAATTTTCC-3′ (SEQ ID NO: 6) 
     CTpFSL 5′-AGTTGGGCAAATGACAGAGC-3′ (SEQ ID NO: 7) 
     CTpFSR 5′-TGAATAACCCGTTGCATTGA-3′ (SEQ ID NO: 8) 
     Systems of detection relating to analysis of regions B and F are described below. Various combinations of primers were tested for specificity and sensitivity in tSDA reactions and in fluorescent real time tSDA reactions. 
     As nucleic acids do not require complete complementarity in order to hybridize, it is to be understood that the probe and primer sequences herein disclosed may be modified to some extent without loss of utility as  C. trachomatis -specific probes and primers. As is known in the art, hybridization of complementary and partially complementary nucleic acid sequences may be obtained by adjustment of the hybridization conditions to increase or decrease stringency (i.e., adjustment of hybridization temperature or salt content of the buffer). Such minor modifications of the disclosed sequences and any necessary adjustments of hybridization conditions to maintain  C. trachomatis -specificity require only routine experimentation and are within the ordinary skill in the art. 
     The amplification products generated using the inventive primers may be detected by a characteristic size, for example on polyacrylamide or agarose gels stained with ethidium bromide. Alternatively, amplified  C. trachomatis  cryptic plasmid target sequences may be detected by means of an assay probe, which is an oligonucleotide tagged with a detectable label. In one embodiment, at least one tagged assay probe may be used for detection of amplified target sequences by hybridization (a detector probe), by hybridization and extension as described by Walker, et al.,  Nucl. Acids Res.,  supra (a detector primer) or by hybridization, extension and conversion to double stranded form as described in EP 0 678 582 (a signal primer). Preferably, the assay probe is selected to hybridize to a sequence in the target which is between the amplification primers, i.e., it should be an internal assay probe. Alternatively, an amplification primer or the target binding sequence thereof may be used as the assay probe. 
     The detectable label of the assay probe is a moiety which can be detected either directly or indirectly as an indication of the presence of the target nucleic acid. For direct detection of the label, assay probes may be tagged with a radioisotope and detected by autoradiography or tagged with a fluorescent moiety and detected by fluorescence as is known in the art. Alternatively, the assay probes may be indirectly detected by tagging with a label which requires additional reagents to render it detectable. Indirectly detectable labels include, for example, chemiluminescent agents, enzymes which produce visible reaction products and ligands (e.g., haptens, antibodies or antigens) which may be detected by binding to labeled specific binding partners (e.g., antibodies or antigens/haptens). Ligands are also useful immobilizing the ligand-labeled oligonucleotide (the capture probe) on a solid phase to facilitate its detection. Particularly useful labels include biotin (detectable by binding to labeled avidin or streptavidin) and enzymes such as horseradish peroxidase or alkaline phosphatase (detectable by addition of enzyme substrates to produce colored reaction products). Methods for adding such labels to, or including such labels in, oligonucleotides are well known in the art and any of these methods are suitable for use in the present invention. 
     Examples of specific detection methods which may be employed include a chemiluminescent method in which amplified products are detected using a biotinylated capture probe and an enzyme-conjugated detector probe as described in U.S. Pat. No. 5,470,723. After hybridization of these two assay probes to different sites in the assay region of the target sequence (between the binding sites of the two amplification primers), the complex is captured on a streptavidin-coated microtiter plate by means of the capture probe, and the chemiluminescent signal is developed and read in a luminometer. As another alternative for detection of amplification products, a signal primer as described in EP 0 678 582 may be included in the SDA reaction. In this embodiment, labeled secondary amplification products are generated during SDA in a target amplification-dependent manner and may be detected as an indication of target amplification by means of the associated label. 
     For commercial convenience, amplification primers for specific detection and identification of  C. trachomatis  cryptic plasmid nucleic acids may be packaged in the form of a kit. Typically, such a kit contains at least one pair of amplification primers according to the present invention. Reagents for performing a nucleic acid amplification reaction may also be included with the  C. trachomatis  cryptic plasmid-specific amplification primers, for example, buffers, additional primers, nucleotide triphosphates, enzymes, etc. The components of the kit are packaged together in a common container, optionally including instructions for performing a specific embodiment of the inventive methods. Other optional components may also be included in the kit, e.g., an oligonucleotide tagged with a label suitable for use as an assay probe, and/or reagents or means for detecting the label. 
     The target binding sequences of the amplification primers confer species hybridization specificity on the oligonucleotides and therefore provide species-specificity to the amplification reaction. Other sequences, as required for performance of a selected amplification reaction, may optionally be added to the target binding sequences disclosed herein without altering the species-specificity of the oligonucleotide. By way of example, the  C. trachomatis  cryptic plasmid-specific amplification primers of the invention may contain a recognition site for the restriction endonuclease BsoBI which is nicked during the SDA reaction. It will be apparent to one skilled in the art that other nickable restriction endonuclease recognition sites may be substituted for the BsoBI recognition site, including but not limited to those recognition sites disclosed in EP 0 684 315. Preferably, the recognition site is for a thermophilic restriction endonuclease so that the amplification reaction may be performed under the conditions of thermophilic SDA (tSDA). Similarly, the tail sequence of the amplification primer (5′ to the restriction endonuclease recognition site) is generally not critical, although the restriction site used for SDA and sequences which will hybridize either to their own target binding sequence or to the other primers should be avoided. Some amplification primers for SDA according to the invention therefore consist of 3′ target binding sequences, a nickable restriction endonuclease recognition site 5′ to the target binding sequence and a tail sequence about 10-25 nucleotides in length 5′ to the restriction endonuclease recognition site. The nickable restriction endonuclease recognition site and the tail sequence are sequences required for the SDA reaction. For other amplification reactions, the amplification primers according to the invention may consist of the disclosed target binding sequences only (e.g., for PCR) or the target binding sequence and additional sequences required for the selected amplification reaction (e.g., sequences required for SDA as described above or a promoter recognized by RNA polymerase for 3SR). 
     In SDA, the bumper primers are not essential for species-specificity, as they function to displace the downstream, species-specific amplification primers. It is only required that the bumper primers hybridize to the target upstream from the amplification primers so that when they are extended they will displace the amplification primer and its extension product. The particular sequence of the bumper primer is therefore generally not critical, and may be derived from any upstream target sequence which is sufficiently close to the binding site of the amplification primer to allow displacement of the amplification primer extension product upon extension of the bumper primer. Occasional mismatches with the target in the bumper primer sequence or some cross-hybridization with non-target sequences do not generally negatively affect amplification efficiency as long as the bumper primer remains capable of hybridizing to the specific target sequence. However, the bumper primers described herein are species-specific for  C. trachomatis  and may therefore also be used as target binding sequences in amplification primers, if desired. 
     Amplification reactions employing the primers of the invention may incorporate thymine as taught by Walker, et al., supra, or they may wholly or partially substitute 2′-deoxyuridine 5′-triphosphate for TTP in the reaction to reduce cross-contamination of subsequent amplification reactions, e.g., as taught in EP 0 624 643. dU (uridine) is incorporated into amplification products and can be excised by treatment with uracil DNA glycosylase (UDG). These abasic sites render the amplification product unamplifiable in subsequent amplification reactions. UDG may be inactivated by uracil DNA glycosylase inhibitor (Ugi) prior to performing the subsequent amplification to prevent excision of dU in newly-formed amplification products. 
     Other systems were developed for performing tSDA using different combinations of primers, bumpers and detectors. However, these other systems were not preferred for various reasons such as lack of adequate specificity, narrow range of optimal conditions and lack of robustness. 
     FIG. 1 indicates the primers, bumpers and detectors for system B. This system is discussed in the Examples. System B is located at the 5′ end of region B. In the initial experiments two upstream primers and two downstream primers were designed along with one upstream bumper and one downstream bumper. These were used in various combinations along with detectors. The primers, bumpers and detectors used in system B are shown below. The hybridization region of the primers is underlined and the BsoBI restriction site is italicized. 
     Upstream Primers: 
     CTpB4.S1 5′-CGATTCCGCTCCAGACTTCTCGGGCGATTACTTGCAGTTG-3′ (SEQ ID NO:9) 
     CTpB4.S1.1 5′-CGATTCCGCTCCAGACTTCTCGGGGATTACTTGCAGTTG-3′ (SEQ ID NO: 10) 
     Upstream Bumper: 
     CTpB4.B1 5′-GAGTAGACGTAATATT-3′ (SEQ ID NO: 11) 
     Downstream Primers: 
     CTpB4.S2 5′-ACCGCATCGAATGCATGTCTCGGGATATCTGCTATTTCATT-3′ (SEQ ID NO: 12) 
     CTpB4.S2.1 5′-ACCGCATCGAATGCATGTCTCGGGATATCTGCTATTTCAT-3′ (SEQ ID NO: 13) 
     Downstream Bumper: 
     CTpB4.B2 5′-CTCTTAAGGTTCTAG-3′ (SEQ ID NO: 14) 
       32 P Detectors: 
     CTpB4.D1 5′-CTCATCGGTTGGAGA-3′ (SEQ ID NO: 15) 
     CTpB4.D2 5′-TCTCCAACCGATGAG-3′ (SEQ ID NO: 16) 
     
