CA2265895A1 - Nucleic acid assay for the detection and differentiation of three chlamydia species - Google Patents

Abstract

This invention relates to diagnostic assays for the detection and differentiation of different species of Chlamydia, and compositions and kits for performing the assays.

Description

HI152030CA 02265895 1999-03-04W0 98/10101 PCT/US97/15556NUCLEIC ACID ASSAY FOR THE DETECTIONAND DIFFERENTIATION OF THREE CHLAMYDIA SPECIESFIELD OF THE INVENTIONThis invention relates to diagnostic assays for thedetection and differentiation of different species ofChlamydia, and compositions and kits for performing theassays.BACKGROUND OF THE INVENTIONChlamydia are widespread intracellular bacteriaknown to cause a variety of infections in humans, marsupials,other mammals and birds. Chlamydia pneumoniae and Chlamydiapsittaci are an important cause of lower respiratory tractinfections. At least 10% of cases of pneumonia in youngadults are associated with C. pneumoniae.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—threateningThus, it wouldC. pneumoniae hasdisease justifying more aggressive treatment.be useful to distinguish these two species for treatmentpurposes.Birds kept as pets have been a significant source ofC. psittaci human infection. Outbreaks of human disease canoccur whenever there is close and continued Contact betweenhumans and infected birds that excrete the organism in fecesand respiratory secretions. The agent is present in tissuesand is often excreted in feces by healthy birds. Theinhalation of infected dried bird feces is a common method ofhuman infection.Laboratory confirmation of psittacosis in humans andbirds is challenging, and treatment is often empiric andpredicated on the clinician eliciting a history of birdexposure. Measurement of complement—fixing antibody titersand, much less commonly, recovery of C. psittaci from patientsU]2035CA 02265895 1999-03-04W0 93/10101 PCTIUS97/155562have been the traditional methods of laboratory confirmationof psittacosis. Neither method is sensitive, and complementfixation is very nonspecific. Microimmunofluorescence (MIF)is a technique which offers greater sensitivity andspecificity than complement fixation (CF) but is not as widelyused (Wong et al. 1994, Journal of Clinical Microbiology 30,1625-30.). MIF interpretation is problematic and requireshighly trained laboratory personnel.Chlamydia trachomatis is the most common sexuallytransmitted disease in the U.S. C. trachomatis causestrachoma and urogenital infections. Diagnosis can bedifficult, since many women in particular have no overtsymptoms of early infection. C. trachomatis infection alsooccurs in newborns and its diagnosis and detection isproblematic. Culture tests are considered the diagnostic goldstandard for detection of Chlamydia.intracellular parasite, culture of this organism istechnically difficult and the organism is famous for itssubstantial laboratory to laboratory variation.Amplification methods such as the polymerase chainreaction ("PCR") are generally sensitive and specificAttemptsHowever, as an obligatetechniques for detection of target DNA sequences.have been made to use PCR to distinguish the three subjectchlamydial species. Kaltenboeck et al. (1992), Journal ofClinical Microbiology 30, 1098-1104 reports the use of a two-step PCR process that targeted major outer membrane protein(MOMP) gene (OmpA) DNA sequence of Chlamydia species and thenused a restriction endonuclease analysis to discriminate thespecies. Tjhie et al. (1993), Journal of MicrobiologicalMethods 18, 137-50, differentiated the species by firstamplifying a region of the OmpA specific to the genus and thenhybridized the amplified product with species specific probesto discriminate among the species.The 16s rRNA (ribosomal RNA) gene has been used as atarget for the detection of Chlamydia sp, but it has beenbelieved that it cannot be used to differentiate between theSee, Tjhie et al., Journal of MicrobiologicalFocus for PCR detection methodsspecies.Methods, l8:l37—l5O (1993).l0l520CA 02265895 1999-03-04W0 98/10101 PCTIUS97/155.563in Chlamydia has been on the OmpA gene and either involvesanalysis by restriction fragments ("RFLP") or two stepprocesses. Id. RFLP can be a complicated and time consumingprocedure, particularly for personnel without significantexperience in the area. Thus, there is a need for a quick,efficient and highly sensitive technique to detect theChlamydia sp. in one assay.SUMMARY OF THE INVENTIONThis invention provides a novel assay for easily andreadily detecting three important Chlamydia sp., i.e., C.trachomatis, C. psittaci, and C. pneumoniae. These threespecies may be detected and differentiated in the same samplealiquot at the same time through the use of amplificationprimers targeted to the 16s rRNA gene specific for each of thespecies. Prior to the assay here, it was not appreciated thatthis gene could be used as a Chlamydia species—specifictarget. The assay described here is surprisingly highlysensitive 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 fordetecting for the presence or absence of Chlamydia pneumoniae,Chlamydia psittaci and Chlamydia trachomatis in a single testaliquot from a nucleic—acid containing sample comprising:(a) contacting the test aliquot with a sense andantisense nucleic acid primer pair such that each pair flanksa region of the 16s rRNA gene specific to one of the threeChlamydia species in an amplification protocol to produceamplification products; and(b) detecting for the presence or absence ofamplification products specific to each of the three Chlamydiaspecies. Preferably, the test aliquot is first contactedwith a pair of sense and antisense primers that flank a regionof the 16s rRNA gene common to all three Chlamydia species.Compositions comprising primer pairs for the methods and kitsto practice the method are also described and claimed.l0l520t\)(I13035CA 02265895 1999-03-04W0 98/ 10101 PCT /U S971 155564DETAILED DESCRIPTIONThis invention provides a novel assay for easily andreadily detecting three important Chlamydia sp., i.e., C.trachomatis, C. psittaci, and C. pneumoniae.. These threespecies may be detected and differentiated in the same samplealiquot at the same time through the use of amplificationprimers targeted to the 16s rRNA gene specific for each of thespecies.The assays of the present invention will allow thelaboratory to test a variety of specimens from