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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• This invention relates to diagnostic assays for the detection and differentiation of different species of Chlamydia , and compositions and kits for performing the, assays .
• Chlamydia are widespread intracellular bacteria known to cause a variety of infections in humans, marsupials, other mammals and birds. Chlamydia pneumoniae and Chlamydia psi t taci 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.
• C. psi t taci 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.
• Chlamydia trachoma tis is the most common sexually transmitted disease in the U.S. C. trachoma tis causes tracnoma and urogenital infections. Diagnosis can be difficult, since many women in particular have no overt symptoms of early infection. C. trachoma tis infection also occurs in newborns and its diagnosis and detection s 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 (“PCR”) are generally sensitive and specific techniques for detection of target DNA sequences.
• PCR polymerase chain reaction
• the 16s rRNA ( ⁇ bosomal RNA) gene has been used as a target for the detection of Chlamydia sp, but t has been believed that it cannot be used to differentiate between the species. See , Tjhie et al . , Journal of Microbiologi cal Me thods , 18:137-150 (1993) . Focus for PCR detection methods m Chlamydia has oeen 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.
• RFLP restriction fragments
• This invention provides a novel assay for easily and readily detecting t.hree important Chlamydia sp . , i . e . , C. trachomatis , C. psi ttaci , 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 .
• amplification primers targeted to the 16s rRNA gene specific for each of the species .
• 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.
• the invention includes a method for detecting for the presence or absence of Chlamydia pneumoniae , Chlamydia psi ttaci and Chlamydia trachoma tis in a single test aliquot from a nucleic-acid containing sample comprising:
• 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.
• DETAILED DESCRIPTION This invention provides a novel assay for easily ana readily detecting three important Chlamydia sp . , i . e . , C. trachoma tis , C. psi t taci , and C. pneumoniae . These three species may be detected and differentiated in the same sample aliquot at the same time througn 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.
• the assay may be able to equally identify C. trachoma tis from endocervical swab samples, C. psi ttaci from cloacal swab samDles from birds and C. pneumoniae from sputum samples
• This assay s 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.
• isolated or “biologically pure” refer to material which is substantially or essentially free from components wnicn normally accompany it as found in its native state
• nucleic acid refers to a deoxyrioonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that nybridize 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 double- strandedness 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.
• Stringent hybridization wash conditions in the conte.xt of nucleic acid hybridization experiments sucn 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 .
• highly stringent wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
• T m 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 T m 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 sunstantially identical. This occurs, e . g. , when a copy zi a nucleic acid s created using the maximum codon degeneracy permitted by the genetic code.
• nucleic acid in the context of two nucleic acid sequences refers to the residues m the two sequences which are the same when aligned for maximum correspondence.
• a nucleic acid is "'suostantially identical to a reference nucleic acid wnen it is at least about 70% identical, preferably at least aoout 80% identical, and optionally anout 90% identical or more.
• primer 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.
• the terminal 3 ' nucleotide 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 t ⁇ phosphates (a nucleotide reagent) and thermostable enzyme in an appropriate buffer and at a suitable temperature
• primer pairs 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.
• 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 7- deazaguanosme or mosine, 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 stringency of the amplification medium.
• the primer snould be substantially homologous to the target strands of each specific sequence to be amplified. This means that the primer muse 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 hyoridize with it and thereby form a template for synthesis of the extension product.
• the invention relates to assays or methods for detecting for the presence or absence of Chlamydia pneumoniae, Chlamydia psi ttaci and Chlamydia trachoma tis in a single test aliquot from a nucleic-acid containing sample comprising:
• 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 than such species are absen .
• the samples may be obtained om any source m wnich the bacteria may be present .
• Any source of nucleic acid, in purified or nonpu ⁇ fied 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 T.ammals or birds.
• Samples from tissues and bodily fluids typically tested could include, but are not limited to, sputum; throat swaps,- 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.
• 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. Clm . 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.
• 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 m
• SEQ ID NOS : 1 and 2 be used: sense 5'-3' ACG GAA TAA TGA CTT CGG (SEQ ID NO : 1 ) 9 antisense 5' -3' TAC CTG GTA CGC TCA ATT (SEQ ID NO:2)
• the resulting amplified product is about 436 bp (base pairs) in length.
• 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 ail 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
• the amplification products are about 412 bp for C. trachoma tis , 221 bp for C. pneumoniae , and 127 bp for C. psi ttaci .
• 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.
• 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 10
• Primers substantially identical to those listed aaove are also preferred. It is most preferred that the primer used be substantially complementary to the same -egions.
• 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 sucn amplification methods, including the polymerase chain reaction (PCR) , the ligase chain reaction (LCR) , Q ⁇ - replicase amplification and other RNA polymerase mediated techniques ( e . g.
• 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 1.5 mM to about 3.5 m .
• 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 11
• Oligonucleotides for use as primers (or probes; are typically synthesized chemically according to the solid phase phosphoramidite t ⁇ ester method described by Beaucage and Carutr-ers (1981), Tetrahedron Letts . , 22 (20) : 1859-1862 , e . g. , using an automated synthesizer, e . g. , as described m eedham-VanDevanter et al . (1984) Nucl ei c Acids Res . , 12:6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill.