       
         
           
               
               
               
            
               
                 Fluorescent Detector: 
                   
                   
               
               
                 CtpB4.FD1   5′-TAGCACCCGAGTGCTTTGACGATTTTCTCCAACCGATGAGTTGAT-3′ 
                 (SEQ ID NO: 17) 
               
               
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                              FAM            ROX 
               
            
           
         
       
     
     In initial screening experiments, primer mixes were prepared to contain one upstream primer and one downstream primer. The four possible primer mixes are: 
     Combination 1 
     CTpB4.S1 (SEQ ID NO: 9) 
     CTpB4.S2 (SEQ ID NO: 12) 
     Combination 2 
     CTpB4.S1.1 (SEQ ID NO: 10) 
     CTpB4.S2 (SEQ ID NO: 12) 
     Combination 3 
     CTpB4.S1 (SEQ ID NO: 9) 
     CTpB4.S2.1 (SEQ ID NO: 13) 
     Combination 4 
     CTpB4.S1.1 (SEQ ID NO: 10) 
     CTpB4.S2.1 (SEQ ID NO: 13) 
     All of the primer mixes also contained the upstream and downstream bumpers. The primers and bumpers are used at a final concentration of 0.5 and 0.05 μM, respectively. 
     The above primer mixes are used in the study of region B of the Cryptic plasmid and are further discussed in the Examples. Other primers which hybridize to region F of the Cryptic plasmid and which also are useful for assaying for  C. trachomatis  are also discussed in the Examples. Later Examples also disclose methods of using primers for region F for use in homogeneous real time fluorescent tSDA. 
     The primers and probes of System B are preferably used in a tSDA real time fluorescence energy transfer method. Strand Displacement Amplification (SDA) is an isothermal method of nucleic acid amplification in which extension of primers, nicking of a hemimodified restriction endonuclease recognition/cleavage site, displacement of single stranded extension products, annealing of primers to the extension products (or the original target sequence) and subsequent extension of the primers occurs concurrently in the reaction mix. This is in contrast to polymerase chain reaction (PCR), in which the steps of the reaction occur in discrete phases or cycles as a result of the temperature cycling characteristics of the reaction. SDA is based upon 1) the ability of a restriction endonuclease to nick the unmodified strand of a hemiphosphorothioate form of its double stranded recognition/cleavage site and 2) the ability of certain polymerases to initiate replication at the nick and displace the downstream non-template strand. After an initial incubation at increased temperature (about 95° C.) to denature double stranded target sequences for annealing of the primers, subsequent polymerization and displacement of newly synthesized strands takes place at a constant temperature. Production of each new copy of the target sequence consists of five steps:  1 ) binding of amplification primers to an original target sequence or a displaced single-stranded extension product previously polymerized,  2 ) extension of the primers by a 5′-3′ exonuclease deficient polymerase incorporating an α-thio deoxynucleoside triphosphate (α-thio dNTP),  3 ) nicking of a hemimodified double stranded restriction site, 4) dissociation of the restriction enzyme from the nick site, and 5) extension from the 3′ end of the nick by the 5′-3′ exonuclease deficient polymerase with displacement of the downstream newly synthesized strand. Nicking, polymerization and displacement occur concurrently and continuously at a constant temperature because extension from the nick regenerates another nickable restriction site. When a pair of amplification primers is used, each of which hybridizes to one of the two strands of a double stranded target sequence, amplification is exponential. This is because the sense and antisense strands serve as templates for the opposite primer in subsequent rounds of amplification. When a single amplification primer is used, amplification is linear because only one strand serves as a template for primer extension. Examples of restriction endonucleases which nick their double stranded recognition/cleavage sites when an α-thio dNTP is incorporated are HincII, HindII, Aval, NciI and Fnu4HI. All of these restriction endonucleases and others which display the required nicking activity are suitable for use in conventional SDA. However, they are relatively thermolabile and lose activity above about 40° C. 
     Targets for amplification by SDA may be prepared by fragmenting larger nucleic acids by restriction with an endonuclease which does not cut the target sequence. However, it is generally preferred that target nucleic acids having the selected restriction endonuclease recognition/cleavage sites for nicking in the SDA reaction be generated as described by Walker, et al. (1992,  Nuc. Acids Res.,  supra) and in U.S. Pat. No. 5,270,184 (hereby incorporated by reference). Briefly, if the target sequence is double stranded, four primers are hybridized to it. Two of the primers (S 1  and S 2 ) are SDA amplification primers and two (B 1  and B 2 ) are external or bumper primers. S 1  and S 2  bind to opposite strands of double stranded nucleic acids flanking the target sequence. B 1  and B 2  bind to the target sequence 5′ (i.e., upstream) of S 1  and S 2 , respectively. The exonuclease deficient polymerase is then used to simultaneously extend all four primers in the presence of three deoxynucleoside triphosphates and at least one modified deoxynucleoside triphosphate (e.g., 2′-deoxyadenosine 5′-O-(1-thiotriphosphate), “dATPαS”). The extension products of S 1  and S 2  are thereby displaced from the original target sequence template by extension of B 1  and B 2 . The displaced, single stranded extension products of the amplification primers serve as targets for binding of the opposite amplification and bumper primer (e.g., the extension product of S 1  binds S 2  and B 2 ). The next cycle of extension and displacement results in two double stranded nucleic acid fragments with hemimodified restriction endonuclease recognition/cleavage sites at each end. These are suitable substrates for amplification by SDA. As in SDA, the individual steps of the target generation reaction occur concurrently and continuously, generating target sequences with the recognition/cleavage sequences at the ends required for nicking by the restriction enzyme in SDA. As all of the components of the SDA reaction are already present in the target generation reaction, target sequences generated automatically and continuously enter the SDA cycle and are amplified. 
     To prevent cross-contamination of one SDA reaction by the amplification products of another, dUTP may be incorporated into SDA-amplified DNA in place of dTTP without inhibition of the amplification reaction. The uracil-modified nucleic acids may then be specifically recognized and inactivated by treatment with uracil DNA glycosylase (UDG). Therefore, if dUTP is incorporated into SDA-amplified DNA in a prior reaction, any subsequent SDA reactions can be treated with UDG prior to amplification of double stranded targets, and any dU containing DNA from previously amplified reactions will be rendered unamplifiable. The target DNA to be amplified in the subsequent reaction does not contain dU and will not be affected by the UDG treatment. UDG may then be inhibited by treatment with Ugi prior to amplification of the target. Alternatively, UDG may be heat-inactivated. In thermophilic SDA, the higher temperature of the reaction itself (≧50° C.) can be used to concurrently inactivate UDG and amplify the target. 
     SDA requires a polymerase which lacks 5′-3′ exonuclease activity, initiates polymerization at a single stranded nick in double stranded nucleic acids, and displaces the strand downstream of the nick while generating a new complementary strand using the unnicked strand as a template. The polymerase must extend by adding nucleotides to a free 3′-OH. To optimize the SDA reaction, it is also desirable that the polymerase be highly processive to maximize the length of target sequence which can be amplified. Highly processive polymerases are capable of polymerizing new strands of significant length before dissociating and terminating synthesis of the extension product. Displacement activity is essential to the amplification reaction, as it makes the target available for synthesis of additional copies and generates the single stranded extension product to which a second amplification primer may hybridize in exponential amplification reactions. Nicking activity is also of great importance, as it is nicking which perpetuates the reaction and allows subsequent rounds of target amplification to initiate. 
     Thermophilic SDA is performed essentially as the conventional SDA described by Walker, et al. (1992,  PNAS  and  Nuc. Acids Res.,  supra), with substitution of the desired thermostable polymerase and thermostable restriction endonuclease. Of course, the temperature of the reaction will be adjusted to the higher temperature suitable for the substituted enzymes and the HincII restriction endonuclease recognition/cleavage site will be replaced by the appropriate restriction endonuclease recognition/cleavage site for the selected thermostable endonuclease. Also in contrast to Walker, et al., the practitioner may include the enzymes in the reaction mixture prior to the initial denaturation step if they are sufficiently stable at the denaturation temperature. Preferred restriction endonucleases for use in thermophilic SDA are BsrI, BstNI, BsmAI, BsII and BsoBI (New England BioLabs), and BstOI (Promega). The preferred thermophilic polymerases are Bca (Panvera) and Bst (New England Biolabs). 
     Homogeneous real time fluorescent tSDA is a modification of tSDA. It employs detector oligonucleotides to produce reduced fluorescence quenching in a target-dependent manner. The detector oligonucleotides contain a donor/acceptor dye pair linked such that fluorescence quenching occurs in the absence of target. Unfolding or linearization of an intramolecularly base-paired secondary structure in the detector oligonucleotide in the presence of the target increases the distance between the dyes and reduces fluorescence quenching. Unfolding of the base-paired secondary structure typically involves intermolecular base-pairing between the sequence of the secondary structure and a complementary strand such that the secondary structure is at least partially disrupted. It may be fully linearized in the presence of a complementary strand of sufficient length. In a preferred embodiment, a restriction endonuclease recognition site (RERS) is present between the two dyes such that intermolecular base-pairing between the secondary structure and a complementary strand also renders the RERS double-stranded and cleavable or nickable by a restriction endonuclease. Cleavage or nicking by the restriction endonuclease separates the donor and acceptor dyes onto separate nucleic acid fragments, further contributing to decreased quenching. In either embodiment, an associated change in a fluorescence parameter (e.g., an increase in donor fluorescence intensity, a decrease in acceptor fluorescence intensity or a ratio of fluorescence before and after unfolding) is monitored as an indication of the presence of the target sequence. Monitoring a change in donor fluorescence intensity is preferred, as this change is typically larger than the change in acceptor fluorescence intensity. Other fluorescence parameters such as a change in fluorescence lifetime may also be monitored. 