differentclinical samples and species with one test. For example, theassay may be able to equally identify C. trachomatis from.endocervical swab samples, C. psittaci from cloacal swabsamples from birds and C. pneumoniae from sputum samples.This assay is a considerable improvement over the use oftraditional stock assay reagents to first test a particularsample for the Chlamydia genus, followed by a second test foreach of the three relevant Chlamydia species.DEFINITIONSUnless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which thisinvention belongs. Singleton et al. (1994) Dictionary ofMicrobiology and Molecular Biology, second edition, John Wileyand Sons (New York); Walker (ed) (1988) The CambridgeDictionary of Science and Technology, The press syndicate ofthe University of Cambridge (New York); and Hale and Marham(1991) The Harper Collins Dictionary of Biology HarperPerennial (New York) all provide one of skill with a generaldictionary of many of the terms used in this invention.Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testingof the present invention, certain preferred methods andmaterials are described. For purposes of the presentinvention, the following terms are defined below.The terms "isolated" or "biologically pure” refer tomaterial which is substantially or essentially free from1520253O35CA 02265895 1999-03-04W0 98/10101 PCT/US97/155565components which normally accompany it as found in its nativestate.The term "nucleic acid" refers to adeoxyribonucleotide or ribonucleotide polymer in eithersingle— or double-stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides thathybridize to nucleic acids in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence optionally includes thecomplementary sequence thereof.Two single-stranded nucleic acids “hybridize” whenthey form a double-stranded duplex. The region of double-strandedness can include the full—length of one or both of thesingle-stranded nucleic acids, or all of one single strandednucleic acid and a subsequence of the other single strandednucleic acid, or the region of double-strandedness can includea subsequence of each nucleic acid. An overview to thehybridization 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 ofnucleic acid probe assays", Elsevier (New York).“stringent hybridization wash conditions“ in thecontext of nucleic acid hybridization experiments such asSouthern and northern hybridizations are sequence dependent,and are different under different environmental parameters.An extensive guide to the hybridization of nucleic acids isfound in Tijssen, supra. Generally, highly stringent washconditions are selected to be about 5°C lower than the thermalmelting point (T ) for the specific sequence at a definedI11ionic strength and pH. The Tm is the temperature (underdefined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Verystringent conditions are selected to be equal to the Tm pointfor a particular probe. Nucleic acids which do not hybridizeto each other under stringent conditions are stillsubstantially identical if the polypeptides which they encodeare substantially identical. This occurs, e.g., when a copyU]1O2O30CA 02265895 1999-03-04W0 98/10101 PCT/US97l155566of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.The term “identical” in the context of two nucleicacid sequences refers to the residues in the two sequenceswhich are the same when aligned for maximum correspondence. Anucleic acid is "substantially identical to a referencenucleic acid when it is at least about 70% identical,preferably at least about 80% identical, and optionally about90% identical or more.The term "primer" as used herein refers to anoligonucleotide, whether occurring naturally as in a purifiedrestriction digest, or produced synthetically, and is capableof hybridizing to a strand of the target sequence. When theterminal 3' nucleotide has hybridized it acts as a point ofinitiation of synthesis under conditions in which synthesis ofan extension of the primer is induced. These conditionstypically include the presence of four different nucleotidetriphosphates (a nucleotide reagent) and thermostable enzymein an appropriate buffer and at a suitable temperature. Whenprimer pairs are referred to herein, the pair is meant toinclude one primer which is capable of hybridizing to thesense strand of a double—stranded target nucleic acid (the"sense primer") and one primer which is capable of hybridizingto the antisense strand of a double—stranded target nucleicacid (the "antisense primer"). The primer pair will bedesigned such that they flank the region of the target nucleicacid to be amplified and will cause the target region to beamplified when placed in an amplification protocol such aspolymerase chain reaction.What is meant by a primer "substantially homologous"or "substantially complementary” to a nucleotide sequence is apolynucleotide or oligonucleotide containing naturallyoccurring nucleotides or their analogs, such as 7—deazaguanosine or inosine, sufficiently complementary tohybridize with the target sequence such that stable andspecific binding occurs between the primer and the targetsequence. The degree of homology required for formation of astable hybridization complex (duplex) varies with thelO203035CA 02265895 1999-03-04W0 98/10101 PCT/U S97/ 155567stringency of the amplification medium. The primer should besubstantially homologous to the target strands of eachspecific sequence to be amplified. This means that the primermust be sufficiently complementary to hybridize with theappropriate strand under standard amplification conditions.Therefore, the primer sequence need not reflect the exactsequence of the template. For example, a noncomplementarynucleotide fragment may be attached to the 5' end of theprimer, with the remainder of the primer sequencecomplementary to the strand.bases or longer sequences can be interspersed into the primerprovided that the primer sequence has sufficientcomplementarity with the sequence of the target sequence tohybridize with it and thereby form a template for synthesis ofthe extension product.THE ASSAYThe invention relates to assays or methods fordetecting for the presence or absence of Chlamydia pneumoniae,Chlamydia psittaci and Chlamydia trachomatis in a single testaliquot from a nucleic—acid containing sample comprising:(a) contacting the test aliquot with a sense andantisense nucleic acid primer pair such that each pair flanksa