AU716766B2 - Nucleic acid assay for the detection and differentiation of three chlamydia species - Google Patents

Description

WO 98/10101 PCT/US97/15556 1 NUCLEIC ACID ASSAY FOR THE DETECTION AND DIFFERENTIATION OF THREE CHLAMYDIA SPECIES FIELD OF THE INVENTION This invention relates to diagnostic assays for the detection and differentiation of different species of Chlamvdia, and compositions and kits for performing the assays.

BACKGROUND OF THE INVENTION Chlamydia are widespread intracellular bacteria known to cause a variety of infections in humans, marsupials, other mammals and birds. Chlamydia pneumoniae and Chlamydia psittaci are an important cause of lower respiratory tract infections. At least 10% of cases of pneumonia in young adults are associated with C. pneumoniae. C. pneumoniae has been responsible for both endemic and epidemic pneumonia.

Though treatment of the two diseases is typically similar, C.

psittaci is generally considered to be a more life-threatening disease justifying more aggressive treatment. Thus, it would be useful to distinguish these two species for treatment purposes.

Birds kept as pets have been a significant source of C. psittaci human infection. Outbreaks of human disease can occur whenever there is close and continued contact between humans and infected birds that excrete the organism in feces and respiratory secretions. The agent is present in tissues and is often excreted in feces by healthy birds. The inhalation of infected dried bird feces is a common method of human infection.

Laboratory confirmation of psittacosis in humans and birds is challenging, and treatment is often empiric and predicated on the clinician eliciting a history of bird exoosure. Measurement of complement-fixing antibody titers and, much less commonly, recovery of C. psittaci from patients WO 98/10101 PCT/US97/15556 2 have been the traditional methods of laboratory confirmation of psittacosis. Neither method is sensitive, and complement fixation is very nonspecific. Microimmunofluorescence (MIF) is a technique which offers greater sensitivity and specificity than complement fixation (CF) but is not as widely used (Wong et al. 1994, Journal of Clinical Microbiology 1625-30.). MIF interpretation is problematic and requires highly trained laboratory personnel.

Chlamydia trachomatis is the most common sexually transmitted disease in the U.S. C. trachomatis causes trachoma and urogenital infections. Diagnosis can be difficult, since many women in particular have no overt symptoms of early infection. C. trachomatis infection also occurs in newborns and its diagnosis and detection is problematic. Culture tests are considered the diagnostic gold standard for detection of Chlamydia. However, as an obligate intracellular parasite, culture of this organism is technically difficult and the organism is famous for its substantial laboratory to laboratory variation.

Amplification methods such as the polymerase chain reaction are generally sensitive and specific techniques for detection of target DNA sequences. Attempts have been made to use PCR to distinguish the three subject chlamydial species. Kaltenboeck et al. (1992), Journal of Clinical Microbiology 30, 1098-1104 reports the use of a twostep PCR process that targeted major outer membrane protein (MOMP) gene (OmpA) DNA sequence of Chlamydia species and then used a restriction endonuclease analysis to discriminate the species. Tjhie et al. (1993), Journal of Microbiological Methods 18, 137-50, differentiated the species by first amplifying a region of the OmpA specific to the genus and then hybridized the amplified product with species specific probes to discriminate among the species.

The 16s rRNA (ribosomal RNA) gene has been used as a target for the detection of Chlamydia sp, but it has been believed that it cannot-be used to differentiate between the species. See, Tjhie et al., Journal of Microbiological Methods, 18:137-150 (1993). Focus for PCR detection methods WO 98/10101 PCT/US97/15556 3 in Chlamydia has been on the OmpA gene and either involves analysis by restriction fragments ("RFLP") or two step processes. Id. RFLP can be a complicated and time consuming procedure, particularly for personnel without significant experience in the area. Thus, there is a need for a quick, efficient and highly sensitive technique to detect the Chlamydia sp. in one assay.

SUMMARY OF THE INVENTION 1 0 This invention provides a novel assay for easily and readily detecting three important Chlamydia sp.,

C.

trachomatis, C. psittaci, and C. pneumoniae. These three species may be detected and differentiated in the same sample aliquot at the same time through the use of amplification primers targeted to the 16s rRNA gene specific for each of the species. Prior to the assay here, it was not appreciated that this gene could be used as a Chlamydia species-specific target. The assay described here is surprisingly highly sensitive and specific and is consistently so. Typically, when multiple targets are amplified in the same sample, sensitivity is sacrificed and the results are usually uneven.

With the present assay, this is not the case.

In particular, the invention includes a method for detecting for the presence or absence of Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia trachomatis in a single test aliquot from a nucleic-acid containing sample comprising: contacting the test aliquot with a sense and antisense nucleic acid primer pair such that each pair flanks a region of the 16s rRNA gene specific to one of the three Chlamydia species in an amplification protocol to produce amplification products; and detecting for the presence or absence of amplification products specific to each of the three Chlamydia species. Preferably, the test aliquot is first contacted with a pair of sense and antisense primers that flank a region of the 16s rRNA gene common to all three Chlamydia species.

