Intergenic spacer target sequence for detecting and distinguishing Chlamydial species or strains

[0002] 1. Field of the Invention

[0003] This invention relates to a target nucleic acid sequence and to probes and primers that detect that target sequence in laboratory and clinical samples containing Chlamydiaceae.

[0004] 2. Description of the Prior Art

[0005] The order Chlamydiales includes at least four families that are proven or suspected pathogens of humans or other animals: Parachlamydiaceae, Simkaniaceae, Waddliaceae, and Chlamydiaceae [Everett, K. D. E., et al. (1998), Int J Syst Bacteriol, Submitted; Rurangirwa, F. R., et al. (1998), Int J Syst Bacteriol, Submitted]. These pathogens have been associated with reproductive, respiratory, cardiovascular, gastrointestinal, or systemic disease, and conjunctivitis, arthritis, and encephalitis in the host animal [Everett, K. D. E., et al. (1997), Int J Syst Bacteriol47: 461-473; Birtles, R. J., et al. (1997), Lancet 349: 925-926; Kahane, S., et al. (1998a), J Infect DisIn press; Kahane, S., et al. (1998b), In Proceedings of the Third Meeting of the European Society for Chlamydia Research, p. 18. Edited by A. Stary., Bologna, Italy: Study Group for STD and Dermatological Microbiology of the Austrian Society for Dermatology and Venerology; Lieberman, D., et al. (1997), Am J Respir Crit Care Med156: 578-82]. Numerous Chlamydiaceae isolates comprising some 60 strains have been reported since the early 1900's. These belong to two genera and nine species, as determined by genetic and phenotypic criteria, and these species can be identified by DNA sequence analysis (Everett & Andersen, 1998). However, a rapid systematic technique to routinely distinguish all nine Chlamydiaceae species is not available.

[0006] Efforts to speciate chlamydial isolates have been ongoing since the early days of chlamydial research. Because chlamydiae only grow inside inclusions in host cells, the only distinguishing criteria that initially could be relied upon were inclusion morphology, sulfadiazine resistance, and the accumulation of glycogen within the cells. Eventually, strains were characterized by PCR-RFLP, type-specific antigens, host associations, and monoclonal antibodies (MAb) [Andersen, A. A. (1991), J Clin Microbiol29: 707-711; Moulder, J. W., Hatch, T. P., Kuo, C. -C., Schachter, J. & Storz, J. (1984), Genus Chlamydia. In Bergey's Manual of Systematic Bacteriology, vol. 1., pp. 729-739. Edited by N. R. Krieg., Baltimore, Md.: The Williams & Wilkins Co.; Stephens, R. S., et al., (1982), J Immunol128: 1083-1089; Wang, S. -P., et al. (1985), J Infect Dis152: 791-800]. Today Chlamydiaceae isolates are generally distinguished by a variety of MAb or PCR tests that recognize only one strain or species at a time. These assays were developed using limited numbers of isolates and were not intended for speciating, per se. A number of problems associated with using these tests for speciation has becoming evident. For example, MAbs that recognize a “species-specific” C. trachomatisepitope (in variable-segment-IV of the major outer membrane protein) also probably recognize Chlamydia suis, according to DNA sequence analyses [Everett, K. D. E., et al., (1998), Int J Syst Bacteriol, Submitted]. Plasmid-based C. trachomatistests originally thought to be species specific, do not detect plasmid-strains [An, Q., et al. (1994), Mol Cell Probes8: 429-435; An, Q., et al. (1992), J Clin Microbiol30: 2814-2821]. Because all of the species cannot be identified with a single test, they can be mistaken for one another or diagnosis can be missed. With only a limited amount of sequence data in hand, PCR tests have been developed that identify up to four species and two subclades [Holland, S. M., et al., (1990), J Infect Dis162: 984-987; Kaltenbock, B., et al., (1997), J Clin Microbiol35: 1835-1841; Meijer, A. et al., (1997), J Clin Microbiol35: 1179-1183; Messmer, T. O., et al. (1997), J Clin Microbiol35; Tong, C. Y., & Sillis, M. (1993), J Clin Pathol 46: 313-317]. For the most part, the primers used in these tests are a reasonable match to most target sequences, but these tests are incapable of distinguishing all nine species. Thus, our ability to examine the epidemiology and pathogenesis of chlamydial species has been critically limited.