region of the 16s rRNA gene specific to one of the threeChlamydia species in an amplification protocol to produceamplification products; and(b) detecting for the presence or absence ofamplification products specific to each of the three Chlamydiaspecies. The best results are seen when the test aliquot iscontacted first with a pair of sense and antisense primersthat flank a region of the 16s rRNA gene common to all threeChlamydia species, such as that region flanked by the primersset out in SEQ ID NOS:l and 2.Thus, the assays of the present inventionadvantageously can be accomplished with a single aliquot froma biological sample of interest suspected of containing one ofthe three Chlamydia sp. or in which one wishes to establishthat such species are absent. The samples may be obtainedAlternatively, noncomplementaryU]1015203O35CA 02265895 1999-03-04W0 98/10101 PCT/US97/15556from any source in which the bacteria may be present. Anysource of nucleic acid, in purified or nonpurified formincluding crude extracts of tissue or cells, can be utilizedas the starting nucleic acid or acids. Samples which aretypically of interest include those from humans, marsupials,other mammals or birds. Samples from tissues and bodilyfluids 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 purifiedor pretreated prior to the assay, but in the case of suchtissues which have significant extraneous protein and otherdebris such as feces, it is preferred to subject the sample tolow speed centrifugation or the like to separate out thedebris before assaying.Once the sample is obtained, it is subjected to anamplification protocol. Optionally, the first step includesan amplification step with amplification primers designed totarget the Chlamydia genus generally by targeting the 16s rRNAgene. The 16s rRNA gene is described as a general target inGaydos 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. Libraryof Medicine, National Institutes of Health, 8600 RockvillePike, Bethesda, Maryland 20894) at Accession Nos. L06lO8 forC. pneumoniae, Accession No. M13769 for C. psittaci, and atAccession No. M59178 for C. trachomatis. These sequences canbe aligned as is known in the art to obtain amplificationprimers which will amplify a generic region. The genericregion, however, must encompass those regions which are to bespecifically targeted to differentiate the three species aswill be discussed below. It is most desirable though that theprimers used for this step target that region of the 16s rRNAgene flanked by the primers set out in SEQ ID NOS:l and 2 orregions which overlap those sequences to which such primershybridize. It is most preferred that the primers described inSEQ ID NOS:l and 2 be used:sense E'—3' ACG GAA TAA TGA CTT CGG (SEQ ID NO:l)l0l5202530CA 02265895 1999-03-0498/10101 PCTIU S97/ 15556‘W09TAC CTG GTA CGC TCA ATT (SEQ ID NO:2)when the above primer pair is used, the resulting amplifiedantisense 5'—3’product is about 436 bp (base pairs) in length.If the above step is employed, then the solutionwith the amplified product is directly contacted with thespecies specific primers. If the above step is not employed,then the test sample aliquot itself is directly contacted withthe species specific primers. A "single test aliquot” is analiquot derived from the test sample in which products fromall three species are amplified together. The speciesspecific primers are sense and antisense primer pairs suchthat each pair of primers flanks and targets a region of the16s rRNA gene unique to one of the Chlamydia sp. Thisinvention describes primers which have been found to beparticularly useful and effective and they are most preferredfor the assays claimed herein. The primer pairs are asfollows:For C. trachomatis:GCA ATT GTT TCG GCA ATT G (SEQ ID NO:3)AGC GGG TAT TAA CCG CCT (SEQ ID NO:4)For C. pneumoniae and C. psittaci:ATA ATG ACT TCG GTT ATT (SEQ ID NO:5)sense S'—3'antisense S'—3'sense 5'-3‘For C. psittaci:TGT TTT AGA TGC CTA AAC AT (SEQ ID NO:6)antisense 5'-3'For C. pneumoniae:antisense 5'-3' CGT CAT CGC CTT GGT GGG CTT (SEQ ID NO:7)When these primers are used, the amplification products areabout 412 bp for C. trachomatis, 221 bp for C. pneumoniae, and127 bp for C. psittaci. These primers can also be used asspecies specific probes. The designated primer pairs can alsobe used alone to detect for one species singly, if so desired.It is preferred to use primers which hybridize to aregion of the nucleic acid which overlaps with the respectiveregions of the target nucleic acid to which the primers listedU1l0l5253035CA 02265895 1999-03-0401 PCT/US97l15556W0 98/101 "10above are complementary. Primers substantially identical tothose listed above are also preferred. It is most preferredthat the primer used be substantially complementary to thesame regions. _The primers and the sample are incubated together inan amplification protocol to obtain, if present, an amplifiednucleic acid product which is indicative of each species beingdetected. Nucleic acid amplification techniques suitable foramplifying sequences with nucleic acid primers are known.Examples of techniques sufficient to direct persons of skillthrough such amplification methods, including the polymerasechain reaction (PCR), the ligase chain reaction (LCR), QB-replicase amplification and other RNA polymerase mediatedtechniques (e.g., 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 toMethods and Applications (Innis et al. eds) Academic PressInc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October1, 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; Landegrenet al., (1988) Science 241, 1077-1080; Van Brunt (1990)Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560;(1990) Gene 89, 117, and Sooknanan and MalekOne of skill will alsoBarringer et al.(1995) Biotechnology 13: 563-564.appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for PCR expansion. See, Ausubel,Sambrook and Berger, all supra. The methods and primersdescribed herein are preferably used in a PCR amplificationprotocol. The amplification buffer will preferably have a pHof about 8.3 to about 9.2 and a MgCl2 concentration of about1.5 mM to about 3.5 mM.The nucleic acid sequence to be amplified andidentified will be the amplification product or "target"sequence that exists between the primer pairs flanking it andwhose presence is indicative of a species of interest.CA 02265895 1999-03-0498/10101 PCTIU S97/ 15556W011Oligonucleotides for use as primers (or probes) aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage andCaruthers (1981), Tetrahedron Letts., 22(2Q):l859—l862, e.g.,using an automated synthesizer, e.g., as described inU]Needham—VanDevanter et al. (1984) Nucleic Acids Res.,l2:6159—6168. Oligonucleotides can also be custom made andordered from a variety of commercial sources known to personsof skill. Purification of oligonucleotides, where necessary,10 is typically performed by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described inPearson and Regnier (1983) J. Chrom. 