Compositions comprising primer pairs for the methods and kits to practice the method are also described and claimed.

WO 98/10101 PCT/US97/15556 4 DETAILED DESCRIPTION This invention provides a novel assay for easily and readily detecting three important Chlamydia sp., C.

trachomatis, C. psittaci, and C. pneumoniae.. These three species may be detected and differentiated in the same sample aliquot at the same time through the use of amplification primers targeted to the 16s rRNA gene specific for each of the species.

The assays of the present invention will allow the laboratory to test a variety of specimens from different clinical samples and species with one test. For example, the assay may be able to equally identify C. trachomatis from endocervical swab samples, C. psittaci from cloacal swab samples from birds and C. pneumoniae from sputum samples.

This assay is a considerable improvement over the use of traditional stock assay reagents to first test a particular sample for the Chlamydia genus, followed by a second test for each of the three relevant Chlamydia species.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons (New York); Walker (ed) (1988) The Cambridge Dictionary of Science and Technology, The press syndicate of the University of Cambridge (New York); and Hale and Marham (1991) The Harper Collins Dictionary of Biology Harper Perennial (New York) all provide one of skill with a general dictionary of many of the terms used in this invention.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, certain preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

The terms "isolated" or "biologically pure" refer to material which is substantially or essentially free from WO 98/10101 PCT/US97/15556 components which normally accompany it as found in its native state.

The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence optionally includes the complementary sequence thereof.

Two single-stranded nucleic acids "hybridize" when they form a double-stranded duplex. The region of doublestrandedness can include the full-length of one or both of the single-stranded nucleic acids, or all of one single stranded nucleic acid and a subsequence of the other single stranded nucleic acid, or the region of double-strandedness can include a subsequence of each nucleic acid. An overview to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-- Hybridization with Nucleic Acid Probes Part I Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier (New York).

"Stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters.

An extensive guide to the hybridization of nucleic acids is found in Tijssen, supra. Generally, highly stringent wash conditions are selected to be about 5 0 C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm point for a particular probe. Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, when a copy WO 98/10101 PCT/US97/15556 6 cf a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

The term "identical" in the context of two nucleic acid sequences refers to the residues in the.two sequences which are the same when aligned for maximum correspondence.

A

nucleic acid is "substantially identical to a reference nucleic acid when it is at least about 70% identical, preferably at least about 80% identical, and optionally about identical or more.

The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest, or produced synthetically, and is capable of hybridizing to a strand of the target sequence. When the terminal 3' nucleotide has hybridized it acts as a point of initiation of synthesis under conditions in which synthesis of an extension of the primer is induced. These conditions typically include the presence of four different nucleotide triphosphates (a nucleotide reagent) and thermostable enzyme in an appropriate buffer and at a suitable temperature. When primer pairs are referred to herein, the pair is meant to include one primer which is capable of hybridizing to the sense strand of a double-stranded target nucleic acid (the "sense primer") and one primer which is capable of hybridizing to the antisense strand of a double-stranded target nucleic acid (the "antisense primer"). The primer pair will be designed such that they flank the region of the target nucleic acid to be amplified and will cause the target region to be amplified when placed in an amplification protocol such as polymerase chain reaction.

What is meant by a primer "substantially homologous" or "substantially complementary" to a nucleotide sequence is a polynucleotide or oligonucleotide containing naturally occurring nucleotides or their analogs, such as 7deazaguanosine or inosine, sufficiently complementary to hybridize with the target sequence such that stable and specific binding occurs between the primer and the target sequence. The degree of homology required for formation of a stable hybridization complex (duplex) varies with the WO 98/10101 PCTIUS97/155C56 7 str-ngency of the amplification medium. The primer should be substantially homologous to the target strands of each specific sequence to be amplified. This means that the primer must be sufficiently complementary to hybridize with the appropriate strand under standard amplification conditions.

Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a noncomplementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence complementary to the strand. Alternatively, noncomplementary bases or longer sequences can be interspersed into the primer provided that the primer sequence has sufficient complementarity with the sequence of the target sequence to hybridize with it and thereby form a template for synthesis of the extension product.

THE ASSAY The invention relates to assays or methods for detecting for the presence or absence of Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia trachomatis in a single test aliquot from a nucleic-acid containing sample comprising: contacting the test aliquot with a sense and antisense nucleic acid primer pair such that each pair flanks a region of the 16s rRNA gene specific to one of the three Chiamydia species in an amplification protocol to produce amplification products; and detecting for the presence or absence of amplification products specific to each of the three Chlamydia species. The best results are seen when the test aliquot is contacted first with a pair of sense and antisense primers that flank a region of the 16s rRNA gene common to all three Chlamydia species, such as that region flanked by the primers set out in SEQ ID NOS:1 and 2.

