WO1990013669A1 - Nucleic acid based diagnostic system and process for the detection of pneumocystis carinii in blood products - Google Patents

Nucleic acid based diagnostic system and process for the detection of pneumocystis carinii in blood products Download PDF

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Publication number
WO1990013669A1
WO1990013669A1 PCT/US1990/002633 US9002633W WO9013669A1 WO 1990013669 A1 WO1990013669 A1 WO 1990013669A1 US 9002633 W US9002633 W US 9002633W WO 9013669 A1 WO9013669 A1 WO 9013669A1
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nucleic acid
carinii
process
polynucleotide
pneumocystis carinii
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PCT/US1990/002633
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French (fr)
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Peter Baram
Kary Banks Mullis
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Xytronyx, Inc.
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Abstract

A process for detecting the presence of a Pneumocystis carinii infection in a mammal is described in which Pneumocystis carinii encoded nucleic acids are detected in the blood of an infected mammal by means of nucleic acid hybridization, preferably using a polymerase chain reaction.

Description

NUCLEIC ACID BASED DIAGNOSTIC SYSTEM AND PROCESS

FOR THE DETECTION OF PNEUMOCYSTIS CARINII

IN BLOOD PRODUCTS

Description

Technical Field

The present invention relates to methods and systems for detecting the presence of a clinical Pneumocystis carinii infection by nucleic acid hybridization using P. carinii specific polynucleotide segments.

Background

Pneumocystis carinii (P. carinii) pneumonia is the most common opportunistic infection in AIDS, and accounts for significant morbidity and mortality in AIDS and other immunoco promised patients. Infection in immunosuppressed patients, such as organ transplant recipients, is an additional major problem caused by P. carinii. Therefore, methods of detecting P. carinii at early stages of infection are necessary to allow effective diagnosis and treatment.

Present methods for detecting P. carinii pneumonia depend on lung biopsy from a patient's lungs with examination of the tissue for indicia of the pathogen or disease symptoms. Immunodiagnostic procedures have been described using antibodies that immunoreact with P. carinii antigens to detect the presence of the antigens in sputum or tracheal aspirate samples. See Lim, U.S. Patent No. 3,992,516.

Attempts at serodiagnosis of P. carinii have been reported widely, detecting either P_j_ carinii antigens in the sera, antibodies to those antigens or both. See Hughes, pp.35-48, in Pneumocystis Carinii Pnβumonitis, CRC Press, Boco Raton, FL, Vol II, 1987.

However, detection of antibody titers against _?__, carinii have been characterized as unreliable in diagnosing pneumonia since a majority of normal individuals have substantial titers. Pifer, Chest, 87:698-700, 1985. Assays for P. carinii antigens in the blood (antigenemia) have also been criticized for reliability in diagnosing pneumonia. Hughes, Chest, 87:700,1985.

The disease caused by P. carinii is almost universally limited to the lungs. Definitive diagnosis of infection in the past has been dependent upon a demonstration of organisms in lung tissue or in an aspirate of lung or respiratory tract. Organisms have been detected in some cases in pharyngeal smears, tracheal aspirates and sputum, gastric aspirates and bronchopulmonary lavages, and in rare cases in bone marrow.

However, aside from the unreliable detection of P. carinii antigens or anti-P. carinii antibodies described above, there has been no reports of P. carinii organisms or other associated materials present in an infected patient's blood.

Limited P. carinii nucleic acid sequence data is presently available. Ribosomal RNA (rRNA) was cloned from rat P. carinii trophozoites and sequenced. Edman et al., Nature, 334:519-522 (1988) . P. carinii rRNA polynucleotides having the least sequence identity with known 16S-like rRNAs were used in situ to hybridize with P. carinii infected lung tissue. Reco binant DNA carrying genomic DNA segments of P. carinii was prepared from rat lung P. carinii cysts and was also used as a probe by southern blot hybridization to detect P. carinii genomic DNA in infected lung tissues from rat and human. Tanabe et al., J. Inf. Diseases. 157:593-596 (1988).

Brief Summary of the Invention

It has now been discovered that mammals harboring a P. carinii clinical infection have detectable P. carinii specific nucleic acids present in their blood.