2S5:l37—l49. Thesequence of the synthetic oligonucleotides can be verifiedusing the chemical degradation method of Maxam and Gilbert15 (1980) in Grossman and Moldave (eds.) Academic Press, NewYork, Methods in Enzymology 65:499-560.One of skill will also recognize many ways ofgenerating alterations in a given nucleic acid sequence. Suchwell-known methods include site-directed mutagenesis, PCR20 amplification using degenerate oligonucleotides, exposure ofcells containing the nucleic acid to mutagenic agents orradiation, chemical synthesis of a desired oligonucleotide(e.g., in conjunction with ligation and/or cloning to generatelarge nucleic acids) and other well-known techniques. See,25 Giliman and Smith (1979) Gene 8:81-97; Roberts et al. (1987)Nature 328:731—734 and Sambrook et al. (1989) MolecularCloning — 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 described30 here are preferably single stranded for maximum efficiency andamplification, but may alternatively be double stranded. Ifdouble stranded, the primer is first treated to separate itsstrands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The35 primer must be sufficiently long to prime the synthesis ofextension products in the presence of an enzyme. The exactlengths of the primers will depend on many factors, includingtemperature, source of primer and use of the method. MostU11015203035CA 02265895 1999-03-048/10101 PCT/US97/ 15556W0 912zypically, amplification primers are between 8 and 100nucleotides in length, and preferably between about 10 and 30nucleotides in length. More typically, the primers arebetween about 18 and 28 nucleic acids in length. A primerpair will include one primer which hybridizes to the sensestrand (the strand of nucleic acid which is translated) of theDNA being targeted and one primer which hybridizes to theantisense strand (the strand of nontranslated nucleic acid) ofthe DNA being targeted. The primer pair will flank the regionto be amplified. Short primer molecules generally requirecooler temperatures to form sufficiently stable hybridcomplexes with template.Amplification-based assays are well known to thoseof skill in the art (see, e.g., Innis, supra.). The nucleicacid sequences and other guidelines provided here aresufficient to teach one of skill to routinely select primersto amplify a portion of the 16s rRNA gene for the Chlamydiasp. of interest. It is expected that one of skill isthoroughly familiar with the theory and practice of nucleicacid 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 (ed ) Methods in Molecular Biology, volume 20; andTijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—-Hybridization with Nucleic Acid Probes,e.g., Part I Chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays",Elsevier, New York provide a basic guide to nucleic acidhybridization. Innis, supra, provides an overview of primerselection. In addition, PCR amplification products areoptionally detected on a polymer array as described in Fodoret al. (1991) Science, 251: 767- 777; Sheldon et al. (1993)Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996)Nature Medicine 2(7): 753-759.One of skill will recognize that the 3’ end of anamplification primer is more important for PCR than the 5‘end. Investigators have reported PCR products where only al0l520I\)U13O35CA 02265895 1999-03-04W0 98/1010 1 PCT/US97/1555613few nucleotides at the 3’ end of an amplification primer werecomplementary to a DNA to be amplified. In this regard,nucleotides at the 5’ end of a primer can incorporatestructural features unrelated to the target nucleic acid; forinstance, for detection purposes. The primers are selected sothat there is no complementarity between any known sequencewhich is likely to occur in the sample to be amplified and anyconstant primer region. One of skill will appreciate thatconstant regions in primer sequences are optional. The primermay 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 complementaryto the known target sequencef Such 5' additions to the primermay include, but are not restricted to: chemically modifiedor biotinylated sequences, restriction endonuclease cloningsites, promoter sequences, regulatory sequences, enzymebinding sites, other genes, or any nucleotide sequence thatserves a specific desired function.Typically, all primer sequences are selected tohybridize only to a perfectly complementary DNA, with thenearest mismatch hybridization possibility from known DNAsequences which are likely to occur in the sample to beamplified having at least about 50 to 70% hybridizationmismatches, and preferably 100% mismatches for the terminal 5nucleotides at the 3’ end of the primer.The primers are selected so that no secondarystructure forms within the primer. Self—complementary primershave poor hybridization properties, because the complementaryportions of the primers self hybridize (i.e., form hairpinstructures). The primers are also selected so that theprimers do not hybridize to each other, thereby preventingduplex formation of the primers in solution, and possibleconcatenation of the primers during PCR. If there is morethan one constant region in the primer, the constant regionsof the primer are selected so that they do not self—hybridizeor form hairpin structures.Where sets of amplification primers (i.e., the 5‘and 3' primers used for exponential amplification) are of a1015203035CA 02265895 1999-03-040 98/10101 PCT/US97Il5556W14single length, the primers are selected so that they haveroughly the same, and preferably exactly the same overall basecomposition (i.e , the same A+T to G+C ratio of nucleicacids). Where the primers are of differing lengths, the A+Tto G+C ratio is determined by selecting a thermal meltingtemperature for the primer—DNA hybridization, and selecting anA+T to G+C ratio and probe length for each primer which hasapproximately the selected thermal melting