[0007] PCR-RFLP may be used to identify bacterial species, and requires a target gene that has appropriately conserved and variable segments. The gene that expresses the major outer membrane protein, ompA (omp1), has now been completely sequenced from more than 50 strains and it is evident that excess sequence diversity in this gene limits its usability in PCR-RFLP identification of the nine chlamydial species (Everett et al., 1998, supra). The 7.5 kbp plasmid is also a poor target for systematic PCR-RFLP identification of Chlamydiaceae species because, in many strains, it is either uncharacterized or absent (Everett et al., 1998, supra).

[0008] Shah et al. (WO 90/15159) reported a series of oligonucleotide probes, 28-36 nucleotides in length, that were specific for C. trachomatis. These probes targeted either the 16S or the 23S rRNA or rDNA.

[0009] Longiaru et al. (U.S. Pat. No. 5,232,829) disclose a number of PCR primers and capture probes for amplifying and detecting C. trachomatis. These oligonucleotides targeted either the 16S or the 23S rRNA or rDNA.

[0010] Yang et al. (U.S. Pat. Nos. 5,512,445 and 5,514,551) show oligonucleotide probes and primers for the amplification and specific detection of C. trachomatis. These oligonucleotides are targeted to regions of the 16S and the 23S rRNA/rDNA.

[0011] Hogan et al. shows a series of probes and primers specific to rRNA of a number of bacteria. The probes and primer for C. trachomatistarget variable regions of the 16S and 23S rRNA.

[0012] Goessens et al. present a comparison of three commercially available amplification assays for detecting C. trachomatisin urine samples. The sensitivity of these assays ranged from 90-96% and the specificities ranged from 98-99%.

[0013] We have now succeeded in identifying the intergenic spacer between the 16S and the 23S genes and a region of the 3′ end of Domain I in the 23S region for 43 strains of Chlamydiaceae and have thereby identified a novel pair of target regions of the genome for assaying and identifying all strains of Chlamydiaceae. The sequence of this target region allows for the construction of suitable probes and primers that can be group-specific for the identification of all species of Chlamydiaceae, or species-specific, or even strain-specific. The appropriate selection of probe or primer set can be used to identify the presence of Chlamydiaceae in a test sample, or to distinguish one strain from another.

[0014] In accordance with this discovery, it is an object of the invention to provide a sensitive, specific, and rapid diagnostic tool for positively identifying Chlamydiaceae in a clinical or laboratory sample.

[0015] It is also an object of the invention to provide a target region for constructing probes and primer sets tailored to the desired specificity for detecting chlamydial infections.

[0016] Another object of the invention is to provide an improved method for diagnosing chlamydial infections in humans and animals.

[0017] Yet another object of the invention is to provide a diagnostic test for chlamydial infection that would distinguish the 9 species of Chlamydiaceae.

[0018] Other objects and advantages of the invention will become readily apparent from the ensuing description.

[0019] FIG. 1 is a map of ribosomal DNA for Chlamydiaceae species showing the location of the target regions of this invention relative to the 16S and the 23S ribosomal genes and further illustrating sequence differences among representative strains of the nine groups of chlamydiae.

[0020] As used herein, the expression “test sample” is intended to mean any clinical, laboratory, environmental or other collected sample of material that is suspected of containing the intended target nucleic acid which is to be detected. Exemplary test samples include swabs, scrapings or collections of food, body fluids or other sources of chlamydial contamination.

[0021] The expression “target nucleic acid” or “target sequence” is intended to include the sequence within Regions A and/or B of any one of rDNA SEQ ID Nos. 1-43 as defined in Table IV, below, as well as corresponding rRNA sequences, DNA or RNA sequences complementary to SEQ ID Nos. 1-43, and any portion of the aforementioned DNA or rRNA sequences that is of sufficient size to permit the desired level of identification. For those instances wherein the purpose of the diagnostic test is to ascertain the presence or absence of Chlamydiaceae spp., the target nucleic acid would preferably be a group-specific consensus sequence. Whereas, in those cases wherein it is desired to distinguish one species or strain of Chlamydiaceae over others, then a species or strain specific sequence would be the target nucleic acid.