• oligonucleotides Purification of oligonucleotides, where necessary, is typically performed by either native acryla ide gel electrophoresis or by anion-exchange HPLC as described m Pearson and Regnier (1983) J " . Chrom. 255:137-1 9.
• the sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) m Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology 65:499-560.
• the primers of use m 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 12
• amplification primers are between 8 and 100 nucleotides in length, and preferably between about 10 and 30 nucleotides in length. More typically, the primers are oetween 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 , e . g. , 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 .
• PCR amplification products are optionally detected on a polymer array as described m Fodor e t al . (1991) Sci ence, 251: 767- 777; Sheldon et al . (1993) Cl ini cal Chemis try 39(4): 718-719, and Kozal et al . (1996) Na ture Medicine 2(7): 753-759.
• 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 whicn is likely to occur in the sample to be amplified and any constant primer region.
• constant regions m 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 biotmylated sequences, restriction endonuclease cloning sites, promoter sequences, regulatory sequences, enzyme binding sites, other genes, or any nucleotide sequence that serves a specific desired function.
• 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 mismatcnes, and preferably 100% mismatches for the terminal 5 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 (i.e., 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 .
• amplification primers i . e . , the 5' and 3 primers used for exponential amplification
• the primers are selected so that they have roughly the same, and preferably exactly the same overall base composition ( i . e . , the same A+T to G+C ratio of nucleic acids) .
• 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.
• 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 digoxigenm. These molecules can be used as the ligand binding component . They can be readily captured by their anti-ligand, e.g. avidin or streptavidin m the case of biot and an anti -digoxigenm antibody, bound on a suitable substrate. These reagents are all readily available, see Clontech Laboratories, Inc., Palo Alto, CA for digoxigenm 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.
• 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, 15
• nucleic acid labels may include enzymes (e.g., LacZ, CAT, norse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker gene products or an ELISA), nucleic acid intercalators (e.g , ethidium bromide) and colonmetric labels such as colloidal gold or colored glass or plastic (e.g.
• enzymes e.g., LacZ, CAT, norse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker gene products or an ELISA
• nucleic acid intercalators e.g , ethidium bromide
• colonmetric labels such as colloidal gold or colored glass or plastic (e.g.
• Labeling agents optionally include e . g , monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids, fluorescent dyes, electron-dense reagents, enzymes
• haptens and proteins for which antisera or monoclonal antibodies are available.
• 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 lmmunoblottmg, 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 the art.
• means for detection include a scintillation counter or photographic film as in autoradiography
• 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, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomuitipliers and the like.
• CCDs charge coupled devices
• enzymatic labels may be detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product.
• simple colorimet ⁇ c labels are often detected simply by observing the color associated with the label.
• 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.
• the solid surface is optionally paper, or a membrane (e.g., nitrocellulose), a microtiter dish ( e . g . , PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick ( e . g. 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.
• 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.
• PVDF polyvinylidene difluoride
• silicones 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.
• substances that form gels such as proteins ( e .
• a plurality of different materials are optionally employed, e . g. , as laminates, to obtain various properties.
• protein coatings such as gelatin can be used to avoid non specific binding, simplify covalent conjugation, enhance signal detection or the like.
• 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, ercapto groups and the like.
• various methods for noncovalently binding an anti-ligand component can be used.
• 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 sp. - - C. pneumoniae, C. psi ttaci and C. trachoma tis 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.
• 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. 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 the invention.
• An exemplary assay method described below is a nested, multiplex polymerase chain reaction (PCR) for detection of chlamydiae 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 s genus specific, and the second-step PCR is multiplexed (i.e. has multiple primer sets in the same tube) and can discriminate between C. pneumoniae, C. psi ttaci , and C. trachoma tis .
• the sensitivity of each of the two PCR steps is 5 infectivity units.
• Chlamydiae were grown as previously described (Wong et al . 1992, Journal of Cl inical 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. 19
• 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 T ⁇ s-HCl, pH 8.3 , 50 mM KC1 , 2.5 mM MgCl 2 , 200 ⁇ M of each deoxynucleoside triphosphate , 0.01% BSA (Sigma Chemical Co, St.
• Genus specific first-step primers 16s rRNA sense 5' -3' ACG GAA TAA TGA CTT CGG (SEQ ID N0:1) antisense 5 '-3' TAC CTG GTA CGC TCA ATT (SEQ ID NO : 2 )
• the sensitivity of each of the primer sets, both the genus and species, is less than 5 mfectivity units.
• the first Georgia group of specimens 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 m the first Georgia group contained C. psi t taci as demonstrated by PCR and/or culture. One of the ill family members of a PCR positive bird had an MIF titer to C. psi ttaci 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. psi ttaci 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 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. psi ttaci compared with culture and traditional histochemical and lmmunohistocnemicai staining.
• a positive human specimen was derived from an outbreak of psittacosis in Southeastern Australia.
• the patient had seroconverted to C. psi t taci by immunofluorescence and complement fixation titers.
• His post mortem lung tissue was also PCR positive for C. psi ttaci m 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. psi ttaci from the tissue, we did find 3 of the 4 lung specimens PCR positive only for C. psi t taci ,- no C. pneumoniae was detected.

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