Therefore, the present invention contemplates a process for detecting the presence of a clinical P. carinii infection in a mammal comprising forming a hybridization reaction admixture by admixing a denatured vascular fluid sample from said mammal with a P. carinii specific polynucleotide probe capable of hybridizing with P. carinii genomic DNA or a RNA transcript thereof. The admixture is maintained under hybridizing conditions for a time period sufficient for said probe to hybridize to any complementary nucleic acid sequence present in said sample to form a hybridized duplex. The presence of any duplex formed in step (b) is then detected, thereby detecting the presence of Pneumocystis carinii infection in said mammal.

In preferred embodiments the polynucleotide probe has a nucleotide sequence that is complementary to a Pneumocystis carinii specific portion of the nucleotide sequence of Pneumocystis carinii ribosomal RNA.

Insofar as the polymerase chain reaction (PCR) is an effective way to increase the sensitivity of a nucleic acid duplex detection step, it is further preferred that the process detects an amplified nucleic acid product produced by PCR.

In other preferred embodiments the hybridized duplex is an RNA-DNA duplex that is detected by first preparing a cDNA molecule from the RNA-DNA duplex and then amplifying the cDNA using a polymerase chain reaction.

Detailed Description of the Invention A. Definitions

Nucleotide: a onomeric unit of DNA or RNA consisting of a sugar moiety (pentose) , a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (l' carbon of the pentose) and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose it is referred to as a nucleotide. A sequence of operatively linked nucleotides is typically referred to herein as a "base sequence" or "nucleotide sequence", and is represented herein by a formula whose left to right orientation is in the conventional direction of 5'-terminus to 3'- terminus.

Polynucleotide: A nucleic acid molecule comprising a polymeric unit of DNA or RNA having a sequence of two or more operatively linked nucleotides that form a single linear strand of nucleotides, also referred to as an oligonucleotide.

DNA Duplex: A double-stranded nucleic acid molecule consisting of two strands of complementary polynucleotide hybridized together by the formation of a hydrogen bond between each of the complementary nucleotides present in a base pair of the duplex. Because the nucleotides that form a base pair can be either a ribonucleotide base or a deoxyribonucleotide base, the phrase "DNA duplex" refers to either a DNA-DNA duplex comprising two DNA strands, or a RNA-DNA duplex comprising one DNA and one RNA strand.

Base Pair (bp) : A partnership of adenine (A) with thymine (T) , or of cytosine (C) with guanine (G) in a double stranded DNA duplex.

Nucleic Acid: A term to refer to any of a class of molecules that includes ribonucleic acid (RNA) , deoxynucleic acid (DNA) in its single or double stranded forms, and polynucleotides.

B. Process for Detecting P. carinii

Infection "Clinical infection" as used herein refers to the presence of clinically detectable symptoms such as dyspnea, fever, cough, cyanosis and diffuse bilateral infiltrate, due to the presence of a high number of P. carinii organisms on or in an individual's lungs. See Pneumocystis carinii Pneumonitis, Chapters 7, 8 and 10, CRC Press, Inc., Boca Raton, Florida, 1987, for a detailed description of the clinical and histological evidence of P. carinii induced disease. In contrast, "latent infection" is used herein to refer to a subclinical P. carinii infection, i.e., the presence of a low number of organisms and no significant clinical or histological evidence of disease.

The present invention contemplates a method for determining the presence of a clinical P. carinii infection in a mammal by assaying for the presence of P. carinii encoded nucleic acids in a vascular fluid sample such as blood, serum, plasma and the like. Typically, the method is performed by subjecting the sample to a hybridization reaction with a P. carinii specific polynucleotide probe and then determining the presence of any hybridization reaction product formed, the presence of the product indicating the presence of a P. carinii infection.