[0022] The term “probe” is used herein in the broadest sense to refer to either a labeled or an unlabeled, single-stranded nucleic acid that will hybridize under predetermined conditions of stringency to the target nucleic acid. Such probes may be RNA or DNA and will typically be at least about 15 bases in length, and preferably about 20-100 bases in length. When used in a hybridization assay, hybrids formed from the probes and the target sequence are usually detected by means of a detectable label affixed directly to the probe. Alternatively, probes can be used as helper probes to facilitate binding of a separate labeled probe to the target nucleotide. It is understood that for hybridization to occur, the probe may or may not be exactly complementary to the target sequence, provided that the hybridization conditions are appropriately selected to permit hybridization even when there are a limited number of mismatches between the respective sequences.

[0023] The term “primer” is used herein in its usual sense to be descriptive of an oligonucleotide (RNA or DNA), usually about 15-30 nucleotides in length, and preferably about 17-26 bases in length, that will participate in a primer extension reaction when catalyzed by a polymerase. These reactions are more commonly referred to as “polymerase chain reactions”. Contemplated herein as primers are only those nucleotides that are properly oriented so as to amplify a region within the target sequence.

[0024] The expression “substantial equivalent thereof” in reference to any target sequence or to the sequence of a probe or primer is intended to mean that minor additions, deletions, or mismatches can be present in the sequence to the extent that such variations do not prevent the hybridization or annealing of the nucleic acids essential to the assay.

[0025] “Stringency” refers to the conditions under which hybridization takes place. At high stringency, only exact matches of DNA and/or RNA will hybridize stably. Under low stringency, 80-90% homologous sequences may still hybridize.

[0026] The expression “sequence-specific oligonucleotide” is used herein to refer to probes or primers having a hybridizing region that is exactly complementary to a segment of the target region.

[0027] The target sequence for use in the invention is principally the region of the intergenic spacer between the 16S and the 23S ribosomal genes including the far downstream (3′) end of the 16S gene and the far upstream (5′) end of the 23S Domain I flanking the intergenic spacer (see FIG. 1) hereafter referred to as Region A, and secondarily a 131-bp region in the 3′ end of Domain I in the 23S rRNA, hereafter referred to as Region B. Region A is sometimes referred to herein as the “region of the intergenic spacer”. The 16S/23S intergenic spacer lends itself to the development of species-specific DNA-based probes for Chlamydiaceae because it contains conserved sequence interspersed with species-specific segments. We have found that this rDNA segment is a phylogenetic marker for the nine chlamydial species and DNA sequence data for all species, including >40 strains.

[0028] To initially identify the target sequences, 2.8 kb partial ribosomal DNA segments from a C. trachomatisstrain R22 and C. psittacistrain NJ1 were amplified by PCR and sequenced. Subsequently, 1320 bp of rDNA, including both the 16S/23S intergenic spacer (232±11 bp) and Domain I (620±2 bp) of the 23S gene, were selected and sequenced from each of 41 additional strains. Using both parsimony and percent distance analyses, these sequences were found to have variable regions that grouped the isolates into two lineages (C. trachomatisand non-C. trachomatis) with nine distinct genotypic groups. The C. trachomatislineage included human, swine, and mouse/hamster groups. The non-C. trachomatislineage included C. pecorum, C. pneumoniae, and C. psittaci-abortion, -avian, -feline, and -guinea pig groups. The 43 strains of Chlamydia spp. studied, included 18 strains of C. psittaci, 7 strains of C. pneumoniae, 6 strains of C. pecorum, and 12 strains of C. trachomatis.