Thus, the assays of the present invention advantageously can be accomplished with a single aliquot from a biological sample of interest suspected of containing one of the three Chlamydia sp. or in which one wishes to establish that such species are absent. The samples may be obtained WO98/10101 PCT/US97/15556 8 from any source in which the bacteria may be present. Any source of nucleic acid, in purified or nonpurified form including crude extracts of tissue or cells, can be utilized as the starting nucleic acid or acids. Samples which are typically of interest include those from humans, marsupials, other mammals or birds. Samples from tissues and bodily fluids typically tested could include, but are not limited to, sputum; throat swabs; nasal pharyngeal swabs; human lung, spleen and liver samples; bronchial alveolar lavage; feces; blood; and cloacal tissue. The samples need not be purified or pretreated prior to the assay, but in the case of such tissues which have significant extraneous protein and other debris such as feces, it is preferred to subject the sample to low speed centrifugation or the like to separate out the debris before assaying.

Once the sample is obtained, it is subjected to an amplification protocol. Optionally, the first step includes an amplification step with amplification primers designed to target the Chlamydia genus generally by targeting the 16s rRNA gene. The 16s rRNA gene is described as a general target in Gaydos et al., J. Clin. Microbiol. 30: 796-800 (1992).

Sequences from the 16s rRNA are also found in GenBank (National Center for Biotechnology Information, Natl. Library of Medicine, National Institutes of Health, 8600 Rockville Pike, Bethesda, Maryland 20894) at Accession Nos. L06108 for C. pneumoniae, Accession No. M13769 for C. psittaci, and at Accession No. M59178 for C. trachomatis. These sequences can be aligned as is known in the art to obtain amplification primers which will amplify a generic region. The generic region, however, must encompass those regions which are to be specifically targeted to differentiate the three species as will be discussed below. It is most desirable though that the primers used for this step target that region of the 16s rRNA gene flanked by the primers set out in SEQ ID NOS:1 and 2 or regions which overlap those sequences to which such primers hybridize. It is most preferred that the primers described in SEQ ID NOS:1 and 2 be used: sense ACG GAA TAA TGA CTT CGG (SEQ ID NO:1) WO 98/10101 PCTIUS9715556 9 antisense TAC CTG GTA CGC TCA ATT (SEQ ID NO:2) When the above primer pair is used, the resulting amplified product is about 436 bp (base pairs) in length.

If the above step is employed, then the solution with the amplified product is directly contacted with the species specific primers. If the above step is not employed, then the test sample aliquot itself is directly contacted with the species specific primers. A "single test aliquot" is an aliquot derived from the test sample in which products from all three species are amplified together. The species specific primers are sense and antisense primer pairs such that each pair of primers flanks and targets a region of the 16s rRNA gene unique to one of the Chlamydia sp. This invention describes primers which have been found to be particularly useful and effective and they are most preferred for the assays claimed herein. The primer pairs are as follows: For C. trachomatis: -sense GCA ATT GTT TCG GCA ATT G (SEQ ID NO:3) antisense AGC GGG TAT TAA CCG CCT (SEQ ID NO:4) For C. pneumoniae and C. psittaci: sense ATA ATG ACT TCG GTT ATT (SEQ ID For C. psittaci: antisense TGT TTT AGA TGC CTA AAC AT (SEQ ID NO:6) For C. pneumoniae: antisense CGT CAT CGC CTT GGT GGG CTT (SEQ ID NO:7) When these primers are used, the amplification products are about 412 bp for C. trachomatis, 221 bp for C. pneumoniae, and 127 bp for C. psittaci. These primers can also be used as species specific probes. The designated primer pairs can also be used alone to detect for one species singly, if so desired.

It is preferred to use primers which hybridize to a region of the nucleic acid which overlaps with the respective regions of the target nucleic acid to which the primers listed WO 98/10101 PCT/US97/15556 above are complementary. Primers substantially identical to those listed above are also preferred. It is most preferred that the primer used be substantially complementary to the same regions.

The primers and the sample are incubated together in an amplification protocol to obtain, if present, an amplified nucleic acid product which is indicative of each species being detected. Nucleic acid amplification techniques suitable for amplifying sequences with nucleic acid primers are known.

Examples of techniques sufficient to direct persons of skill through such amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Q0replicase amplification and other RNA polymerase mediated techniques NASBA) are found, for example, in Berger, Sambrook et al. (1989) Molecular Cloning A Laboratory Manual (2nd Ed) Vol. 1-3; and Ausubel, as well as Mullis et al., (1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564. One of skill will also appreciate that essentially any RNA can be converted into a double stranded DNA suitable for PCR expansion. See, Ausubel, Sambrook and Berger, all supra. The methods and primers described herein are preferably used in a PCR amplification protocol. The amplification buffer will preferably have a pH of about 8.3 to about 9.2 and a MgCl 2 concentration of about mM to about 3.5 mM.

The nucleic acid sequence to be amplified and identified will be the amplification product or "target" sequence that exists between the primer pairs flanking it and whose presence is indicative of a species of interest.