1. Denatured Blood Sample In practicing the present invention it is advantageous to first treat the vascular fluid sample, preferably serum or plasma, to be assayed with a denaturing agent that dissociates nucleic acid/protein complexes, thereby providing access to the nucleic acids for hybridization. Useful denaturing agents include organic solvents, chaotropic salts, surfactants and the like. Exemplary organic solvents include acetone, alcohol, acetone/alcohol admixtures and the like. Exemplary chaotropic salts include sodium iodide, guanidinium chloride, guanidinium isothiocyanate and the like. Exemplary surfactants include anionic and nonionic materials. Illustrative anionic surfactants include sodium dodecyl sulfate (SDS) , ammonium lauryl sulfate (ALS) , potassium lauryl sulfate, sodium myristyl sulfate and the like. Preferably, the vascular fluid sample is treated with acetone by admixing about 5 to about 100 volumes of acetone with 1 volume of sample. The acetone/sample admixture is then maintained at 100 degrees Centigrade (100C) for a time period sufficient to evaporate the acetone and water present, thereby forming a dried, denatured sample. Nucleic acids present in the dried sample are solubilized by admixture with an aqueous solvent, preferably sterile RNase-free water. Subsequent to denaturation, the nucleic acids present in the sample can be further isolated from proteinaceous material present in the sample by methods well known in the art. See, for example, Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory p. 458 (1982).

Typically, the nucleic acid isolation method used will depend, as is well known in the art, on the type of nucleic acid, i.e., RNA or DNA, to be detected.

2. Polynucleotide Probes

The denatured vascular fluid sample is then probed by hybridization with a P. carinii specific polynucleotide probe. Polynucleotide probes (segments) are polynucleotides of about 10 to 500 nucleotide bases in length, preferably 15 to 25 nucleotides in length, having a nucleotide base sequence that is substantially complementary with P. carinii genomic DNA or P. carinii RNA transcripts.

By "substantially complementary" and its grammatical equivalents is meant that there is sufficient nucleotide base sequence similarity between a subject polynucleotide probe and a nucleic acid sequence having exact complementarity with a P. carinii nucleic acid sequence that the subject polynucleotide is capable of hybridizing with a P. carinii specific nucleic acid under hybridizing conditions. Therefore, the polynucleotide probe need not contain the exact sequence that is complementary to the nucleotide base sequence of the nucleic acid (i.e., the target sequence) with which the polynucleotide probe is to hybridize so long as the probe contains substantial complementarity with the target sequence.

For example, a non-complementary polynucleotide portion may be attached to the 5' end of the complementary polynucleotide portion, with the remainder of the nucleotide base sequence being at least substantially complementary to the target sequence. Alternatively, non-complementary bases or groups of bases can be interspersed into the polynucleotide, provided that the polynucleotide has sufficient nucleic acid sequence complementarity with the target sequence to hybridize therewith and form a DNA duplex comprised of polynucleotide probe and P. carinii specific nucleic acid.

By "P. carinii specific" is meant that the nucleic acid sequence referred to contains a sequence of nucleotides that has substantial complementarity with P. carinii genomic DNA or RNA transcripts thereof but does not have substantial complementarity with non-P. carinii genomic DNA or RNA transcripts. Therefore, P. carinii specific polynucleotides do not hybridize with non-P. carinii genomic DNA or RNA transcripts. The identification of P. carinii specific polynucleotides typically involves a comparison of P. carinii genomic nucleotide sequence with other known non-P. carinii genomic sequences to locate regions of P. carinii that do not have substantial complementarity with non-P. carinii sequences. Computer-aided searches of published nucleotide sequence data banks, such as GENBANK, EMBL and the like, are well known and provide rapid means to compare nucleotide sequences and locate nucleotide sequences having substantial complementarity (i.e., that are homologous), and locate nucleotide sequences that do not have substantial complementarity (i.e., that are unique) .

Once a nucleotide sequence comparison has located regions of P. carinii that do not have substantial complementarity to the non- P. carinii nucleotide sequences in a published data bank, it is preferred to verify the uniqueness of the nucleotide sequence in a hybridization assay to identify a P. carinii specific polynucleotide. A verifying hybridization assay is one in which the P. carinii nucleotide sequence is subjected to hybridizing conditions as described herein in the presence of nucleic acid sequences derived from P. carinii. and also from the group of diverse non-P. carinii organisms that consist of essentially of Escherichia coli, Saccharomyces cerevisiae. Homo sapiens, P. carinii specific nucleotide sequences are ones that hybridize to P. carinii nucleic acids but do not hybridize to nucleic acids from the above-identified non-P. carinii organisms.