     A detector oligonucleotide for homogeneous real time fluorescent tSDA is an oligonucleotide which comprises a single-stranded 5′ or 3′ section which hybridizes to the target sequence (the target binding sequence) and an intramolecularly base-paired secondary structure adjacent to the target binding sequence. The detector oligonucleotides of the invention further comprise a donor/acceptor dye pair linked to the detector oligonucleotide such that donor fluorescence is quenched when the secondary structure is intramolecularly base-paired and unfolding or linearization of the secondary structure results in a decrease in fluorescence quenching. Cleavage of an oligonucleotide refers to breaking the phosphodiester bonds of both strands of a DNA duplex or breaking the phosphodiester bond of single-stranded DNA. This is in contrast to nicking, which refers to breaking the phosphodiester bond of only one of the two strands in a DNA duplex. 
     The detector oligonucleotides of the invention for homogeneous real time fluorescent tSDA comprise a sequence which forms an intramolecularly base-paired secondary structure under the selected reaction conditions for primer extension or hybridization. The secondary structure is positioned adjacent to the target binding sequence of the detector oligonucleotide so that at least a portion of the target binding sequence forms a single-stranded 3′ or 5′ tail. As used herein, the term “adjacent to the target binding sequence” means that all or part of the target binding sequence is left single-stranded in a 5′ or 3′ tail which is available for hybridization to the target. That is, the secondary structure does not comprise the entire target binding sequence. A portion of the target binding sequence may be involved in the intramolecular base-pairing in the secondary structure, it may include all or part of a first sequence involved in intramolecular base-pairing in the secondary structure, it may include all or part of a first sequence involved in intramolecular base-pairing in the secondary structure but preferably does not extend into its complementary sequence. For example, if the secondary structure is a stem-loop structure (e.g., a “hairpin”) and the target binding sequence of the detector oligonucleotide is present as a single-stranded 3′ tail, the target binding sequence may also extend through all or part of the first arm of the stem and, optionally, through all or part of the loop. However, the target binding sequence preferably does not extend into the second arm of the sequence involved in stem intramolecular base-pairing. That is, it is desirable to avoid having both sequences involved in intramolecular base-pairing in a secondary structure capable of hybridizing to the target. Mismatches in the intramolecularly base-paired portion of the detector oligonucleotide secondary structure may reduce the magnitude of the change in fluorescence in the presence of target but are acceptable if assay sensitivity is not a concern. Mismatches in the target binding sequence of the single-stranded tail are also acceptable but may similarly reduce assay sensitivity and/or specificity. However, it is a feature of the present invention that perfect base-pairing in both the secondary structure and the target binding sequence do not compromise the reaction. Perfect matches in the sequences involved in hybridization improve assay specificity without negative effects on reaction kinetics. 
     When added to the amplification reaction, the detector oligonucleotide signal primers of the invention are converted to double-stranded form by hybridization and extension of an amplification primer as described above. Strand displacement by the polymerase also unfolds or linearizes the secondary structure and converts it to double-stranded from by synthesis of a complementary strand. The RERS, if present, also becomes double-stranded and cleavable or nickable by the restriction endonuclease. As the secondary structure is unfolded or linearized by the strand displacing activity of the polymerase, the distance between the donor and acceptor dye is increased, thereby reducing quenching of donor fluorescence. The associated change in fluorescence of either the donor or acceptor dye may be monitored or detected as an indication of amplification of the target sequence. Cleavage or nicking of the RERS generally further increases the magnitude of the change in fluorescence by producing two separate fragments of the double-stranded secondary amplification product, each having one of the two dyes linked to it. These fragments are free to diffuse in the reaction solution, further increasing the distance between the dyes of the donor/acceptor pair. An increase in donor fluorescence intensity or a decrease in acceptor fluorescence intensity may be detected and/or monitored as an indication that target amplification is occurring or has occurred, but other fluorescence parameters which are affected by the proximity of the donor/acceptor dye pair may also be monitored. A change in fluorescence intensity of the donor or acceptor may also be detected as a change in a ratio of donor and/or acceptor fluorescence intensities. For example, a change in fluorescence intensity may be detected as a) an increase in the ratio of donor fluorophore fluorescence after linearizing or unfolding the secondary structure and donor fluorophore fluorescence in the detector oligonucleotide prior to linearizing or unfolding, or b) as a decrease in the ratio of acceptor dye fluorescence after linearizing or unfolding and acceptor dye fluorescence in the detector oligonucleotide prior to linearizing or unfolding. 
     It will be apparent that, in addition to SDA, the detector oligonucleotides of the invention may be adapted for use as signal primers in other primer extension amplification methods (e.g., PCR, 3SR, TMA or NASBA). For example, the methods may be adapted for use in PCR by using PCR amplification primers and a strand displacing DNA polymerase which lacks 5′→3′ exonuclease activity (e.g., Sequencing Grade Taq from Promega or exo −  Vent or exo −  Deep Vent from New England BioLabs) in the PCR. The detector oligonucleotide signal primers hybridize to the target downstream from the PCR amplification primers, are displaced and are rendered double-stranded essentially as described for SDA. In PCR any RERS may optionally be selected for use in the detector oligonucleotide, as there are typically no modified deoxynucleoside triphosphates present which might induce nicking rather than cleavage of the RERS. As thermocycling is a feature of amplification by PCR, the restriction endonuclease is preferably added at low temperature after the final cycle of primer annealing and extension for end-point detection of amplification. However, a thermophilic restriction endonuclease which remains active through the high temperature phases of the PCR reaction could be present during amplification to provide a real-time assay. As in SDA systems, linearization of the secondary structure and separation of the dye pair reduces fluorescence quenching, with a change in a fluorescence parameter such as intensity serving as an indication of target amplification. 
     The change in fluorescence resulting from unfolding or linearizing of the detector oligonucleotides may be detected at a selected endpoint in the reaction. However, because linearized secondary structures are produced concurrently with hybridization or primer extension, the change in fluorescence may also be monitored as the reaction is occurring, i.e., in “real-time”. This homogeneous, real-time assay format may be used to provide semiquantitative or quantitative information about the initial amount of target present. For example, the rate at which fluorescence intensity changes during the unfolding or linearizing reaction (either as part of target amplification or in non-amplification detection methods) is an indication of initial target levels. As a result, when more initial copies of the target sequence are present, donor fluorescence more rapidly reaches a selected threshold value (i.e., shorter time to positivity). The decrease in acceptor fluorescence similarly exhibits a shorter time to positivity, detected as the time required to reach a selected minimum value. In addition, the rate of change in fluorescence parameters during the course of the reaction is more rapid in samples containing higher initial amounts of target than in samples containing lower initial amounts of target (i.e., increased slope of the fluorescence curve). These or other measurements as is known in the art may be made as an indication of the presence of target or as an indication of target amplification. The initial amount of target is typically determined by comparison of the experimental results to results for known amounts of target. 
     Assays for the presence of a selected target sequence according to the methods of the invention may be performed in solution or on a solid phase. Real-time or endpoint homogeneous assays in which the detector oligonucleotide functions as a primer are typically performed in solution. Hybridization assays using the detector oligonucleotides of the invention may also be performed in solution (e.g., as homogeneous real-time assays) but are also particularly well-suited to solid phase assays for real-time or endpoint detection of target. In a solid phase assay, detector oligonucleotides may be immobilized on the solid phase (e.g., beads, membranes or the reaction vessel) via internal or terminal labels using methods known in the art. For example, a biotin-labeled detector oligonucleotide may be immobilized on an avidin-modified solid phase where it will produce a change in fluorescence when exposed to the target under appropriate hybridization conditions. Capture of the target in this manner facilitates separation of the target from the sample and allows removal of substances in the sample which may interfere with detection of the signal or other aspects of the assay. 
     The following Examples illustrate specific embodiments of the invention described herein. As would be apparent to skilled artisans, various changes and modifications are possible, and are contemplated within the scope of the invention described. 
     EXAMPLE 1 
     Assay of Primer Combinations for Robustness 
     All four primer combinations were tested against a matrix of conditions to determine which of the combinations worked best across the most conditions. The reaction conditions which were varied were as follows: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Amplification temperature 
                 52° and 54° C. 
               