[0029] In FIG. 1, the locations of primers that were conserved in all Chlamydia strains and that were particularly useful in the PCR and sequence analysis are indicated, as are conserved restriction sites. The vertical solid bars in the figure indicate sequence differences. The upper part of FIG. 1 is a sequence map comparison for the 2.8-kb ribosomal segment from C. psittaciNJ1 and swine strain C. trachomatisR22. The shaded regions of the 16S and 23S ribosomal genes plus the intergenic spacer were used for the double stranded sequence analysis performed with the 43 Chlamydiaceae strains. The lower part of FIG. 1 illustrates sequence differences among representative strains of the nine groups of chlamydiae (strains A/Har-13^T, R22, MoPn, 6BC^T, B577, FML-12, FP, GPIC, and E58^T) . Each of the 43 Chlamydiaceae strains clustered with one of these strains on the basis of the ribosomal sequence. Either a G, A, T, or C or a gap was scored by absolute difference from the consensus sequence (one, two, or three bases different out of nine sequences) or by a finding of non consensus sequence in the nine representative strains (i.e. the plurality for consensus was six). The baseline indicates absolute consensus in all nine strains; no consensus is shown by the tallest solid bars; three differences from the consensus are indicated by the next-highest bars; and one or two differences are indicated by the shortest bars.

[0030] The 1320-bp regions sequenced for the additional 41 chlamydial strains mentioned above were aligned with the sequences of the 2.8-kb fragment of C. psittaciNJ1 and C. trachomatisR22 (Table I, below). These aligned fragments allowed identification of consensus regions and regions of variability from which to identify target regions for use in this invention. Target Regions A and B are identified in the Table.

[0031] The strategy for identifying useful probes or primers would be in accord with standard guidelines as well-known in the art. In general, the consensus regions would be the source of constructing group-specific probes or primers; whereas the regions of variability would be targeted for species or strain specific probes and primers. Other considerations would include the number or percentage of contiguous nucleotides within the targeted region that are identical to regions found in non-targeted organisms; the length of the selected target region; and the melt temperature of the selected target region. The methods for construction and use of probes and primers are well-established in the art.

[0032] A strategy for constructing an oligonucleotide useful as a probe or primer is initiated by predetermining the length of the oligonucleotide. As previously indicated, probes will typically be at least about 15 bases, and preferably about 20-100 bases, in length. Primers are more typically about 15-30 bases, and preferably about 17-26 bases, in length. The nucleotide sequence complementary to the target rRNA or rDNA is determined, and the oligodeoxyribonucleotide or oligoribonucleotide is synthesized as the inverse of the complementary sequence. In this way, the probe or primer is in the correct orientation for binding to native nucleic acid in the target sample.

[0033] As illustrated in Example 2, below, the taxonomic level of distinction between any two isolates can be predetermined by appropriate selection of the level of specificity of the primers or probes. A summary of the source Region for selecting the primers or probes for a desired level of specificity is given in Table V, below.

[0034] Bacterial Strains

[0035] Forty-three strains of Chlamydiaceae from both animal and human hosts were used (Table II). All organisms were previously characterized using microimmuno-fluorescence, serology, ompi sequence analysis, RFLP, DNA hybridization, and/or in vitro growth.

[0036] DNA Preparation, Amplification, and Cloning

[0037] Genomic DNAs from intact chlamydiae were prepared by incubating the chlamydiae at 37° C. in 50 to 100 μl of 50 mM dithiothreitol (DTT), 30 mM Tris/10 mM EDTA, pH 9.0, for 1 hour, adding an equal volume of 1% Nonidet® P-40 and incubating for 1 hour, adding RNase and incubating at 37° C. for 1 hour, extracting with phenol/chloroform (68), and finally, extracting with chloroform. Chlamydial material obtained as DNA or as lysates was treated with RNase prior to PCR amplification. Approximately 0.25 μg of template DNA was used in each 50 μl PCR reaction; higher concentrations of template DNA were used for specimens of DNA that had not been freshly prepared. The PCR Reagent Kit® (Perkin Elmer, Foster City, Calif.) was used to amplify a single-band PCR product of 2.8 kb from each strain in the Hybaid OmniGene Temperature Cycler® (Middlesex, England) with the following parameters: 30 cycles of 25 sec at 95° C., 15 sec at 55° C., 100 sec at 72° C., and a final 7 min extension at 72° C. The 16S oligonucleotide primer used in this amplification, U16SF, matched three known 16S chlamydial sequences (GenBank accession numbers M13769 [C. psittaci6BC^T], M59178 [C. trachomatisL2/434/BU], and L06108 [C. pneumoniaeTW-183^T]) at approximately position 990 of the 1540 bp gene. The 23S oligonucleotide primer used in this amplification, U23SR, complemented a highly conserved sequence at bacterial-23S-gene position 2000, far enough into the 2900 bp gene to obtain PCR products representing bonafide rRNA operons and to include a Chlamydiaceae-specific sequence, as well as possible intra-23S spacers. Primer sequences were:

[0038] U16SF: GCATGTGGTTTAATTCGATGCAACGCGAAGAACC (SEQ ID No. 44); and

[0039] U23SR: GAATTTCGCTACCTTAGGACCGTTATAGTTAC (SEQ ID No. 45).

[0040] Clones of the 2.8 kb ribosomal PCR products were prepared for DNA sequencing using the TA Cloning Kit® (Invitrogen, San Diego, Calif.).

[0041] DNA Sequence Analysis

[0042] Oligonucleotide primer synthesis and DNA sequencing were performed by the Iowa State University DNA Sequencing and Synthesis Facility, Ames, Iowa. The full-length, cloned 2.8 kb PCR products from strain R22 and from strain NJ1 were subjected to double-stranded DNA sequence analysis. After comparison of these sequences, the homologous 2.8 kb PCR products were prepared from 41 additional strains (Table II). Clones and PCR products were characterized by cutting with the restriction enzyme EcoRI. EcoRI cut all 2.8 kb chlamydial PCR products at a single site, yielding fragments of 1.7 kb and 1.1 kb. A number of the 2.8 kb cloned PCR products obtained during clone screening could not be cut with EcoRI, and DNA sequence analysis of over 500 bases of each, using a Universal primer to the cloning vector, identified them as Mycoplasma orale ribosomal sequence.

[0043] A 1320 bp segment in each of these forty-one 2.8 kb products was subjected to double stranded DNA sequencing. Thirty-three of the sequences were obtained from clones of PCR product in pCRII vector (Invitrogen). Others were obtained from direct sequencing of 2.8 kb PCR products that were extracted with phenol/chloroform and chloroform [Sambrook, J., et al., (1989) Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, N.Y.] and concentrated over Microcon® 100 microconcentrators (Amicon Corp., Beverly, Mass.). R27, CT1, and GPIC were sequenced in their entirety from both PCR products and clones. Because C. trachomatishas been reported to have two ribosomal operons [Engel, J. N., et al. (1987), J. Bacteriol. 169:5678-5685; Fukushi, H., et al. (1993), Int. J. Syst. Bacteriol. 43:613-617], the highly variable segments of C. trachomatisstrains A/Har-13^T, B/TW-5/OT, D/UW-3/CX, F/IC/CAL3, L2/434/BU, and R22 were sequenced from both clones and PCR products to look for ambiguous sequence data. Sequences for ten chlamydial strains (1710S, EBA, FPcello, IPA, N16, M56, L71, Par1, S45, TW-183^T) were obtained from PCR-product template alone. The 1320-bp rDNA segment was found be to highly variable among the 43 Chlamydiaceae strains.

[0044] Several primers were found to be particularly useful for PCR amplification and sequencing, because they matched or complemented all of the chlamydial sequences. The locations of these primers are shown in FIG. 1. Forward (upstream) primers are #16SF2 (CCGCCCGTCACATCATGG, SEQ ID No. 46) and #16SF3 (TCGTAACAAGGTAGCCC, SEQ ID No. 47). Complementary primers are #23R (TACTAAGATGTTTCAGTTC, SEQ ID No. 48), #23R2 (AAAAGGCACGCCGTCAACC, SEQ ID No. 49), #23R3 (GATATCTCCAAGTTTGATT, SEQ ID No. 50), and #23R4 (GAGCTGTTACGCACTCTTT, SEQ ID No. 51). Primer #16SF2 is located 150 bases before the 3-prime end of the 16S gene, primer #23R about 200 bases after the start of the 23S gene, primer #23R2 at the 3-prime end of Domain I, primer #23R3 about 12 bases beyond the end of the 1320 bp sequenced segment, and #23R4 about 1200 bp into the 23S gene. Double-stranded DNA sequencing data were compiled from approximately 70,000 bp of PCR products and/or clones. To gauge the accuracy of sequence analysis, over 10,000 bp were examined using both cloned and direct PCR templates.