Thus the invention contemplates a process for detecting the presence of a P. carinii infection in a mammal which comprises forming a hybridization reaction admixture by admixing a denatured vascular fluid sample from the mammal with a polynucleotide probe capable of hybridizing with P. carinii specific nucleic acids. Examples of useful P. carinii specific polynucleotides are those having a sequence with substantial complementarity to P. carinii specific portions of the sequence of the ____ carinii ribosomal RNA sequence reported by Edman et al., Nat re, 334:519-522, 1988.

Where the process of the present invention detects an amplified nucleic acid product, the hybridization reaction admixture is formed by admixing two distinct polynucleotide probes with a denatured vascular fluid sample. At least one of the two admixed polynucleotide probes are P. carinii specific, and they both have nucleotide sequences selected to allow for amplification of hybridized P. carinii nucleic acid sequences by the polymerase chain reaction, as discussed further below. That is, the two admixed polynucleotides comprise first and second polynucleotide probes having nucleotide sequences with the following characteristics: (1) at least one of the polynucleotides is P. carinii specific; (2) the two polynucleotides are substantially complementary to one or the other strand of P. carinii genomic DNA, wherein the first polynucleotide is complementary to the plus strand and the second polynucleotide is complementary to the minus strand of a P. carinii genomic DNA nucleic acid sequence; (3) the two polynucleotides are substantially complementary to nucleotide sequences that are located a distance of about 0 to about 300 nucleotide bases apart from one another on the P. carinii genomic DNA, preferably about 20 to about 200 bases apart; and (4) the two polynucleotides are substantially complementary to nucleotide sequences whose relative location on the geno ic DNA are such that the target for the first polynucleotide is located 3' on the plus strand relative to the location of the target for the second polynucleotide. Preferred polynucleotide probes for use where the contemplated process detects amplified nucleic acid products are the pair of polynucleotides PN03 and PN40, or alternatively the pair of polynucleotides PN20 and PR02. The nucleotide base sequences of PN03, PN20, PN40 and PR02 are shown in Table 1, and correspond with exact complementarity to portions of plus and minus strands of P. carinii genomic DNA sequences that encode the ribosomal RNA reported by Edman et al. , supra.

Table 1 POLYNUCLEOTIDES

Polynucleotide Designation Nucleotide Base Sequence1

PN03 AAT AAC CCA TCA CCA GTC CGA

PN20 ATT TAG ATA CCT TA

PN40 CAG AGC CAG CAA GTT CAT TT

PRO2 TTA CCG CGG CTG GCA C

1 Nucleotide base sequences are shown in a 5' to 3' direction (left to right) .

Polynucleotide segments can be prepared by a variety of methods including de novo chemical synthesis of polynucleotides and derivation of nucleic acid fragments from native nucleic acid sequences existing as genes, or parts of genes, in a genome, plasmid, or other vector, such as by restriction endonuclease digest of larger nucleic acid fragments and strand separation or by enzymatic synthesis using a nucleic acid template.

De novo chemical synthesis of polynucleotides can be carried out, for example, by the phosphotriester method described by Matteucci et al., J. Am. Che . Soc.. 103:3185 (1981), or as described in U.S. Pat. No. 4,356,270.

Derivation of a polynucleotide from nucleic acids involves the cloning of a nucleic acid into an appropriate host by means of a cloning vector, replication of the vector and therefore multiplication of the amount of the cloned nucleic acid, and then the isolation of subfragments of the cloned nucleic acids.

For a description of subcloning nucleic acid fragments, see Maniatis et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, pp 390-401 (1982); and see U.S. Pat. Nos. 4,416,988 and 4,403,036.

3. Hybridization Conditions The process for detecting P. carinii infection in a mammal includes maintaining the hybridization reaction admixture under hybridizing conditions for a time period sufficient for the polynucleotide probe to hybridize to any complementary nucleic acid sequence present in the vascular fluid sample to form a hybridized DNA duplex.

The phrase "hybridizing conditions" and its grammatical equivalents, when used in connection with a maintenance time period, indicates subjecting the hybridization reaction admixture, in the context of the concentrations of reactants and accompanying reagents in the admixture, to time and temperature conditions sufficient to allow the polynucleotide to anneal with the target (P. carinii specific) nucleic acid sequence and form a double stranded DNA duplex. Such time and temperature conditions required to accomplish hybridization depend, as is well known in the art, on the length of the polynucleotide segment to be hybridized, the stringency of hybridization desired, and the presence of salts or additional reagents in the hybridization reaction admixture as may affect the kinetics of hybridization. Methods for optimizing hybridization conditions for a given hybridization reaction admixture are well known in the art.