               
                   
                 Potassium phosphate, pH 7.6 
                 35 and 45 mM 
               
               
                   
                 Magnesium acetate 
                  5 and 6 mM 
               
               
                   
                 Dimethyl sulfoxide 
                  3 and 7% (vol/vol) 
               
               
                   
                 Glycerol 
                  3 and 7% (vol/vol) 
               
               
                   
                   
               
            
           
         
       
     
     The reactions (50 μL) also contained 9 units Bst polymerase, 165 units BsoBI restriction endonuclease, 1200 ng human placental DNA, 0.2 mM dATP and dGTP, 1.4 mM dC s TP, 0.5 mM dUTP, 0.1 mg/mL acetylated bovine serum albumin, 1.8% trehalose, 0.36 mM dithiothreitol (DTT) and 1×10 6  copies of target plasmid DNA. The target plasmid was plasmid pCT16 which contains regions B and F of the Cryptic plasmid (from Serovar J) inserted into pUC 18. 
     Samples were prepared to contain phosphate, DMSO, glycerol, human DNA and target DNA in a volume of 30 μL. The samples were heat denatured for 2 minutes in a boiling water bath, briefly centrifuged, and placed in a 45° C. Thermal-Lok Dry Bath (U.S.A. Scientific, Fla.). After 2-5 minutes of equilibration, the following components were added in a volume of 10 μL: potassium phosphate, primers, bumpers, dGTP, dATP, dC s TP, BSA, DTT, trehalose, magnesium and 1 unit of uracil-N-DNA glycosylase (UDG). Samples were incubated for 30 minutes. The samples were then transferred to a Thermal-Lok dry bath set to the desired amplification temperature. The following reagents were added in a volume of 10 μL: potassium phosphate, dUTP, BSA, DTT, trehalose, magnesium, Bst, BsoBI and uracil-N DNA glycosylase inhibitor (UDI). Thermal SDA occurred over the next thirty minutes. Reactions were stopped by boiling samples for 5 minutes. Amplified products were detected by the extension of  32 P-labeled probe CTpB4.D1 (SEQ ID NO: 15) hybridized specifically to the central region of the amplicon. Detected samples were run on an 8% polyacrylamide gel and visualized by phosphorimage or by overnight exposure to autoradiographic film. 
     FIGS. 2A-D show the phosphorimages of the matrix of test conditions. The primer combination is indicated at the bottom of each lane. Under certain conditions only some of the primer combinations will work, while other conditions are favorable for any of the four combinations. Though combinations 1 and 2 appear to work better under a number of conditions, it is not clearly evident that one combination is superior to all others. 
     In the same experiment, four of the samples (52° C., 35 mM KPO 4 , 5 mM MgOAC, 3%DMSO, 7% Glycerol) were detected with a complementary detector, CTpB4.D2 (SEQ ID NO: 16). The results are shown in FIG.  3 . The detector which hybridized to the sense strand gave a 50-fold increase in intensity of the phosphorimage which indicates that better sensitivity is achieved with this detector as compared to its complement. Thus, all subsequent examples with System B used detector CTpB4.D2 (SEQ ID NO: 16). 
     EXAMPLE 2 
     Assay for Conditions Giving Greatest Sensitivity 
     In order to select a primer combination and buffer conditions with the greatest sensitivity, each of the above 4 primer combinations was tested at the two best conditions seen in Example 1. These two sets of conditions are as follow: 
     Condition 1 
     35 mM potassium phosphate, pH 7.6 
     6 mM magnesium acetate 
     3% dimethyl sulfoxide 
     3% glycerol 
     Amplification at 54° C. 
     Condition 2 
     35 mM potassium phosphate, pH 7.6 
     6 mM magnesium acetate 
     3% dimethyl sulfoxide 
     7% glycerol 
     Amplification at 54° C. 
     All other reaction conditions were as described in Example 1. The phosphorimage of the gel is shown in FIG.  4 . These results indicate that all combinations of primers worked better under condition 2, and that better sensitivity (10 2 ) was seen with primer combinations  1  and  2  under both conditions. Primer combination 2 was selected for further study. Thus, all subsequent examples for System B use primer combination 2 (CTpB4.S1.1 (SEQ ID NO: 10) and CTpB4.S 2  (SEQ ID NO: 12)). 
     EXAMPLE 3 
     Assay of the Specificity and Crossreactivity of System B 
     An assay was performed to test the ability of primer system B to amplify the multiple serovars of  C. trachomatis.  Fourteen serovars were tested using condition 2 described in Example 2. Each serovar was tested at 10 4  and 10 3  genome copies (the actual number of cryptic plasmid copies present in the sample is unknown). Thirteen of the fourteen serovars amplified at the 10 4  level, and 7 of the fourteen amplified at the 10 3  level (see FIG.  5 ). Serovar B did not amplify. Serovar B is 100% identical to the primers and bumpers and it is believed that the lack of amplification was due to an impure sample or low copy number of the plasmid. Two other species of Chlamydia and one strain of  Neisseria gonorrhoeae  were tested at an input level of 10 7  genome copies. None of these produced a specific amplified product. 
     EXAMPLE 4 
     Assay of the Sensitivity in the Presence of Human DNA 
     An assay was performed to test the system sensitivity in the presence of human DNA, using Condition 2 as described in Example 2 above. Target plasmid was titrated down to 10 copies in the presence of human DNA ranging from 300 to 3000 ng. The results are shown in FIG.  6 . Reactions contained 10 3 , 10 2  (duplicates), and 10 (duplicates) copies of target plasmid DNA for each level of hDNA. A negative control (no target plasmid) was also run at the 1200 ng hDNA level. The system showed 10 copy sensitivity from 600 to 2500 ng of human DNA and 10 2  sensitivity at 300 and 3000 ng of human DNA. 
     EXAMPLE 5 
     Assay for Crossreactivity with Other Microorganisms 
     An assay was performed to test whether there would be crossreactivity with potential genitourinary contaminants. Condition 2 as outlined in Example 2 was used. A total of thirty-five organisms sampling thirty species were tested. These organisms are listed in Table 1. Nine pools were prepared to each contain three or four species of DNA at 10 7  genome copies per species. Each pool was amplified in the absence of target plasmid or in the presence of 200 copies of plasmid pCT16. The pools with added target served as amplification controls. None of the pools without added target showed amplification while all the pools with added plasmid target amplified specific product. These results are shown in FIG.  7 . 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Pool 
                 Organism 
                 Strain 
               
               
                   
               
             
            
               
                 1 
                 
                   Neisseria gonorrhoeae 
                 
                 ATCC 19424 
               
               
                   
                 
                   Neisseria gonorrhoeae 
                 
                 ATCC 35541 
               
               
                   
                 
                   Neisseria gonorrhoeae 
                 
                 BDMS 2900 
               
               
                   
                 
                   Neisseria gonorrhoeae 
                 
                 BDMS 1632 
               
               
                 2 
                 
                   Neisseria lactamica 
                 
                 ATCC 23970 
               
               
                   
                 
                   Neisseria lactamica 
                 
                 ATCC 23972 
               
               
                   
                 
                   Neisseria meningitidis 
                 
                 ATCC 13090 
               
               
                   
                 
                   Neisseria meningitidis 
                 
                 ATCC 13077 
               
               
                 3 
                 
                   Staphylococcus aureus 
                 
                 ATCC 12598 
               
               
                   
                 
                   Streptococcus faecalis 
                 
                 ATCC 29212 
               
               
                   
                 Group A strep 
                 ATCC 16915 
               
               
                   
                 Group B strep 
                 ATCC 12386 
               
               
                 4 
                 
                   Salmonella typhimurium 
                 
                 ATCC 13311 
               
               
                   
                 
                   Salmonella minnesota 
                 
                 ATCC 9700  
               
               
                   
                 
                   Escherichia coli 
                 
                 ATCC 11775 
               
               
                   