temperature.The amplified product can be detected by means wellknown in the art. The product may be isolated and identifiedby size and through the use of a ligand."ligand binding end" refers to_a component which may directlyThe term "ligand" oror indirectly be detected or captured by another component,the "anti—ligand" which permits the physical or chemicalseparation of compositions bearing the ligand from those whichdo not. The ligand will be attracted to an anti—ligandmolecule such that molecules which do not bear the ligand willnot be captured or otherwise attracted to the anti—ligand.The ligand will need to be one which may be attached directlyor indirectly to nucleic acid sequences. Examples of directligand binding include the use of biotin labeled nucleotidesor the use of digoxigenin. These molecules can be used as theligand binding component. They can be readily captured bytheir anti—ligand, e.g. avidin or streptavidin in the case ofbiotin and an anti-digoxigenin antibody, bound on a suitablesubstrate. These reagents are all readily available, seeClontech Laboratories, Inc., Palo Alto, CA for digoxigeninreagents, for example.The ligand could alternatively be a specific nucleicacid sequence with the anti-ligand being the complement of thesequence or an antibody specific for the sequence. The ligandcould include labeled molecules which may be manipulated on asubstrate so that they are physically or chemically separatedfrom non—ligand bearing molecules. Alternatively, the ligandmolecule can have affinity for an anti—ligand molecule whichis labeled or inherently detectable. These compositions canbe further detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example,[I1l0l5202530'35CA 02265895 1999-03-040 98/10101 PCT/US97/l5556“715useful nucleic acid labels may include enzymes (e.g., Lacz,CAT, horse radish peroxidase, alkaline phosphatase and others,commonly used as detectable enzymes, either as marker geneproducts or in an ELISA), nucleic acid intercalators (e.g.,ethidium bromide) and colorimetric labels such as colloidalgold or colored glass or plastic (e.g. polystyrene,polypropylene, latex, etc.) beads, substrates, cofactors,inhibitors, fluorescent moieties (e.g., fluorescein and itsderivatives, Texas red, rhodamine and its derivatives, dansyl,umbelliferone and the like), chemiluminescent moieties (e.g.luciferin and 2,3—dihydrophthalazinediones), magneticparticles, and the like. Labeling agents optionally includee.g., monoclonal antibodies, polyclonal antibodies, proteins,or other polymers such as affinity matrices, carbohydrates orlipids, fluorescent dyes, electron—dense reagents, enzymes(e.g., as commonly used in an ELISA), or haptens and proteinsfor which antisera or monoclonal antibodies are available. Awide variety of labels suitable for labeling nucleic acids andconjugation techniques are known and are reported extensivelyin both the scientific and patent literature, and aregenerally applicable to the present invention for the labelingof nucleic acids, or amplified nucleic acids for detection andisolation by the methods of the invention. The choice oflabel depending on the sensitivity required, ease ofconjugation of the compound, stability requirements, availableinstrumentation, and disposal provisions. Separation anddetection of nucleic acids proceeds by any known method,including immunoblotting, tracking of radioactive orbioluminescent markers, Southern blotting, northern blotting,southwestern blotting, northwestern blotting, or other methodswhich track a molecule based upon size, charge or affinity.Means of detecting labels are well known to those ofskill in the art. Thus, for example, where the label is aradioactive label, means for detection include a scintillationcounter or photographic film as in autoradiography. Where thelabel is a fluorescent label, it may be detected by excitingthe fluorochrome with the appropriate wavelength of light anddetecting the resulting fluorescence, e g., by microscopy,l01.5203035CA 02265895 1999-03-04PCT/US97/15556W0 98/1010116visual inspection, via photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDS) orphotomultipliers and the like.Similarly, enzymatic labels may be.detected byproviding appropriate substrates for the enzyme and detectingthe resulting reaction product. Finally, simple colorimetriclabels are often detected simply by observing the colorassociated with the label. Thus, in various dipstick assays,conjugated gold often appears pink, while various conjugatedbeads appear the color of the bead.Substrates to be used as an environment for thecapture and separation of the ligand bound molecules fromthose without ligand depend on the ligand being used and thedesired format.paper, or a membrane (e.g., nitrocellulose), a microtiter dish(e.g., PVC, polypropylene, or polystyrene), a test tube (glassor plastic), a dipstick (e.g. glass, PVC, polypropylene,polystyrene, latex, and the like), a microcentrifuge tube, ora glass, silica, plastic, metallic or polymer bead or othersubstrate as described herein. The desired anti—ligand may becovalently bound, or noncovalently attached to the substratethrough nonspecific bonding.A wide variety of organic and inorganic polymers,both natural and synthetic may be employed as the material forthe solid surface. Illustrative polymers includepolyethylene, polypropylene, poly(4—methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate),rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride(PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials whichare 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 (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymerswhich form several aqueous phases, such as dextrans,polyalkylene glycols or surfactants, such as phospholipids,long chain (12-24 carbon atoms) alkyl ammonium salts and theFor instance, the solid surface is optionallyl015202530CA 02265895 1999-03-04PCT/US97/15556W0 98I10l0l17like are also suitable. Where the solid surface is porous,various pore sizes may be employed depending upon the natureof the system.In preparing the surface, a plurality of differentmaterials are optionally employed, e.g., as laminates, toobtain various properties. For example, protein coatings,such as gelatin can be used to avoid non specific binding,simplify covalent conjugation, enhance signal detection or thelike.is desired, the surface will usually be polyfunctional or becapable of being