Typical hybridizing conditions include the use of solutions buffered to pH values between 5 and 9, and are carried out at temperatures from 18 to 70 degrees C, preferably about 55 to about 65 degrees C, for time periods from 0.5 minutes to 24 hours. For small reaction volumes and rapid hybridization reactions, such as is described in Example 3, hybridization temperatures can vary as the temperature changes such as when the sample is maintained first at 100C to denature a duplex DNA, then cooled to room temperature (RT) for hybridization, then heated and maintained at 30C for polymerization.

Hybridization can be carried out in a homogeneous or heterogeneous format as is well known. The homogeneous hybridization reaction occurs entirely in solution, in which both the polynucleotide probe and the nucleic acid sequences to be hybridized (target) are present in soluble forms in solution. A heterogeneous reaction involves the use of a matrix that is insoluble in the reaction medium to which either the probe or target nucleic acid is bound.

Typical and preferred are the homogeneous hybridization reactions such as are described in Example 3 in which the DNA duplex detection step is conducted by amplifying the formed DNA duplex in a polymerase chain reaction (PCR) , described further herein. Typical heterogeneous hybridization reactions include the use of nitro-cellulose sheets as the matrix to which target nucleic acids are bound.

4. Detecting a DNA Duplex After the formation of a hybridized

DNA duplex, the process of the present invention contemplates detecting the presence of a polynucleotide probe-containing DNA duplex formed in the hybridization reaction, and thereby detecting the presence of P. carinii infection in the mammal from whom the sample was obtained.

The detection of formed DNA duplexes can be accomplished by a variety of means, and although there are preferred embodiments disclosed herein for DNA duplex detection, it is to be understood that other well known duplex detection means readily apparent to one skilled in the art are suitable for use in the presently contemplated process and associated diagnostic system. In one embodiment, the DNA duplex detection step comprises detecting an amplified nucleic acid product. An amplified nucleic acid product is the product of an amplification process well known in the art that is referred to as the polymerase chain reaction (PCR) . The amplified product is produced when PCR is carried out in the presence of a DNA duplex containing a P. carinii specific polynucleotide probe.

Therefore embodiments that contemplate detecting an amplified nucleic acid product include the steps of first amplifying by PCR any DNA duplex formed in the hybridization reaction to form an amplified nucleic acid product, and then detecting the presence of any amplified nucleic acid product formed.

PCR is carried out in cycles, in which each cycle typically comprises the following steps:

(1) A PCR reaction admixture is maintained for a time period and at a temperature sufficient to denature any DNA duplex, resulting in strand separation and the formation of single stranded nucleic acids. Denaturing a DNA duplex to form single stranded nucleic acids is well known and can be accomplished by a variety of conditions. In a PCR format, denaturation of duplex is typically carried out quickly, in about 10 seconds to 5 minutes, preferably about 1 minute, and at temperatures to accomplish rapid denaturization (duplex melting) , at about 90C to 100C, preferably 95C.

(2) The denatured admixture from step (1) above is maintained for a time period and at a temperature sufficient to produce hybridization conditions, wherein P. carinii specific polynucleotide probes hybridize with complementary target P. carinii specific nucleotide sequences to form DNA duplexes. Hybridizing conditions were described earlier and are suitable for use in the PCR format. However, it is preferred and convenient to conduct hybridization in short periods of time, in 5 seconds to 3 minutes, preferably in 1 minute, and in the temperature range of 30C to 75C, preferably about 55C to 65C.

(3) The hybridized admixture from step (2) above is then maintained for a time period and at a temperature sufficient for a DNA polymerase primer extension reaction to occur to produce primer extension products as is well known. Conditions for conducting a primer extension reactions are well known. In a PCR format the maintenance is carried out quickly to conveniently facilitate numerous cycles, in about 1 second to 5 minutes, preferably about 2 minutes, and at about 55C to 75C, preferably about 65C. Conducting several cycles of PCR results in the formation of amplified nucleic acid products. The PCR is typically conducted with at least 15 cycles, and preferably with about 20 to 35 cycles.