                 
                   Klebsiella pneumoniae 
                 
                 ATCC 13883 
               
               
                 5 
                 
                   Proteus mirabilis 
                 
                 ATCC 29906 
               
               
                   
                 
                   Moraxella lacunata 
                 
                 ATCC 17967 
               
               
                   
                 
                   Haemophilus influenzae 
                 
                 ATCC 33533 
               
               
                   
                 
                   Acinetobacter lwoffii 
                 
                 ATCC 19001 
               
               
                 6 
                 
                   Gardenerella vaginalis 
                 
                 ATCC 14018 
               
               
                   
                 
                   Mycoplasma orale 
                 
                 ATCC 73714 
               
               
                   
                 
                   Trichomonas vaginalis 
                 
                 ATCC 30001 
               
               
                   
                 
                   Candida albicans 
                 
                 ATCC 44808 
               
               
                 7 
                 Herpes Simplex Virus-1 
                 McIntyre 
               
               
                   
                 HSV-1 
               
               
                   
                 Herpes Simplex Virus-2 
                 G 
               
               
                   
                 HSV-2 
               
               
                   
                 
                   Peptostreptococcus 
                 
                 ATCC 27340 
               
               
                   
                 
                   productus 
                 
               
               
                 8 
                 
                   Neisseria flavescens 
                 
                 ATCC 13120 
               
               
                   
                 
                   Neisseria sicca 
                 
                 ATCC 29193 
               
               
                   
                 
                   Neisseria cinerae 
                 
                 ATCC 14685 
               
               
                   
                 
                   Neisseria elongata 
                 
                 ATCC 25295 
               
               
                 9 
                 
                   Neisseria mucosa 
                 
                 ATCC 19696 
               
               
                   
                 
                   Neisseria subflava 
                 
                 ATCC 14799 
               
               
                   
                 
                   Branhamella catarrhalis 
                 
                 ATCC 25240 
               
               
                   
                 
                   Kingella kingae 
                 
                 ATCC 23330 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 6 
     Primers, Bumpers and Detectors from Cryptic Plasmid Region F for Performing tSDA 
     Sequence data of the F region of the Cryptic plasmid from four human serovars of  C. trachomatis  was available from the Genbank database. Four other serovars were sequenced in this region. A composite alignment was used to design tSDA primer sets to overcome sequence variations. The primers, bumpers and detectors chosen to be used with Region F are set out below. BsoBI sites within the tSDA primers are italicized and the hybridization region is underlined. Detector probes D1L and D2L were used in radiolabel extension assays, D3L and D4L were used in capture/detector assays, and FD1 was used as a fluorescent labeled detector. 
     Bumpers 
     CtpF8.BL 5′-CAGCAAATAATCCTTGG-3′ (SEQ ID NO: 18) 
     CtpF8.BR 5′-CATTGGTTGATGAATTATT-3′ (SEQ ID NO: 19) 
     tSDA Primers 
     CtpF8.AL1 5′-CGATTCCGCTCCAGACTTCTCGGGACAAAATCAACACCTG-3′ (SEQ ID NO: 20) 
     CtpF8.AL2 5′-CGATTCCGCTCCAGACTTCTCGGGACAAAATCAACACCTGT-3′ (SEQ ID NO: 21) 
     CtpF8.AR1 5′-ACCGCATCGAATGCATGTCTCGGGGAGACTGTTAAAGATA-3′ (SEQ ID NO: 22) 
     CtpF8.AR2 5′-ACCGCATCGAATGCATGTCTCGGGGAGACTGTTAAAGATAT-3′ (SEQ ID NO: 23) 
     CtpF8.AR3 5′-ACCGCATCGAATGCATGTCTCGGGGAGACTGTTAAAGATATT-3′ (SEQ ID NO: 24) 
     32P Detectors 
     CtpF8.D1L 5′-GTCGCAGCCAAAATG-3′ (SEQ ID NO: 25) 
     CtpF8.D2L 5′-ACAGCTTCTGATGGAA-3′ (SEQ ID NO: 26) 
     Chemiluminescent Capture/Detector Probes 
     CtpF8.D3L 5′-GTCGCAGCCAAAAT-(alkaline phosphatase×3)-3′ (SEQ ID NO: 27) 
     CtpF8.D4L 5′-(biotin×3)-GACAGCTTCTGATGGAA-3′ (SEQ ID NO: 28) 
     
       
         
           
               
               
               
            
               
                 Homogenous (Fluorescent) Detector Probe 
                   
                   
               
               
                               FAM           ROX 
               
               
                                |             | 
               
               
                 CtpF8.FD1   5′-TAGCACCCGAGTGCTCGCAGCCAAAATGACAGCTTCTGATGGAA-3′ 
                 (SEQ ID NO: 29) 
               
            
           
         
       
     
     EXAMPLE 7 
     Primer Screen for Region F 
     Primers of different lengths (and consequently different Tm) were screened in an experiment varying temperature (50° C. or 52° C.), potassium phosphate (25 mM or 35 mM), glycerol (3.5% or 7%), DMSO (3% or 8%) and human placental DNA (300 ng or 1200 ng). Other components for the tSDA reactions were: 6 mM magnesium acetate, 160 units BsoBI, 9 units Bst polymerase, 0.2 mM dATP, 1.4 mM d s CTP, 0.2 mM dGTP, 0.5 mM dUTP, 0.5 μM each primer, 0.05 μM each bumper, 1 unit UDG, 5 units UDI, 100 μg/mL BSA, 0.36 mM DTT, 1.86% trehalose. The target was 10 6  genomes of  C. trachomatis  LGVII. Decontamination was at 45° C. for 30 minutes and amplification was at either 50° C. or 52° C. for 30 minutes. 
     All primer combinations ( 6 ) were tested. Amplified products were detected in an extension reaction using the radiolabelled detector probe CtpF8.D1L (SEQ ID NO: 20). After separation of amplified products on an 8% acrylamide gel, quantification of intensity of radiolabeled products was performed with a Molecular Dynamics 445SI Phospholmager and ImageQuant v1.1 software. 
     All primer combinations produced detectable amplification products under at least five of the eight conditions tested and listed below. 
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Temp 
                 50° 
                 50° 
                 50° 
                 50° 
                 52° 
                 52° 
                 52° 
                 52° 
               
               
                 Phosphate 
                 25 mM 
                 25 mM 
                 35 mM 
                 35 mM 
                 25 mM 
                 25 mM 
                 35 mM 
                 35 mM 
               
               
                 Glycerol 
                 3.5% 
                 7% 
                 3.5% 
                 7% 
                 3.5% 
                 7% 
                 3.5% 
                 7% 
               
               
                 DMSO 
                 3% 
                 8% 
                 8% 
                 3% 
                 8% 
                 3% 
                 3% 
                 8% 
               
               
                 hpDNA 
                 1200 ng 
                 300 ng 
                 300 ng 
                 1200 ng 
                 1200 ng 
                 300 ng 
                 300 ng 
                 1200 ng 
               
               
                   
               
            
           
         
       
     
     One primer combination, AL1/AR1, produced strong amplification in six of the conditions tested. The best amplification was observed when using 35 mM potassium phosphate, 3% DMSO, 7% glycerol, 1200 ng human DNA and 50° C. amplification. This primer combination, along with the bumpers, make up primer set F. 
     EXAMPLE 8 
     Sensitivity Titration for Primer Set F 
     A titration of a plasmid, pCT16, constructed to contain one copy of the target region from  Chlamydia trachomatis  serovar J, was tested from 10 6  copies down to 1 copy. Also, a titration of a genomic DNA preparation from  C. trachomatis  elementary bodies was carried out for comparison. The conditions used for the tSDA were the same as those described in Example 7: 35 mM potassium phosphate, 3% DMSO, 7% glycerol, 6 mM magnesium acetate, 1200 ng human placental DNA and a 50° C. amplification temperature. After amplification, products were detected using CtpF8.D1L (SEQ ID NO: 25) in a radiolabelled probe extension reaction and quantified using ImageQuant v1.1 software. The results are shown in Table 2. Any intensity below 2×background is reported as zero. The data demonstrate that the tSDA primer set F can detect 10 copies ({fraction (3/3)} replicates) and can detect 1 copy (⅓ replicates) of the target region. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 Genome Copies of 
                   
               
               
                 Copies of pCT16 
                 Intensity 
                 Serovar LGV II 
                 Intensity (pixels) 
               
               
                   
               
             
            
               
                 10 6   
                 54,336,500 
                 10 6   
                 64,616,550 
               
               
                 10 5   
                 62,185,822 
                 10 5   
                 76,821,582 
               
               
                 10 4   
                 61,722,424 
                 10 4   
                 57,716,379 
               