WO 98/10101 PCTIS97/15556 11 Oligonucleotides for use as primers (or probes) are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., S using an automated synthesizer, as described in Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill. Purification of oligonucleotides, where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom. 255:137-149. The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology 65:499-560.

One of skill will also recognize many ways of generating alterations in a given nucleic acid sequence. Such well-known methods include site-directed mutagenesis,

PCR

amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques. See, Giliman and Smith (1979) Gene 8:81-97; Roberts et al. (1987) Nature 328:731-734 and Sambrook et al. (1989) Molecular Cloning A Laboratory Manual (2nd Ed) Vol. 1-3; Innis, Ausbel, Berger, Needham VanDevanter and Mullis (all supra).

The primers of use in the assay methods described here are preferably single stranded for maximum efficiency and amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.

Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of an enzyme. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method. Most WO 98/10101 PCT/US97/15556 12 typically, amplification primers are between 8 and 100 nucleotides in length, and preferably between about 10 and nucleotides in length. More typically, the primers are between about 18 and 28 nucleic acids in length. A primer pair will include one primer which hybridizes to the sense strand (the strand of nucleic acid which is translated) of the DNA being targeted and one primer which hybridizes to the antisense strand (the strand of nontranslated nucleic acid) of the DNA being targeted. The primer pair will flank the region to be amplified. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with template.

Amplification-based assays are well known to those of skill in the art (see, Innis, supra.). The nucleic acid sequences and other guidelines provided here are sufficient to teach one of skill to routinely select primers to amplify a portion of the 16s rRNA gene for the Chlamydia sp. of interest. It is expected that one of skill is thoroughly familiar with the theory and practice of nucleic acid hybridization and primer selection. Gait, ed.

Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford (1984); W.H.A. Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K.L. Dueholm J. Org. Chem. 59, 5767-5773 (1994); S. Agrawal Methods in Molecular Biology, volume 20; and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York provide a basic guide to nucleic acid hybridization. Innis, supra, provides an overview of primer selection. In addition, PCR amplification products are optionally detected on a polymer array as described in Fodor et al. (1991) Science, 251: 767- 777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) Nature Medicine 753-759.

One of skill will recognize that the 3' end of an amplification primer is more important for PCR than the end. Investigators have reported PCR products where only a WO 98/10101 PCT/US97/15556 13 few nucieotides at the 3' end-of an amplification primer were complementary to a DNA to be amplified. In this regard, nucleotides at the 5' end of a primer can incorporate structural features unrelated to the target nucleic acid; for instance, for detection purposes. The primers are selected so that there is no complementarity between any known sequence which is likely to occur in the sample to be amplified and any constant primer region. One of skill will appreciate that constant regions in primer sequences are opt-onal. The primer may be constructed so that it contains a label at its 5' end, examples of which are discussed below, or other nucleotides.

Preferably such 5' nucleotide sequences are not complementary to the known target sequence. Such 5' additions to the primer may include, but are not restricted to: chemically-modified or biotinylated sequences, restriction endonuclease cloning sites, promoter sequences, regulatory sequences, enzyme binding sites, other genes, or any nucleotide sequence that serves a specific desired function.

Typically, all primer sequences are selected to hybridize only to a perfectly complementary DNA, with the nearest mismatch hybridization possibility from known DNA sequences which are likely to occur in the sample to be amplified having at least about 50 to 70% hybridization mismatches, and preferably 100% mismatches for the terminal nucleotides at the 3' end of the primer.

The primers are selected so that no secondary structure forms within the primer. Self-complementary primers have poor hybridization properties, because the complementary portions of the primers self hybridize form hairpin structures). The primers are also selected so that the primers do not hybridize to each other, thereby preventing duplex formation of the primers in solution, and possible concatenation of the primers during PCR. If there is more than one constant region in the primer, the constant regions of the primer are selected so that they do not self-hybridize or form hairpin structures.

Where sets of amplification primers the and 3' primers used for exponential amplification) are of a WO 98/10101 PCTIU~S97/15556 14 single length, the primers are selected so that they have roughly the same, and preferably exactly the same overall base composition the same A+T to G+C ratio of nucleic acids). Where the primers are of differing lengths, the A+T to G+C ratio is determined by selecting a thermal melting temperature for the primer-DNA hybridization, and selecting an A+T to G+C ratio and probe length for each primer which has approximately the selected thermal melting temperature.

The amplified product can be detected by means well known in the art. The product may be isolated and identified by size and through the use of a ligand. The term "ligand" or "ligand binding end" refers to a component which may directly or indirectly be detected or captured by another component, the "anti-ligand" which permits the physical or chemical separation of compositions bearing the ligand from those which do not. The ligand will be attracted to an anti-ligand molecule such that molecules which do not bear the ligand will not be captured or otherwise attracted to the anti-ligand.