polyfunctionalized. Functional groups whichmay be present on the surface and used for linking can includecarboxylic acids, aldehydes, amino groups, cyano groups,ethylenic groups, hydroxyl groups, mercapto groups and thelike. In addition to covalent bonding, various methods fornoncovalently binding an anti-ligand component can be used.For additional information regarding suitable ligand—anti—ligand and labeling technology as it relates to nucleic acids,see, for example, Essential Molecular Biology, ed. T.A. BrownIRL Press (1993); In Situ Hybridization Protocols, ed. K.H.Andy Choo, Humana Press (1994).KitsFurther contemplated are kits for the assaysdescribed here. Combinations of reagents useful in themethods set out above, particularly the primers, can bepackaged together with instructions for using them in thedescribed assays. A preferred kit would contain three pairsof primers, each pair comprising a sense and antisense nucleicacid primer which flank regions of the 16s rRNA gene specificto each of the three Chlamydia sp.-— C. pneumoniae, C.psittaci and C. trachomatis and instructions for performingthe assay with a single test aliquot. Primers which arespecific for the genus, Chlamydia, are also preferablyincluded, as is described above. Also, as indicated by thedescription provided herein, a single primer may serve as amember of more than one pair of primers.buffers and the like could further be included in the kits.AmplificationIf covalent bonding between a compound and the surface,CA 02265895 1999-03-04W0 98/10101 PCT/US97/1555618All of the references cited herein are cited forgeneral background purposes and are hereby incorporated byreference. The following examples are merely illustrative ofthe invention and are not to be construed as.a limitation ofthe invention.(J1EXAMPLESAn exemplary assay method described below is anested, multiplex polymerase chain reaction (PCR) fordetection of chlamydiae in human and avian specimens. The}...|Cassay resulted in increased sensitivity to circumventinhibitors of PCR present in clinical specimens. The targetsequence is the 16s rRNA gene. The first-step PCR is genusspecific, and the second-step PCR is multiplexed (i.e. has.15 multiple primer sets in the same tube) and can discriminatebetween C. pneumoniae, C. psittaci, and C. trachomatis. Thesensitivity of each of the two PCR steps is 5 infectivityunits. We used PCR and serologic evidence during an outbreakof psittacosis to infer that C. psittaci had been transmitted20 from birds purchased in pet stores to humans. We also usedthis method to test both live and dead birds from pet storesfor infection with C. psittaci. PCR results were comparedwith culture methods. The application of PCR to avianspecimens significantly increased the detection rate of C.25 psittaci compared with culture methods.Growth and purification of organismsChlamydiae were grown as previously described (Wong et al.1992, Journal of Clinical Microbiology 30, 1625-30.).30 Briefly, chlamydiae were propagated by centrifugation ofthawed stock cultures onto exponentially growing Hep—2 cellmonolayers. Chlamydial elementary bodies were harvested 72hours after inoculation by disrupting the host monolayer withglass beads in fresh Isocove's Modified Dulbecco's Medium with35 10% fetal calf serum. The disrupted cell suspension wassonicated, and partially purified by centrifugation at 500 x gfor 10 minutes, followed by centrifugation on renografin.1O20253035CA 02265895 1999-03-04W0 98/10101 PCT/U S97/ 1555619SerologyComplement fixation and microimmunofluorescence methods werepreviously described (Wong et al. 1994, Journal of ClinicalMicrobiology 32, 2417-21.).Preparation of clinical specimensClinical specimens included unclotted blood, throat swabs,feces, tissue, and cloacal swabs. These specimens wereprepared for PCR using the QiaAmp Blood and QiaAmp Tissue kits(Qiagen Inc, 9600 Desoto Ave, Chatsworth, CA 91311, USA). Allspecimens except blood and tissue underwent a differentialcentrifugation of 500 X g for 5 minutes to pellet debris priorto DNA extraction using the Qiagen kits.PCR amplificationSamples for PCR were prepared in a class II laminar flow hood,and amplification and analysis of PCR products were eachperformed in separate locations. Reaction volumes of 50 plcontaining 10 mM Tris—HCl, pH 8.3, 50 mM KCl, 2.5 mM MgC12,200 uM of each deoxynucleoside triphosphate, 0.01% BSA (SigmaChemical Co, St. Louis, MO), 1.25 units of Taq polymerase(Boehringer Mannheim), 0.2 pM of each outer primer, and 5 plof sample were overlaid with one drop of mineral oil andplaced in a Perkin—Elmer Thermalcycler Model 480 (Perkin—ElmerCetus Corp., Norwalk, Conn.), for 1 cycle of 95°C for 2minutes, followed by 35 cycles of 94°C for one minute, 55°Cfor 30 seconds, and 72°C for one minute. The nested or innerPCR reaction mixture was similar to the first except that itcontained 1 pl of the product of the outer PCR and 0.2 pM ofeach inner primer. The cycling conditions were identical.Genus specific first-step primers 163 rRNA:ACG GAA TAA TGA CTT CGG (SEQ ID NO:l)TAC CTG GTA CGC TCA ATT (SEQ ID NO:2)sense 5'—3'antisense 5'—3'Genus product: 436 bpSpecies specific second step-primers 16s rRNA:GCA ATT GTT TCG GCA ATT G (SEQ ID NO:3>C. tr sense 5'—3'C. tr antisense 5'—3' AGC GGG TAT TAA CCG CCT (SEQ IDNO:4)C. pn\psi sense 5'—3' ATA ATG ACT TCG GTT GTT ATT (SEQ IDNO:S)1O15203035CA 02265895 1999-03-045 6W0 98/10101 PCT/US97/1 5520C. psi antisense 5'-3' TGT TTT AGA TGC CTA AAC AT (SEQ IDNO : 5 )C. pn antisense 5‘-3' CGT CAT CGC CTT GGT GGG CTT (SEQID NO : 7 }Both the outer and inner PCRs were optimized withthe Opti-Prime PCR optimization Kit (Stratagene, La Jolla,CA). Amplification products were separated by electrophoresisthrough 2.5% agarose gels [l.5% Nusieve GTG agarose (FMCBioproducts, Rockland, ME) and 1.0% agarose (BioRadLaboratories, Richmond, CA)] in Tris—borate—EDTA and werevisualized by ethidium bromide fluorescence.All of the PCR primers were both sensitive andspecific. Neither the outer nor inner primer sets cross-reacted with other respiratory pathogens. Table 1 lists themicroorganisms tested for specificity.The sensitivity of each of the primer sets, both thegenus and species, is less than 5 infectivity units. Themultiplex PCR, containing all 5 primers for detection of eachChlamydia organism, was run as a second-step PCR using 1 pl ofthe first—step PCR product. Five infectivity units of eachChlamydia species