[0045] Primer Extension Analysis

[0046] Total RNA was prepared from lysed C. psittaci6GBC^T, C. psittaciNJ1, and C. trachomatisR22 by repeated phenol/chloroform extraction and DNase treatment, for template in primer extension analysis. Primer extension to identify the 5-prime end of the 23S rRNA was carried out using a conserved primer complementing the 23S chlamydial rRNA (#23R: TACTAAGATGTTTCAGTTC, SEQ ID No. 48) and the GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit® (Perkin Elmer). [³²P]dCTP was incorporated into the rTth reverse transcriptase product with the following changes to the manufacturer's recommended conditions: the reverse transcription mix was prepared using unlabeled dCTP at half of the recommended concentration, MnCl₂and rTth enzyme were omitted, and the mix was heated for 5 min in a boiling water bath. The heated mix was cooled rapidly on ice and 40μCi of [³²P]dCTP (ICN, Irvine, Calif.) along with MnCl₂and rTth enzyme were added.

[0047] Extension was carried out for 1 cycle in a GeneAmp PCR System 9600® (Perkin Elmer) for 10 min at 50° C., 15 min at 70° C., and 1 min at 95° C. For each chlamydial strain, one sequence ladder was prepared with [³²P]dCTP label and another with [³⁵S]dATP (ICN) using the CircumVent Thermal Cycle Dideoxy DNA Sequencing Kit® (New England Biolabs, Beverly, Mass.).

[0048] DNA Analysis Programs and Output

[0049] Sequence analysis programs described in the Program Manual for the Wisconsin Package, Version 8, September 1994 (Genetics Computer Group, 575 Science Drive, Madison, Wis. USA 53711) were used for DNA and RNA analysis. These programs were Reformat, SeqEd, BestFit, Gap, Reverse, Assemble, PileUp, Pretty, LineUp, Distances, FastA, RNAfold, and Squiggles. Phylogenetic distances were determined by Robin M. Bush and Walter Fitch using parsimony analysis (PAUP v3.1) (83). Branching order reliability was evaluated by 100 replications of bootstrap resampling (21). Sequence data were examined in four ways: 1) a PileUp multiple sequence alignment was prepared, including appropriate gaps and substitutions (see Table I); 2) using the PileUp alignment, distance analysis tables of Jukes-Cantor pairwise corrected percent differences (42) were generated for the intergenic spacer and for Domain I [Tables 2, 3, and 4 summarize these data]; 3) outgroup sequence data were obtained from GenBank for Pirellula marina[accession number X07408] and by DNA sequencing of Simkania “Z” and “Z1”; and 4) the sequences were analyzed using PAUP, the most parsimonious consensus trees were prepared, and branching order reliability was tested by 100 replications of bootstrap sampling [FIGS. 4 and 5]. Finally, biological, serological, and host range data and the rooted Domain I tree were compiled (Table II).

[0050] GenBank Accession Numbers

[0051] DNA sequence data for the ribosomal segments described in this study appear in the GenBank database with accession numbers U68419 through U68460, U76710, and U76711.

[0052] Characterization of Sequence Diversity Within a 2.8 kb Portion of the Ribosomal Operon

[0053] The 2.8 kb cloned ribosomal PCR products from swine strain R22 and turkey strain NJ1 of Chlamydia contained approximately one-third of the 16S gene, the intergenic spacer, and two-thirds of the 23S gene. Comparison of the two sequences is shown in FIG. 1. The 23S gene start site (TACAGACCAAGT . . . ; SEQ ID No. 52) was located four bases upstream of the homologous Escherichia colisite (data not shown). The 5-prime ends of the two segments each contained 563 bp of DNA homologous to 16S gene sequences, differing from each other by only 3%. A search of the GenBank database with the 16S segments indicated that R22 was most closely related to C. trachomatisand NJ1 was most closely related to C. psittaci. The 16S/23S intergenic spacer sequences of R22 and NJ1 were 243 bp and 224 bp in length, respectively, and differed by 22.0%. The 1960-base, 23S gene segments differed by 9.6%. Much of the variability in the 23S segment was in the 5-prime end homologous to Domain I of the E. coli23S gene. The Domain I sequences of R22 and NJ1 (622 bp and 621 bp, respectively) differed by 17% while the remaining portion of the 23S segments (1338 bp) varied by 6.4%. No intragenic spacers were identified within the chlamydial 23S genes.