The ligand will need to be one which may be attached directly or indirectly to nucleic acid sequences. Examples of direct ligand binding include the use of biotin labeled nucleotides or the use of digoxigenin. These molecules can be used as the ligand binding component. They can be readily captured by their anti-ligand, e.g. avidin or streptavidin in the case of biotin and an anti-digoxigenin antibody, bound on a suitable substrate. These reagents are all readily available, see Clontech Laboratories, Inc., Palo Alto, CA for digoxigenin reagents, for example.

The ligand could alternatively be a specific nucleic acid sequence with the anti-ligand being the complement of the sequence or an antibody specific for the sequence. The ligand could include labeled molecules which may be manipulated on a substrate so that they are physically or chemically separated from non-ligand bearing molecules. Alternatively, the ligand molecule can have affinity for an anti-ligand molecule which is labeled or inherently detectable. These compositions can be further detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, WO 98/10101 PCT/US97/15556 useful nucleic acid labels may include enzymes LacZ, CAT, horse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker gene products or in an ELISA), nucleic acid intercalators S ethidium bromide) and colorimetric labels such as colloidal gold or colored glass or plastic polystyrene, polypropylene, latex, etc.) beads, substrates, cofactors, inhibitors, fluorescent moieties fluorescein and its derivatives, Texas red, rhodamine and its derivatives, dansyl, umbelliferone and the like), chemiluminescent moieties (e.g.

luciferin and 2, 3 -dihydrophthalazinediones), magnetic particles, and the like. Labeling agents optionally include monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids, fluorescent dyes, electron-dense reagents, enzymes as commonly used in an ELISA), or haptens and proteins for which antisera or monoclonal antibodies are available.

A

wide variety of labels suitable for labeling nucleic acids and conjugation techniques are known and are reported extensively in both the scientific and patent literature, and are generally applicable to the present invention for the labeling of nucleic acids, or amplified nucleic acids for detection and isolation by the methods of the invention. The choice of label depending on the sensitivity required, ease of conjugation of the compound, stability requirements, available instrumentation, and disposal provisions. Separation and detection of nucleic acids proceeds by any known method, including immunoblotting, tracking of radioactive or bioluminescent markers, Southern blotting, northern blotting, southwestern blotting, northwestern blotting, or other methods which track a molecule based upon size, charge or affinity.

Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, by microscopy, WO 98/10101 PCTIUS97/15556 16 visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.

Similarly, enzymatic labels may be. detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels are often detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

Substrates to be used as an environment for the capture and separation of the ligand bound molecules from those without ligand depend on the ligand being used and the desired format. For instance, the solid surface is optionally paper, or a membrane nitrocellulose), a microtiter dish PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick glass, PVC, polypropylene, polystyrene, latex, and the like), a microcentrifuge tube, or a glass, silica, plastic, metallic or polymer bead or other substrate as described herein. The desired anti-ligand may be covalently bound, or noncovalently attached to the substrate through nonspecific bonding.

A wide variety of organic and inorganic polymers, both natural and synthetic may be employed as the material for the solid surface. Illustrative polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like. Other materials which are appropriate depending on the assay include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cements and the like. In addition, substances that form gels, such as proteins gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides can be used. Polymers which form several aqueous phases, such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long-chain (12-24 carbon atoms) alkyl ammonium salts and the WO 98/10101 PCT/US97/15556 17 like are also suitable. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.

In preparing the surface, a plurality of different materials are optionally employed, as laminates, to obtain various properties. For example, protein coatings, such as gelatin can be used to avoid non specific binding, simplify covalent conjugation, enhance signal detection or the like. If covalent bonding between a compound and the surface is desired, the surface will usually be polyfunctional or be capable of being polyfunctionalized. Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. In addition to covalent bonding, various methods for noncovalently binding an anti-ligand component can be used.

For additional information regarding suitable ligand-antiligand and labeling technology as it relates to nucleic acids, see, for example, Essential Molecular Biology, ed. T.A. Brown IRL Press (1993); In Situ Hybridization Protocols, ed. K.H.

Andy Choo, Humana Press (1994) Kits Further contemplated are kits for the assays described here. Combinations of reagents useful in the methods set out above, particularly the primers, can be packaged together with instructions for using them in the described assays. A preferred kit would contain three pairs of primers, each pair comprising a sense and antisense nucleic acid primer which flank regions of the 16s rRNA gene specific to each of the three Chlamydia C. pneumoniae,

C.

psittaci and C. trachomatis and instructions for performing the assay with a single test aliquot. Primers which are specific for the genus, Chlamydia, are also preferably included, as is described above. Also, as indicated by the description provided herein, a single primer may serve as a member of more than one pair of primers. Amplification buffers and the like could further be included in the kits.

WO 98/10101 PCTliUS97/15556 18 All of the references cited herein are cited for general background purposes and are hereby incorporated by reference. The following examples are merely illustrative of the invention and are not to be construed as-a limitation of S the invention.