               
                 10 3   
                 44,104,645 
                 10 3   
                 14,191,912 
               
               
                 10 3   
                 37,944,957 
               
               
                 10 2   
                  6,427,450 
                 10 2   
                  1,534,883 
               
               
                 10 2   
                 14,184,075 
               
               
                 10 2   
                  9,067,676 
               
               
                 10     
                  2,004,088 
                 10     
                    32,110 
               
               
                 10     
                   717,772 
               
               
                 10     
                  2,178,641 
               
               
                  1     
                   101,144 
                  1     
                    31,291 
               
               
                  1     
                     0 
               
               
                  1     
                     0 
               
               
                  0     
                     0 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 9 
     Specificity of Primer Set F for  C. trachomatis  Serovars 
     To determine whether system F could amplify all of the  C. trachomatis  serovars, whole genomic DNA preparations from thirteen serovars were tested. Serovar LGV I was not available for testing. These were tested under the same amplification conditions as in Example 8 and using 10 3  genomes. Each assay was performed in duplicate. The results are shown in Table 3. Any intensity less than 2×above the background level was reported as zero. 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Serovar 
                 Intensity (pixels) 
                 Intensity (pixels) 
               
               
                   
               
             
            
               
                 A 
                 218,413 
                  56,556 
               
               
                 B 
                     0 
                     0 
               
               
                 Ba 
                 187,056 
                 122,053 
               
               
                 C 
                 178,899 
                 126,053 
               
               
                 D 
                  51,968 
                 205,425 
               
               
                 E 
                 256,928 
                 715,980 
               
               
                 F 
                 309,899 
                 402,525 
               
               
                 G 
                     0 
                  53,155 
               
               
                 H 
                 465,217 
                 553,424 
               
               
                 I 
                 265,694 
                 446,492 
               
               
                 J 
                 250,981 
                 281,921 
               
               
                 K 
                  60,013 
                 148,717 
               
               
                 LIII 
                 220,077 
                 119,152 
               
               
                 Negative 
                     0 
                     0 
               
               
                   
               
            
           
         
       
     
     The data in Table 3 along with the data in Example 8 demonstrate that primer set F can detect at least 13 of the 15 serovars of  C. trachomatis  at 10 3  genomes. Only 14 of the 15 were tested because serovar LGV I was unavailable. The one serovar which gave a negative result is Serovar B. Serovar B genomic DNA was not detected by any of the amplification systems specific for  C. trachomatis  (see the above Examples), suggesting that the serovar B DNA preparation is suspect. 
     EXAMPLE 10 
     Further Specificity Testing of Primer Sets B and F for  C. trachomatis  Serovars 
     Because of the susceptibility of serovar B DNA and the unavailability of serovar LGV 1 DNA as reported in Example 9 above, the same conditions of Example 9 were used with only these two serovar DNAs to determine whether systems B and F could also amplify  C. trachomatis  serovars B and LGV 1. Each assay was performed in duplicate for 10 3 and 10 4  genomes. The results are shown in Table 4. Any intensity less than 2×above the background level was reported as zero. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Serovar 
                 # of Genomes 
                 System F 
                 System B 
               
               
                   
                   
               
             
            
               
                   
                 B 
                 10 4   
                 14,592  
                   71 
               
               
                   
                 B 
                 10 4   
                 8,072 
                 1,286 
               
               
                   
                 B 
                 10 3   
                 9,859 
                    0 
               
               
                   
                 B 
                 10 3   
                   134 
                   52 
               
               
                   
                 LGV 1 
                 10 4   
                 8,888 
                 8,260 
               
               
                   
                 LGV 1 
                 10 4   
                 15,663  
                 5,371 
               
               
                   
                 LGV 1 
                 10 3   
                 1,032 
                   140 
               
               
                   
                 LGV 1 
                 10 3   
                 6,728 
                    0 
               
               
                   
                   
               
            
           
         
       
     
     Thus, it was concluded that System F could amplify and detect both serovar B and serovar LGV 1 at 10 3  genomes, and that System B could amplify and detect serovar B at 10 4  genomes and serovar LGV 1 at 10 3  genomes. 
     EXAMPLE 11 
     Optimization of Reaction Conditions for Primer Set F 
     Primer set F was used in a series of assays utilizing a variety of reaction conditions. The assays used a target level of 100 copies of pCT16. Amplification was seen under all of the following reaction condition ranges: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Amplification temperature 
                  50-54° C. 
               
               
                   
                 Potassium phosphate 
                  25-35 mM 
               
               
                   
                 DMSO 
                  3-7% (vol/vol) 
               
               
                   
                 Glycerol 
                  3-7% (vol/vol) 
               
               
                   
                 Magnesium acetate 
                  5-6 mM 
               
               
                   
                 Human placental DNA 
                 300-3050 ng 
               
               
                   
                   
               
            
           
         
       
     
     In several instances the above ranges were the limits of what was tested and it is likely that amplification will occur with reaction conditions beyond the ranges shown above. The conditions identified as producing the greatest amplification were: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Amplification temperature 
                  52° C. 
               
               
                   
                 Potassium phosphate 
                  35 mM 
               
               
                   
                 DMSO 
                   7% (vol/vol) 
               
               
                   
                 Glycerol 
                   7% (vol/vol) 
               
               
                   
                 Magnesium acetate 
                   5 mM 
               
               
                   
                 Human placental DNA 
                 1200 ng 
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 12 
     Assay of Crossreactivity of Primer Set F 
     There are potential cross-reactant bacteria and viruses commonly found in genitourinary clinical specimens. Thermal SDA reactions were performed using primer set F using the best assay conditions of Example 11. The potential cross-reactant specimens were tested at 10 7  genomes. These were tested in pooled samples of four species, with a control of the same pool spiked with 100 copies of pCT16 to show that no inhibition of amplification occurred. The results are shown in Table 5. None of the cross-reactants was amplified or detected by primer set F. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Organism 
                 Strain 
                 Result 
                 Organism 
                 Strain 
                 Result 
               
               
                   
               
             
            
               
                 
                   Chlamydia psittaci 
                 
                 TWAR 
                 — 
                 
                   Branhamella catarrhalis 
                 
                 ATCC 25240 
                 — 
               
               
                 
                   Chlamydia pneumoniae 
                 
                 Borg 
                 — 
                 
                   Moraxella lacunata 
                 
                 ATCC 17967 
                 — 
               
               
                 
                   Neisseria gonorrhoeae 
                 
                 BDMS 1632 
                 — 
                 
                   Kingella kingae 
                 
                 ATCC 23330 
                 — 
               
               
                 
                   Neisseria gonorrhoeae 
                 
                 ATCC 19424 
                 — 
                 
                   Salmonella typhimurium 
                 
                 ATCC 13311 
                 — 
               
               
                 
                   Neisseria gonorrhoeae 
                 
                 BDMS 2900 
                 — 
                 
                   Salmonella minnesota 
                 
                 ATCC 9700 
                 — 
               
               
                 
                   Neisseria gonorrhoeae 
                 
                 ATCC 35541 
                 — 
                 
                   Staph. aureus 
                 
                 ATCC 12598 
                 — 
               
               
                 
                   Neisseria meningitidis 
                 
                 ATCC 13090 
                 — 
                 
                   Acinetobacter lwoffi 
                 
                 ATCC 19001 
                 — 
               
               
                 
                   Neisseria meningitidis 
                 
                 ATCC 13077 
                 — 
                 
                   E. coli 
                 
                 ATCC 11775 
                 — 
               
               
                 
                   Neisseria lactamica 
                 
                 ATCC 23970 
                 — 
                 
                   Klebsiella pneumoniae 
                 
                 ATCC 13883 
                 — 
               
               
                 
                   Neisseria lactamica 
                 
                 ATCC 23972 
                 — 
                 
                   Gardnerella vaginalis 
                 
                 ATCC 14018 
                 — 
               
               
                 
                   Neisseria flavescens 
                 
                 ATCC 13120 
                 — 
                 Streptococcus Group A 
                 ATCC 16915 
                 — 
               
               
                 
                   Neisseria sicca 
                 
                 ATCC 29193 
                 — 
                 Streptococcus Group B 
                 ATCC 12386 
                 — 
               
               
                 
                   Neisseria subflava 
                 
                 ATCC 14799 
                 — 
                 
                   Proteus mirabilis 
                 
                 ATCC 29906 
                 — 
               
               
                 
                   Neisseria cinerea 
                 
                 ATCC 14685 
                 — 
                 
                   Haemophilus influenzae 
                 
                 ATCC 33533 
                 — 
               
               
                 
                   Neisseria elongata 
                 
                 ATCC 25295 
                 — 
                 
                   Mycoplasma orale 
                 
                 ATCC 23714 
                 — 
               
               
                 