[0054] Analysis of the Ribosomal Intergenic Spacer

[0055] The 16S/23S intergenic spacer sequences from chlamydiae identified in Table II are indicated in the multiple sequence alignment in Table I. The intergenic spacers spanned 232±11 bases depending on the strain. Nine single-base differences were unique to only one or two strains, and approximately 80 sequence position differences were group-clustered, conserved by species, subspecies, or several species. Among these 80 positions were three highly variable segments of approximately 20 bp each between base 12 and base 115 that were distinctly group-cluster specific. Foldrna/Squiggles analysis of the intergenic spacer indicated that each of the α segments could form a hairpin structure with a 10 bp stem centered between bases 23 and 24 (analyses not shown).

[0056] A comparison of the intergenic spacers showed that there were 143 base positions (56.1%) that were absolute identities among all chlamydial strains. This compared to 74.2% absolute identity for Domain I and 91% for 16S.

[0057] Development of Useful Primers

[0058] Primers having various levels of specificity were identified as described below. The conditions for PCR amplifications using these primers are uniform, except for annealing temperatures which are given below in each instance. Typically, the phases of each cycle are 30 sec at 94° C., 15 sec annealing, and 30 sec at 72° C. Cycle numbers may be relatively low (30) or higher, depending on the amount of template. Equimolar quantities of primer are recommended in each reaction. For all of these assays, a heat-activated DNA polymerase such as AmpliTaq Gold® is recommended for the purpose of obtaining optimal specificity.

[0059] U23F: 5′ GATGCCTTGGCATTGATAGGCGATGAAGGA 3′ (SEQ ID No. 53)

[0060] 23SIGR: 5′ TGGCTCATCATGCAAAAGGCA 3′ (SEQ ID No. 54)

[0061] (annealing temperature 61° C.)

[0062] These primers PCR amplify the 23S signature sequence of all Chlamydiaceae, of Simkaniaceae, and of many other bacteria. Signature sequence data is used for rapid identification of each of the nine species in Chlamydiaceae. These primers can be used for direct DNA sequencing of the PCR product that they produce. The broad specificity of these primers ensures that if a chlamydia-like bacterium is isolated, sequence data can be obtained that will allow rapid identification of the organism.

[0063] IGF: 5′ GACTAGGTTGGGCAAG 3′ (SEQ ID No. 55)

[0064] IGR: 5′ AGCTCTTA(t/g/a) (c/t)AACTTGGTCTGTA 3′ (SEQ ID No. 56)

[0065] (annealing temperature 57° C.)

[0066] These primers will amplify in PCR the intergenic spacer of all Chlamydiaceae spp. and are specific only for Chlamiydiaceae. That is, they should not amplify an appropriately-sized product from other bacterial templates. IGF is located within the 16S/23S intergenic spacer and is the forward primer. IGR complements the start of the 23S rRNA gene and is the reverse primer. PCR amplification of Chlamydiaceae template with these primers generates an approximately 230-base PCR product that can be directly sequenced for identification or that may serve as a probe in hybridization experiments. Using this PCR product, specific restriction analysis can also be conducted for species or strain identification.

[0067] SIGF: 5′-ATAATAATAGACGTTTAAGA-3′ (SEQ ID No. 57);

[0068] (annealing temperature 52° C.)

[0069] This oligonucleotide, located a little more than 100 bases downstream of the IGF primer, also recognizes all Chlamydiaceae spp. SIGF will anneal to denatured template DNA or to PCR products. It can be used for sequence analysis or as a hybridization probe. Because this oligonucleotide is A/T rich and may amplify nonspecifically from A/T-rich sites, its potential as a primer is diminished. To use SIGF to confirm the identity of template DNAs or PCR products like IGF/IGR, target DNA is bound to a substrate, denatured, and probed with labeled SIGF. Detectable SIGF hybridization indicates that the target is derived from Chlamydiaceae.