EXAMPLES

An exemplary assay method described below is a nested, multiplex polymerase chain reaction (PCR) for detection of chlamydiae in human and avian specimens. The assay resulted in increased sensitivity to circumvent inhibitors of PCR present in clinical specimens. The target sequence is the 16s rRNA gene. The first-step PCR is genus specific, and the second-step PCR is multiplexed has multiple primer sets in the same tube) and can discriminate between C. pneumoniae, C. psittaci, and C. trachomatis. The sensitivity of each of the two PCR steps is 5 infectivity units. We used PCR and serologic evidence during an outbreak of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. PCR results were compared with culture methods. The application of PCR to avian specimens significantly increased the detection rate of C.

psittaci compared with culture methods.

Growth and purification of organisms Chlamydiae were grown as previously described (Wong et al.

1992, Journal of Clinical Microbiology 30, 1625-30.).

Briefly, chlamydiae were propagated by centrifugation of thawed stock cultures onto exponentially growing Hep-2 cell monolayers. Chlamydial elementary bodies were harvested 72 hours after inoculation by disrupting the host monolayer with glass beads in fresh Isocove's Modified Dulbecco's Medium with 10% fetal calf serum. The disrupted cell suspension was sonicated, and partially purified by centrifugation at 500 x g for 10 minutes, followed by centrifugation on renografin.

WO 98/10101 PCT/US97/15556 19 Serology Complement fixation and microimmunofluorescence methods were previously described (Wong et al. 1994, Journal of Clinical Microbiology 32, 2417-21.).

Preparation of clinical specimens Clinical specimens included unclotted blood, throat swabs, feces, tissue, and cloacal swabs. These specimens were prepared for PCR using the QiaAmp Blood and QiaAmp Tissue kits (Qiagen Inc, 9600 DeSoto Ave, Chatsworth, CA 91311, USA). All specimens except blood and tissue underwent a differential centrifugation of 500 x g for 5 minutes to pellet debris prior to DNA extraction using the Qiagen kits.

PCR amplification Samples for PCR were prepared in a class II laminar flow hood, and amplification and analysis of PCR products were each performed in separate locations. Reaction volumes of 50 l containing 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 2.5 mM MgC1 2 200 AM of each deoxynucleoside triphosphate, 0.01% BSA (Sigma Chemical Co, St. Louis, MO), 1.25 units of Taq polymerase (Boehringer Mannheim), 0.2 AM of each outer primer, and 5 zl of sample were overlaid with one drop of mineral oil and placed in a Perkin-Elmer Thermalcycler Model 480 (Perkin-Elmer Cetus Corp., Norwalk, Conn.), for 1 cycle of 95 0 C for 2 minutes, followed by 35 cycles of 94 0 C for one minute, 55 0

C

for 30 seconds, and 72 0 C for one minute. The nested or inner PCR reaction mixture was similar to the first except that it contained 1 Al of the product of the outer PCR and 0.2 AM of each inner primer. The cycling conditions were identical.

Genus specific first-step primers 16s rRNA: sense ACG GAA TAA TGA CTT CGG (SEQ ID NO:1) antisense TAC CTG GTA CGC TCA ATT (SEQ ID NO:2) Genus product: 436 bp Species specific second step-primers 16s rRNA: C. tr sense GCA ATT GTT TCG GCA ATT G (SEQ ID NO:3) C. tr antisense AGC GGG TAT TAA CCG CCT (SEQ ID NO:4) C. pn\psi sense ATA ATG ACT TCG GTT GTT ATT (SEQ ID WO 98/10101 PCT/US97/15556 C. psi antisense TGT TTT AGA TGC CTA AAC AT (SEQ ID NO:6) C. pn antisense CGT CAT CGC CTT GGT GGG CTT (SEQ ID NO:7) Both the outer and inner PCRs were optimized with the Opti-Prime PCR optimization Kit (Stratagene, La Jolla, CA). Amplification products were separated by electrophoresis through 2.5% agarose gels Nusieve GTG agarose (FMC Bioproducts, Rockland, ME) and 1.0% agarose (BioRad Laboratories, Richmond, CA)] in Tris-borate-EDTA and were visualized by ethidium bromide fluorescence.

All of the PCR primers were both sensitive and specific. Neither the outer nor inner primer sets crossreacted with other respiratory pathogens. Table 1 lists the microorganisms tested for specificity.

The sensitivity of each of the primer sets, both the genus and species, is less than 5 infectivity units. The multiplex PCR, containing all 5 primers for detection of each Chlamydia organism, was run as a second-step PCR using 1 1l of the first-step PCR product. Five infectivity units of each Chlamydia species were used in the genus, first-step PCR.

Due to illness among owners of sick birds purchased from pet stores, we were asked to test three separate groups of sick birds and a few human throat swabs and blood specimens for C. psittaci. We used our two-step PCR to test all specimens sent to us. Table 2 is a summary of the three groups of bird specimens tested for culture and PCR. All of the culture positive specimens were also PCR positive.