                   Neisseria mucosa 
                 
                 ATCC 19696 
                 — 
                 Herpes simplex V-1 
                 McINTYRE 
                 — 
               
               
                 
                   Streptococcus faecalis 
                 
                 ATCC 27340 
                 — 
                 Herpes simpiex V-2 
                 Strain G 
               
               
                 
                   Candida albicans 
                 
                 ATCC 44808 
                 — 
                 
                   Trichomonas vaginalis 
                 
                 ATCC 30001 
                 — 
               
               
                 
                   Peptostreptococcus productus 
                 
                 ATCC 27340 
                 — 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 13 
     Homogeneous Real Time Fluorescent tSDA 
     Detection of  C. trachomatis  in Systems B and F 
     The primer sets F and B were also useful in performing homogeneous real time fluorescent tSDA reactions. Such reactions use a detector which includes a hairpin sequence and an acceptor and a donor fluorescent molecules. One such detector which works well for use with the system F primer set is CTpF8.FD1 5′-TAGCACCCGAGTGCTCGCAGCCAAA ATGACAGCTTCTGATGGAA-3′ (SEQ ID NO: 29). A detector which works well for use with system B is CTpB4.FD1 5′-TAGCACCCGAGTGCTTTGACGATTTTCTCCAACCGA TGAGTTGAT-3′ (SEQ ID NO: 17). The first 15 bases of these sequences include a hairpin structure as well as a BsoBI restriction site. Bases 2-6 can base pair with bases 15-11. Furthermore, base  1  is modified by addition of FAM and base 15 is modified by addition of ROX. This hairpin structure (the first 15 bases of SEQ ID NO: 29) is 5′-(FAM)*TAGCACCCGAGTGCT*(ROX)-3′ (SEQ ID NO: 30). Bases 6-11 are shown in italics and represent the BsoBI restriction site. 
     The protocol for performing homogeneous real time fluorescent tSDA is similar to that for regular tSDA. One such protocol, which has been adapted for larger volume reactions for use with microtiter well formats, but which follows the tSDA protocol in ratios of components is as follows: 
     1. Equilibrate a Thermal-Lok bath to 45° C. and equilibrate another Thermal-Lok bath to 52° C. using an Electro-Therm digital thermometer after filling wells with 1 mL of water. Also equilibrate a dry Thermal-Lok bath to 54° C. 
     2. Prepare the tSDA reaction buffer and prepare the decontamination mix. Keep these at room temperature. 
     3. Add 65 μL of tSDA reaction buffer to each 0.5 mL Eppendorf Safelok tube. 
     4. Add 5 μL of template diluted in 10 ng/μL human DNA per tube. 
     5. Place tubes in boiling waterbath for 2 minutes. 
     6. Transfer tubes to microfuge and pulse spin. 
     7. Transfer tubes to the 45° C. Thermal-Lok for 5 minutes. 
     8. Add 15 μL of decontamination mix to each tube, vortex briefly, and incubate for 20 minutes. 
     9. Prepare amplification mix and store at room temperature. 
     10. Place microtiter wells in metal tray and place on top of 54° C. Thermal-Lok to preheat. 
     11. Transfer tubes to 52° C. Thermal-Lok for 5 minutes. 
     12. Transfer 85 μL of the reaction into appropriate microtiter well. For end point reactions only, leave sample in tube. 
     13. Add 15 μL of amplification mix to each well. Seal wells with a self adhesive acetate sheet. For end point reactions, add 15 μl of amplification mix to each tube, vortex and continue incubating in thermal-lok for 60 minutes. 
     14. Place the tray into a fluorescent plate reader at 52° C. and collect RFU data over 60 minutes. For end point reactions, transfer contents of tubes to microtiter wells and take a single reading for each well. 
     EXAMPLE 14 
     Sensitivity of Systems B and F Using Homogeneous Detection 
     To test the sensitivity of using primer systems F and B with the CtpF8.FD1 and CTpB4.FD1 detectors in a homogeneous real time fluorescent tSDA reaction, a series of such reactions was performed using plasmid pCT16 as the target at different copy numbers. For System B, the end point protocol described in Example 12 was used. The final reaction conditions were: 35 mM potassium phosphate 3% DMSO, 7% glycerol, 6 mM magnesium acetate, 40 units Bst, 640 units BsoBI, 2400 ng human placental DNA, 0.2 mM dATP and dGTP, 1.4 mM dCsTP, 0.5 mM dUTP, 0.1 mg/ml acetylated bovine serum albumin, 1.8% trehalose, 0.36 mM DTT, and 200 mM CTpB4.FD1 (SEQ ID NO: 17). Amplification was done at 54° C. and decontamination enzyme (UDG) and inhibitor (UDI) was used. The following results were obtained by reading the samples after 60 minutes incubation (excitation 485 nm, emission 538 nm): 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                   
                 Endpoint RFU/ 
               
               
                 Plasmid Copy 
                 End Point RFU 
                 Mean Background 
               
               
                   
               
             
            
               
                 500 
                 2.52 
                  4.8 
               
               
                   
                 7.27 
                 14.0 
               
               
                   
                 14.35  
                 27.6 
               
               
                 200 
                 6.72 
                 12.9 
               
               
                   
                 2.14 
                  4.1 
               
               
                   
                 11.64  
                 22.4 
               
               
                  50 
                 1.20 
                  2.3 
               
               
                   
                 0.66 
                  1.3 
               
               
                   
                 0.43 
                  0.8 
               
               
                 0, Background 
                 0.57 
               
               
                   
                 0.51 
               
               
                   
                 0.48 
               
               
                   
               
            
           
         
       
     
     The results indicate a sensitivity of 50 copies of pCT16 for one out of three replicates, when positive is defined as 2×above mean background. 
     For system F, decontamination and amplification mixes were prepared as previously described in Example 12 but at a 10×concentration level for all reagents. Fifteen microliters of decontamination mix or ten microliters of amplification mix were spotted into the bottoms of microtiter wells and dried in a low humidity drying chamber. Decontamination wells and amplification wells were packaged separately with desiccant until use. 
     Wells containing decontamination spots were prewarmed at 45° C. on a Thermal-Lok heat block for five minutes. Samples containing purified water, potassium phosphate, glycerol, DMSO, human placental DNA, and plasmid pCT16 were boiled for two minutes and spun quickly in a microcentrifuge to pool. One hundred fifty microliters of each sample were pipetted into the prewarmed decontamination wells, sealed with adhesive film and incubated for twenty minutes at 45° C. The wells were moved to a second Thermal-Lok heat block set to achieve a temperature of 52° C. in the wells. Coincidentally, an equivalent number of the above described spotted amplification wells were placed on the same heat block to prewarm. After five to ten minutes, the film was removed from the decontamination wells, and one hundred microliters of sample was removed from each well and pipetted into an amplification well (one amplification well per sample). The final reaction conditions in the micro wells after the rehydration of the amplification spot were: 35 mM potassium phosphate, 7% glycerol, 7% DMSO, 5 mM magnesium acetate, 0.2 mg/mL acetylated BSA, 1.86% trehalose, 0.36 mM dithiothreitol, 0.7 mM α-thiolated dCTP, 0.25 mM dUTP, 0.1 mM dATP, 0.1 mM dGTP, 40 units Bst polymerase, 640 units BsoBI, 1 unit Uracil DNA glycosylase, 5 units uracil DNA glycosylase inhibitor, 0.5 μM ARI (primer), 0.3 μM AL1 (primer), 0.05 μM BL1 and BR1 (bumpers), 0.2 μM FD1 (detector oligo) and 2400 ng human placental DNA. Amplification wells were sealed using a fresh sheet of adhesive film, and immediately transferred to a PerSeptives plate reader which had been set to maintain a constant incubation temperature of 52° C. Wells were read immediately and at one minute intervals for the following sixty minutes. The samples were excited at 485 nm and the emitted fluorescence was monitored at 530 nm. Table 6 shows the results. These results indicate a system sensitivity of 10 copies of target DNA. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 Plasmid Copy 
                   
                   
                   
               
               
                   
                 # 
                 Initial RFU 
                 Final RFU 
                 Difference 
               
               
                   
                   
               
             
            
               
                   
                  0 
                 298 
                 334 
                  36 
               
               
                   
                  0 
                 361 
                 407 
                  46 
               
               
                   
                 10 
                 370 
                 726 
                 356 
               
               
                   
                 10 
                 295 
                 299 
                  4 
               
               
                   
                 50 
                 390 
                 718 
                 328 
               
               
                   
                 50 
                 357 
                 1076  
                 719 
               
               
                   
                 100  
                 299 
                 1164  
                 865 
               
               
                   
                 100  
                 391 
                 1706  
                 1315  
               
               
                   
                   
               
            
           
         
       
     
     EXAMPLE 15 
     Sensitivity of System F Using Homogeneous Detection 
     A second set of experiments was performed to check the sensitivity of system F. Whereas Example 14 used plasmid pCT16 as the target, for this second set of experiments, elementary bodies of  C. trachomatis  LGV2 were used as the target. Reactions were performed as in Example 13. The final reaction conditions were as described in Example 14 except the following: 1.4 mM thiolated dCTP, 0.5 mM dUTP, 0.2 mM dATP and dGTP. RFU data was collected every minute for the 60 minute amplification time. The difference in RFU between time 0 and time 60 is shown in Table 7 for two replicates at each level. Again, this fluorescent detector based system F can amplify and detect above background level as few as 10 elementary bodies of  C. trachomatis.   
     