[0070] Detection of all Members of Chlamydiaceae Using High Throughput, Fluorescent Probe Detection with the Perkin Elmer TaqMan® System

[0071] The TaqMan® system will detect either DNA or RNA targets that match or complement the Chlamydiaceae-specific primers and probe identified below as SEQ ID Nos. 71-73, occurring in Region B. When matching target nucleic acids are present, PCR or RT-PCR using oligonucleotides produces a fluorescent reporter that is detected using an argon lamp or laser in the TaqMan® System. Readings can be taken at the end of each amplification cycle or at the endpoint. This system provides superb quantitative and qualitative target identification. For endpoint detection after 80-min of cycling, a microtiter plate with 90 samples and 6 negative controls can be analyzed in less than 10 minutes.

[0072] With the recent introduction of genetic analysis techniques for the identification of organisms, bacterial nomenclature and speciation has undergone a revolution. Based on the information provided in this study and other genetic data, it has been possible to apply the new genetic principles to the designation of species and genera in Chlamydiaceae. Although not yet approved by the International Journal of Systematic Bacteriology, Table III outlines a proposal for chlamydial speciation that is currently being considered. The proposed nomenclature for these genera and species is used throughout Test 4.

[0073] Genus and Species Specific Primers for PCR Amplification

[0074] The following primers can be used in PCR amplifications followed by gel detection to identify the 2 proposed genera in Chlamydiaceae and also to identify the 9 species. Each set of Chlamydiaceae-specific primers consists of a forward (F) and a reverse (R) primer. There is one R primer for the genus Chlamydia and a different R primer for the genus Chlamydophila. If simply knowing the genus of the unknown template is desired, amplifications are done using primer IGF (above) and either the Chlamydia R primer and or the Chlamydophila R primer:

[0075] Chlamydia R 5′ GCCGGAGCTTTTCGCA 3′ (SEQ ID No. 58); (annealing temperature 71° C.).

[0076] Chlamydophila R 5′ GCAGGTAAACGCATCCTTCA 3′ (SEQ ID No. 59); (annealing temperature 72° C.). A positive identification of the genus is the production of an approximately 300-bp PCR product.

[0077] To identify a species that belongs to one of these genera, the original template or a small amount of PCR product from the genus-specific amplification can be used in the following PCR assays. If a specimen is a mixed sample of both genera, use of the genus-specific amplification product will serve to significantly purify the desired target and is recommended for use with the following tests. The PCR products generated with these primers are nearly 300 bases in length, including sequence from the 16S/23S intergenic spacer and an approximately 80-base segment of the 23S rRNA gene (the abortus/psittaciproducts, however, are approximately 100 bases long). Predicted annealing temperatures are indicated. When two primers are used together, the lower of the 2 annealing temperatures should be used.

[0078] For Chlamydia-specific PCR amplification, the Chlamydia R primer and the following forward (F) primers are used:

[0079] For identification of a Chlamydia sample, a C. suisF primer amplification should be used as a control for every test. If the C. suisyields more PCR product than the other F primers, the sample is C. suis. If the other primers yield more product, the sample is C. muridarumor C. trachomatis, respectively.

[0080] For Chlamnydophila-specific PCR amplification, the Chlamiydophila R primer and the following forward (F) primers are used:

[0081] It is noted in the abortus/psittaciF primer, above, that the fourth occurrence of cytosine (underlined) is a deliberate mismatch to the actual abortus/psittacisequence. This one-base mismatch is intended to destabilize the primer from annealing to caviae/pecorum/felis/pneumoniaetemplates, but is not sufficient to destabilize annealing to the abortus/psittacitemplate.

[0082] To identify Chlamydophila species, the R primer is used in four separate PCR amplifications: pecorum F, pneumoniae F, felis/caviae F, and abortus/psittaci F. There is no template crossreaction between these four tests. If the felis/caviaeor abortus/psittaciproducts are produced, then secondary (2°) PCR amplifications are run using a small amount of the initial PCR product, to determine which of the two species is present.