Of the human sera from 4 individuals tested in the first Georgia group, one had an MIF titer to C. psittaci of 1:512. All of the throat swabs from these individuals were PCR negative. There were no human specimens sent for testing from the W. Virginia group; all isolates came from birds. The MIF titers to C. psittaci of ill people exposed to PCR positive birds in the W. Virginia group ranged from 1:16 to 1:512.

WO 98/10101 PCTIUS97/15556 21 It is our experience that sometimes not enough product is made in the first-step PCR to be visualized on the agarose gel, due to inhibitors still present in the sample even after preparation\purification of the clinical specimen for PCR. We find that positive specimens can most quickly and efficiently be identified with a second-step PCR using a tiny fraction of the first-step PCR product.

The focus of this investigation was to determine if there had been transmission of Chlamydia psittaci from shipments of birds known to be infected to owners of these birds. Sensitive and specific tests to confirm psittacosis are lacking. We developed a new nested, multiplex PCR that simultaneously distinguishes and detects C. pneumoniae,

C.

psittaci, and C. trachomatis. This PCR was applied to all of the specimens available to us in the study.

In the first Georgia group of specimens, a few of the exposed humans had unclotted blood and throat swabs collected for culture and PCR. Fresh bird droppings were also collected from the corresponding households. Droppings from half of the birds tested in the first Georgia group contained C. psittaci as demonstrated by PCR and/or culture. One of the ill family members of a PCR positive bird had an MIF titer to C. psittaci of 1:512. We came into the investigation when the people had been ill for some time and were recovering and\or had sought medical care and had been treated with antibiotics.

Thus among the few throat swabs collected, there were none that were PCR positive.

The second Georgia group of specimens were tissues from 26 dead birds that were from the same pet stores as the initial group of sick birds. They were collected by the Animal Disease Eradication Veterinarian for the State of Georgia and were tested at the University of Georgia for the presence of C. psittaci by necropsy, gross inspection, traditional histochemical staining (Machiavelo and Gimenez stains), and immunohistochemical staining. Due to poor handling of carcasses prior to testing, many could not be tested properly using these techniques, and many of the birds tested negative. Tissue specimens from these birds unsuitable WO 98/10101 PCTIS97/15556 22 for testing by conventional methods were sent to us for testing using PCR and culture techniques. In this investigation, the application of PCR to avian specimens significantly increased the rate of detection of C. psittaci compared with culture and traditional histochemical and immunohistochemical staining.

When pet store employees and owners of sick birds or dead birds in W. Virginia became ill with a psittacosis like illness, we were asked to test 45 birds (droppings, cloacal swabs, or tissue) from pet stores in the region for C.

psittaci. People with high MIF titers to C. psittaci had been exposed to sick birds that were PCR positive.

Our PCR distinguished C. psittaci from C. pneumoniae and detected more positive specimens than any other technique employed in this study. In addition, it worked when other tests failed due to poor specimen quality. Culture positive specimens were always PCR positive, and people with a high MIF titer to C. psittaci had been exposed to birds that were PCR positive. The strongest inference of transmission from birds to humans were the positive PCR results from birds that belonged to humans with a psittacosis like illness.

A positive human specimen was derived from an outbreak of psittacosis in Southeastern Australia. We received four specimens of post mortem lung tissue from one patient for detection and characterization of C. psittaci.

The patient had seroconverted to C. psittaci by immunofluorescence and complement fixation titers. His post mortem lung tissue was also PCR positive for C. psittaci in Australia using a species-specific PCR, but they could not exclude C. pneumoniae as the etiologic agent because they could not detect for it by PCR. The tissue was sent to us for isolation of the organism and for detection of C. pneumoniae.

While we were unable to culture C. psittaci from the tissue, we did find 3 of the 4 lung specimens PCR positive only for C.

psittaci; no C. pneumoniae was detected.

Our assay distinguished C. psittaci from C.

pneumoniae, an important factor to clinicians making a diagnosis. The Australian attending physician of the patient WO 98/10101 PCTI~S97/1556 23 from whom we tested the post mortem lung specimens would not agree to a diagnosis of psittacosis until C. pneumoniae was ruled out, due to cross reactivity inherent in many of the tests employed in diagnosis. The PCR assay detected more positive specimens than the other techniques employed in this study. Other tests likely failed to detect positives due to poor specimen quality. Culture positive specimens were always PCR positive, and people with a high MIF titer to C. psittaci had been exposed to birds that were PCR positive. The strongest evidence of transmission from birds to humans was the positive PCR results in birds belonging to humans with laboratory-confirmed psittacosis.

wo 98/10101 WO 9810101PCT/US97/15556 24 Table 1 Acinetcbacter species Alcaligenes faecalis Bordetella pertussis Corynebacteriun diphtheriae Corynebacteri ur coryne form Corynebacterun maruchotis Corynebacterium straitiun Corynebacteriurn xerosais Ehrlichia chaffiensis Flavobacteriurn meningoseptic H-aerophilus influenzae strai 819D, KC 528E, KC 529F E6756, E378 21 G5048, BC F124 D9110 E 4684 G676, G3375 urn ns KC 818A, KC 10502, KC'1051C, KC Kin gella kin gae Legionella pneurnophila serogroup 1 Mycobacteriurn tuberculosis Proteus iirabilis Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pneumoniae Table 2 PCR POSITIVE CULTURE POSITIVE' Group I GA Group II GA West Virginia 50-% 2 of 4) 19'1 (5 of 26) 13% (6 of 45) 25*1 (1 of 4) (2 of 26) 2%1 (1 of 1All culture positive specimens were also PCR positive.