       
         
           
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                 Change in RFU 
                 Change in RFU 
               
               
                 Number of Elementary Bodies 
                 Replicate 1 
                 Replicate 2 
               
               
                   
               
             
            
               
                 300 
                 1142  
                 769 
               
               
                 250 
                 1430  
                 1015  
               
               
                 200 
                 836 
                 813 
               
               
                 150 
                 401 
                 307 
               
               
                 100 
                 262 
                 573 
               
               
                  80 
                 717 
                 407 
               
               
                  60 
                 381 
                 424 
               
               
                  40 
                 257 
                 461 
               
               
                  20 
                 468 
                 349 
               
               
                  10 
                 158 
                  66 
               
               
                 Positive control pCT16 - 150 copies 
                 586, 215, 524 
               
               
                 Negative control 
                  75 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 16 
     Specificity of System F Using Homogenous Detection 
     The specificity of the CtpF811.FD1 system as used in homogeneous real time fluorescent tSDA was tested. Reactions were performed as in Example 14. Cross reactant pools were tested at 10 7  genomes per reaction. Amplifications were performed in a PerSeptive Biosystems CytoFluor 4000 plate reader. RFU data was collected every minute for the 60 minute amplification time. The difference in RFU from time 0 to time 60 is shown in Table 8. None of the pools show amplification or detection of the possible cross-reactants which were tested. 
     
       
         
           
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Pool 
                 Cross-Reactant 
                 Change in RFU 
               
               
                   
               
             
            
               
                 1 
                   Neisseria gonorrhoeae  ATCC 19424 
                 25 
               
               
                   
                   Neisseria gonorrhoeae  ATCC 35541 
               
               
                   
                   Neisseria gonorrhoeae  BDMS 2900 
               
               
                   
                   Neisseria gonorrhoeae  BDMS 1632 
               
               
                 2 
                   Neisseria meningitidis  ATCC 13077 
                 24 
               
               
                   
                   Neisseria meningitidis  ATCC 13090 
               
               
                 3 
                   Staph. aureus  ATCC 12598 
                 13 
               
               
                   
                 Streptococcus grp A ATCC 16915 
               
               
                   
                 Streptococcus grp B ATCC 12386 
               
               
                   
                   Streptococcus faecalis  ATCC 29212 
               
               
                 4 
                   Salmonella minnesota  ATCC 9700 
                 20 
               
               
                   
                   Salmonella typhimurium  ATCC 13311 
               
               
                   
                   E. coli  ATCC 11775 
               
               
                   
                   Klebsiella pneumoniae  ATCC 13883 
               
               
                 5 
                   Proteus mirabilis  ATCC 29906 
                 13 
               
               
                   
                   Moraxella lacunata  ATCC 17967 
               
               
                   
                   Haemophilus influenzae  ATCC 33533 
               
               
                 6 
                   Gardenerella vaginalis  ATCC 14018 
                 28 
               
               
                   
                   Mycoplasma orale  ATCC 23714 
               
               
                   
                   Trichomonas vaginalis  ATCC 30001 
               
               
                   
                   Candida albicans  ATCC 44808 
               
               
                 7 
                 HSV 1 McIntyre 
                 13 
               
               
                   
                 HSV 2 G 
               
               
                   
                   Peptostreptococcus productus  ATCC 44808 
               
               
                 8 
                   Neisseria flavescens  ATCC 13120 
                 −1 
               
               
                   
                   Neisseria sicca  ATCC 29193 
               
               
                   
                   Neisseria cinera  ATCC 14685 
               
               
                   
                   Neisseria elongata  ATCC 25295 
               
               
                 9 
                   Neisseria mucosa  ATCC 19696 
                 23 
               
               
                   
                   Neisseria subflava  ATCC 14799 
               
               
                   
                   Branhamella catarrhalis  ATCC 25240 
               
               
                   
                   Kingella kingae  ATCC 23330 
               
               
                 10  
                   Neisseria meningitidis  ATCC 14632 
                  0 
               
               
                   
                   Neisseria meningitidis  ATCC 13077 
               
               
                   
                   Neisseria meningitidis  ATCC 13102 
               
               
                   
                   Neisseria meningitidis  ATCC 13113 
               
               
                   
                   Neisseria meningitidis  ATCC 35559 
               
               
                 11  
                   Neisseria lactamica  ATCC 44418 
                 15 
               
               
                   
                   Neisseria lactamica  ATCC 49142 
               
               
                   
                   Neisseria lactamica  ATCC 23971 
               
               
                   
                   Neisseria lactamica  ATCC 23972 
               
               
                 12  
                   Neisseria lactamica  ATCC 23970 
                 30 
               
               
                 13  
                 Positive control pCT16 - 100 copies 
                 401, 150 
               
               
                 14  
                 Negative control 
                 22, 27 
               
               
                   
               
            
           
         
       
     
     While the invention has been described with some specificity, modifications apparent to those with ordinary skill in the art may be made without departing from the scope of the invention. Various features of the invention are set forth in the following claims. 
     
       
         
           
             30 
           
           
             
               22 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             1
GAAGATCGAG TAGACGTAAT AT                                              22 
           
           
             
               23 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             2
ATAGCAGATA TATCTAGAAC CTT                                             23 
           
           
             
               22 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             3
TTGGATCGAA ATGTAATACC GA                                              22 
           
           
             
               22 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             4
ACTTCTGATT TTCAAGGTGG AT                                              22 
           
           
             
               22 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             5
CAAAACTGCG TCTTTGCTGA TA                                              22 
           
           
             
               21 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             6
GGGTGTGACT GTGAATTTTC C                                               21 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             7
AGTTGGGCAA ATGACAGAGC                                                 20 
           
           
             
               20 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             8
TGAATAACCC GTTGCATTGA                                                 20 
           
           
             
               40 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             9
CGATTCCGCT CCAGACTTCT CGGGCGATTA CTTGCAGTTG                           40 
           
           
             
               39 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             10
CGATTCCGCT CCAGACTTCT CGGGGATTAC TTGCAGTTG                            39 
           
           
             
               16 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             11
GAGTAGACGT AATATT                                                     16 
           
           
             
               41 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             12
ACCGCATCGA ATGCATGTCT CGGGATATCT GCTATTTCAT T                         41 
           
           
             
               40 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             13
ACCGCATCGA ATGCATGTCT CGGGATATCT GCTATTTCAT                           40 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             14
CTCTTAAGGT TCTAG                                                      15 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             15
CTCATCGGTT GGAGA                                                      15 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             16
TCTCCAACCG ATGAG                                                      15 
           
           
             
               45 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             17
TAGCACCCGA GTGCTTTGAC GATTTTCTCC AACCGATGAG TTGAT                     45 
           
           
             
               17 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             18
CAGCAAATAA TCCTTGG                                                    17 
           
           
             
               19 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             19
CATTGGTTGA TGAATTATT                                                  19 
           
           
             
               40 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             20
CGATTCCGCT CCAGACTTCT CGGGACAAAA TCAACACCTG                           40 
           
           
             
               41 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             21
CGATTCCGCT CCAGACTTCT CGGGACAAAA TCAACACCTG T                         41 
           
           
             
               40 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             22
ACCGCATCGA ATGCATGTCT CGGGGAGACT GTTAAAGATA                           40 
           
           
             
               41 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             23
ACCGCATCGA ATGCATGTCT CGGGGAGACT GTTAAAGATA T                         41 
           
           
             
               42 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             24
ACCGCATCGA ATGCATGTCT CGGGGAGACT GTTAAAGATA TT                        42 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             25
GTCGCAGCCA AAATG                                                      15 
           
           
             
               16 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             26
ACAGCTTCTG ATGGAA                                                     16 
           
           
             
               14 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             27
GTCGCAGCCA AAAT                                                       14 
           
           
             
               17 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             28
GACAGCTTCT GATGGAA                                                    17 
           
           
             
               44 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             29
TAGCACCCGA GTGCTCGCAG CCAAAATGAC AGCTTCTGAT GGAA                      44 
           
           
             
               15 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               unknown 
             
             30
TAGCACCCGA GTGCT                                                      15