were used in the genus, first-step PCR.Due to illness among owners of sick birds purchasedfrom pet stores, we were asked to test three separate groupsof sick birds and a few human throat swabs and blood specimensfor C. psittaci. we used our two-step PCR to test allspecimens sent to us. Table 2 is a summary of the three groupsof bird specimens tested for culture and PCR. All of theculture positive specimens were also PCR positive.Of the human sera from 4 individuals tested in thefirst Georgia group, one had an MIF titer to C. psittaci of1:512.PCR negative. There were no human specimens sent for testingAll of the throat swabs from these individuals werefrom the W. Virginia group; all isolates came from birds. TheMIF titers to C. psittaci of ill people exposed to PCRpositive birds in the W. Virginia group ranged from 1:16 to1:512.l0203035CA 02265895 1999-03-04W0 98/10101 PCT/US97/1555621It is our experience that sometimes not enoughproduct is made in the first—step PCR to be visualized on theagarose gel, due to inhibitors still present in the sampleeven after preparation\purification of the clinical specimenfor PCR. We find that positive specimens can most quickly andefficiently be identified with a second-step PCR using a tinyfraction of the first—step PCR product.The focus of this investigation was to determine ifthere had been transmission of Chlamydia psittaci fromshipments of birds known to be infected to owners of thesebirds.are lacking.simultaneously distinguishes and detects C. pneumoniae, C.Sensitive and specific tests to confirm psittacosisWe developed a new nested, multiplex PCR thatpsittaci, and C. trachomatis. This PCR was applied to all ofthe specimens available to us in the study.In the first Georgia group of specimens, a few ofthe exposed humans had unclotted blood and throat swabscollected for culture and PCR. Fresh bird droppings were alsocollected from the corresponding households. Droppings fromhalf of the birds tested in the first Georgia group containedC. psittaci as demonstrated by PCR and/or culture. One of theill family members of a PCR positive bird had an MIF titer toC. psittaci of 1:512. We came into the investigation when thepeople had been ill for some time and were recovering and\orhad sought medical care and had been treated with antibiotics.Thus among the few throat swabs collected, there were nonethat were PCR positive.The second Georgia group of specimens were tissuesfrom 26 dead birds that were from the same pet stores as theinitial group of sick birds. They were collected by theAnimal Disease Eradication Veterinarian for the State ofGeorgia and were tested at the University of Georgia for thepresence of C. psittaci by necropsy, gross inspection,traditional histochemical staining (Machiavelo and Gimenezstains), and immunohistochemical staining. Due to poorhandling of carcasses prior to testing, many could not betested properly using these techniques, and many of the birdstested negative. Tissue specimens from these birds unsuitable1520253035CA 02265895 1999-03-04W0 98/10101 PCTIUS97/1555622for testing by conventional methods were sent to us fortesting using PCR and culture techniques. In thisinvestigation, the application of PCR to avian specimenssignificantly increased the rate of detection of C. psittacicompared with culture and traditional histochemical andimmunohistochemical staining.when pet store employees and owners of sick birds ordead birds in W. Virginia became ill with a psittacosis likeillness, we were asked to test 45 birds (droppings, cloacalswabs, or tissue) from pet stores in the region for C.psittaci. People with high MIF titers to C. psittaci had beenexposed to sick birds that were PCR positive.Our PCR distinguished C. psittaci from C. pneumoniaeand detected more positive specimens than any other techniqueemployed in this study. In addition, it worked when othertests failed due to poor specimen quality.specimens were always PCR positive, and people with a high MIFtiter to C. psittaci had been exposed to birds that were PCRCulture positivepositive. The strongest inference of transmission from birdsto humans were the positive PCR results from birds thatbelonged to humans with a psittacosis like illness.A positive human specimen was derived from anoutbreak of psittacosis in Southeastern Australia. Wereceived four specimens of post mortem lung tissue from onepatient for detection and characterization of C. psittaci.The patient had seroconverted to C. psittaci byimmunofluorescence and complement fixation titers. His postmortem lung tissue was also PCR positive for C. psittaci inAustralia using a species—specific PCR, but they could notexclude C. pneumoniae as the etiologic agent because theycould not detect for it by PCR. The tissue was sent to us forisolation 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 adiagnosis. The Australian attending physician of the patientlOCA 02265895 1999-03-04W0 98/10101 PCT/US97/1555623from whom we tested the post mortem lung specimens would notagree to a diagnosis of psittacosis until C. pneumoniae wasruled out, due to cross reactivity inherent in many of thetests employed in diagnosis. The PCR assay detected morepositive specimens than the other techniques employed in thisOther tests likely failed to detect positives due toCulture positive specimens were alwaysstudy.poor specimen quality.PCR positive, and people with a high MIF titer to C. psittacihad been exposed to birds that were PCR positive. Thestrongest evidence of transmission from birds to humans wasthe positive PCR results in birds belonging to humans withlaboratory—confirmed psittacosis.1520253035CA 02265895 1999-03-04W0 98/10101 PCT/US97/1555624Table 1Acinetcbacter speciesAlcaligenes faecalisBordetella pertussisCorynebacterium diphtheriaeCorynebacterium coryneform E6756, E378Bl G5048, BC F124D9llO(A), E 4684G676, G3375Corynebacterium maruchotisCorynebacterium straitiumCorynebacterium xerosisEhrlichia chaffiensisFlavobacterium meningosepticumHaemophilus influenzae strains KC 818A, KC IOSOB, KC lO5lC, KC819D, KC 528E, KC 529FKingella kingaeLegionella pneumophila serogroup 1Mycobacterium tuberculosisProteus mirabilisPseudomonas aeruginosaStaphylococcus aureusStreptococcus pneumoniaeTable 2PCR POSITIVE CULTURE POSITIVEIGroup I GA 50% ( 2 of 4) 25% (1 of 4)Group II GA 19% (S of 26) 8% (2 of 26)West Virginia 13% (6 of 45) 2% (I of 45)1 All culture positive specimens were also PCR positive.