Claims (17)

1. A method for detecting for the presence or absence of Chlamydia pneumoniae, chlamydia psittaci and Chlamydia trachomatis in a single test aliquot from a nucleic acid containing sample comprising: contacting the test aliquot with a first sense and antisense nucleic acid primer pair, a second sense and antisense nucleic acid primer pair, and a third sense and antisense nucleic acid primer pair, such that each primer pair flanks a region of the 16s rRNA gene specific to one of the three Chlamydia species in an amplification protocol to produce amplification products; and detecting for the presence or absence of amplification products specific to each of the three Chlamydia species.

2. The method of claim 1, further wherein before step the test aliquot is contacted with a fourth sense and antisense primer pair that flanks a region of the 16s rRNA gene common to all three Chlamydia species.

3. The method of claim 2, further wherein the primers of the fourth primer pair are those set out in SEQ ID NOS:1 and 2.

4. The method of claim 1, further wherein the primers of the first, second, and third primer pairs used in step are those set out in SEQ ID NOS:3-7. The method of claim 1, further wherein the sample is sputum.

6. The method of claim 1, further wherein the sample is a nasal pharyngeal swab. AMENDED SHEET Pb*=J 97/155-56 IPEA/US 0 2 NOV 1998 PCT/US97/15556 WO 98/10101

7. The method of claim 1, further wherein the sample is bronchial alveolar lavage.

8. The method of claim 1, further wherein the sample has not been purified before subjecting it to step (a) AMENDED SHEET WO 98/10101 PCT/US97/15556 26 1 9. The method of claim 2, further wherein the 2 sample has not been purified before the step added by claim 2. 1

10. The method of claim 1, further wherein the 2 amplification protocol occurs at a pH from about 8.3 to about 3 9.2. 1

11. The method of claim 1, further wherein the 2 amplification protocol takes place in a buffer with MgCl 2 in a 3 concentration of about 1.5 to about 1

12. The method of claim 1, further wherein the 2 sample is blood. 1 13. The method of claim 1, further wherein the 2 sample is feces. 1 14. The method of claim 1, further wherein the 2 sample is cloacal tissue. 1 15. A composition comprising an isolated pair of 2 sense and antisense nucleic acid primers complementary to the 3 16s rRNA gene and specific for C. trachomatis. 1 16. A composition according to claim 15, comprising 2 those primers set out in SEQ ID NOS:3 and 4. 1 17. A composition comprising an isolated pair of 2 sense and antisense nucleic acid primers complementary to the 3 16s rRNA gene and specific for C. psittaci. 1 18. A composition according to claim 17, comprising 2 those primers set out in SEQ ID NOS:5 and 6. 1 19. A composition comprising an isolated pair of 2 sense and antisense nucleic acid primers complementary to the 3 16s rRNA gene and specific for C. pneumoniae. 27 A composition according to claim 19, comprising those primers set out in SEQ ID NOS:5 and 7.

21. A kit for detecting for the presence or absence of Chlamydia pneumoniae, C. psittaci and C. trachomatis comprising: three pairs of primers, each pair comprising a sense and antisense nucleic acid primer which flank regions of the 16s rRNA gene specific to one of the three Chlamydia species; and instructions for performing the assay with a single test aliquot.

22. The kit of claim 21, comprising the primers set out in SEQ ID NOS:1-7.

23. A method for detecting for the presence or absence of Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia trachomatis in a single test aliquot from a nulceic-acid containing sample, substantially as hereinbefore described with reference to any one of the examples.

24. A composition comprising an isolated pair of sense and antisense nucleic acid S 15 primers complementary to the 16s rRNA gene and specific for C. trachomatis, substantially as hereinbefore described with reference to any one of the examples.

25. A composition comprising an isolated pair of sense and antisense nucleic acid primers complementary to the 16s rRNA gene and specific for C. psittaci, substantially as hereinbefore described with reference to any one of the examples.

26. A composition comprising an isolated pair of sense and antisense nucleic acid primers complementary to the 16s rRNA gene and specific for C. pneumoniae, o. substantially as hereinbefore described with reference to any one of the examples.

27. A kit for detecting for the presence or absence of Chlamydia pneumoniae, C. psittaci and C. trachomatis, substantially as hereinbefore described with reference to any one of the examples. Dated 31 March, 1999 The Government of the United States of America, represented by The secretary, Department of Health and Human Services Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [N:/libc]00329:bav