Claims (22)

WHAT IS CLAIMED IS:

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:
(a) 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 (b) 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 (a) 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 (a) are those set out in SEQ ID NOS:3-7.

5. 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.

25a

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).

9. The method of claim 2, further wherein the sample has not been purified before the step added by claim 2.

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

11. The method of claim 1, further wherein the amplification protocol takes place in a buffer with MgCl2 in a concentration of about 1.5 to about 3.5.

12. The method of claim 1, further wherein the sample is blood.

13. The method of claim 1, further wherein the sample is feces.

14. The method of claim 1, further wherein the sample is cloacal tissue.

15. A composition comprising an isolated pair of sense and antisense nucleic acid primers complementary to the 16s rRNA gene and specific for C. trachomatis.

16. A composition according to claim 15, comprising those primers set out in SEQ ID NOS:3 and 4.

17. A composition comprising an isolated pair of sense and antisense nucleic acid primers complementary to the 16s rRNA gene and specific for C. psittaci.

18. A composition according to claim 17, comprising those primers set out in SEQ ID NOS:5 and 6.

19. A composition comprising an isolated pair of sense and antisense nucleic acid primers complementary to the 16s rRNA gene and specific for C. pneumoniae.

20. A composition according to claim 19, comprising those primer pairs set out in SEQ ID NOS:5 and 7.

21. A kit for detecting the presence or absence of Chlamydia pneumoniae, chlamydia psittaci and Chlamydia trachomatis comprising:
(a) 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 (b) instructions for performing the assay with a single test aliquot.

22. The kit of claim 21, wherein the three pairs of primers are set out in SEQ ID NOS:3-7, and the kit further comprises a fourth pair of primers as set out in SEQ ID
NOS:1-2.