For general methods and conditions for performing a polymerase chain reaction see U.S. Pat. Nos. 4,683,202 and 4,683,195.

In practicing the detection of a DNA duplex it is preferred that the duplex is an RNA- DNA duplex formed between P. carinii ribosomal RNA (rRNA) present in the vascular fluid sample and a polynucleotide. In that case, the detection of an amplified nucleic acid product is preceded by the amplification of the RNA-DNA duplex.

Amplification of an RNA-DNA duplex involves first the preparation of a complementary DNA (cDNA) molecule from the RNA-DNA duplex and then the subsequent amplification of the cDNA molecule to form a large number of copies of cDNA. The preparation of cDNA from an RNA- DNA duplex is also well known. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982. cDNA preparation from an RNA molecule having a DNA polynucleotide probe (e.g., a primer) hybridized thereto typically involves admixing the RNA-DNA duplex with the deoxynucleotides dGTP, dATP, dTTP and dCTP in the presence of magnesium salts and an aqueous buffer, and maintaining the admixture in the presence of a reverse transcriptase (RT) enzyme, preferably avian myeloblastosis virus RT (AMV-RT) , at a temperature and for a time period sufficient to form at least one cDNA copy of the RNA molecule present in the duplex. Preferred cDNA preparation conditions are described in Example 1.

Detecting the presence of a DNA duplex in a process of the present invention can be accomplished by a variety of means. In one approach for detecting the presence of a DNA duplex, a polynucleotide probe that is hybridized in the DNA duplex includes a label or indicating group that will render the duplex detectable. Typically such labels include radioactive atoms, chemically modified nucleotide bases, and the like.

Radioactive elements operatively linked to or present as part of a polynucleotide probe provide a useful means to facilitate the detection of a DNA duplex. A typical radioactive element is one that produces beta ray emissions. Elements that emit beta rays, such as 3H, 14C, 32P and 35S represent a class of beta ray emission- producing radioactive element labels. A radioactive polynucleotide probe is typically prepared by enzymatic incorporation of radioactively labeled nucleotides into a nucleic acid using DNA polymerase, and then the labeled nucleic acid is denatured to form a radiolabeled polynucleotide probe.

Alternatives to radioactively labeled polynucleotides are polynucleotides that are chemically modified to contain metal complexing agents, biotin-containing groups, fluorescent compounds, and the like.

One useful metal complexing agent is a lanthanide chelate formed by a lanthanide and an aromatic beta-diketone, the lanthanide being bound to the nucleic acid or polynucleotide via a chelate forming compound such as an EDTA-analogue so that a fluorescent lanthanide complex is formed. See U.S. Patent No. 4,374,120, No. 4,569,790 and published Patent Application Nos. EP0139675 and WO87/02708.

Biotin or acridine ester-labeled oligonucleotides and their use to label polynucleotides have been described. See U.S. Patent No. 4,707,404, published Patent Application EP0212951 and European Patent No. 0087636. Useful fluorescent marker compounds include fluorescein, rhodamine, Texas Red, NBD and the like.

A labeled polynucleotide present in a DNA duplex renders the duplex itself labeled and therefore distinguishable over other nucleic acids present in a sample to be assayed. Detecting the presence of the label in the duplex and thereby the presence of the duplex, typically involves separating the DNA duplex from any labeled polynucleotide probe that is not hybridized to a DNA duplex. Techniques for the separation of single stranded polynucleotide, such as non- hybridized labeled polynucleotide probe, from DNA duplex are well known, and typically involve the separation of single stranded from double stranded nucleic acids on the basis of their chemical properties. More often separation techniques involve the use of a heterogeneous hybridization format in which the non-hybridized probe is separated, typically by washing, from the DNA duplex that is bound to an insoluble matrix. Exemplary is the Southern blot technique, in which the matrix is a nitrocellulose sheet and the label is 32P. Southern, J. Mol. Biol.. 98:503 (1975). In another approach for detecting the presence of a DNA duplex, and the one used herein as exemplary of a preferred embodiment, the DNA duplex is amplified as described herein and the resulting amplified nucleic acid product is detected. The presence of amplified product indicates the presence of P. carinii infection.

Detection of amplified nucleic acid product can be accomplished by any of a variety of well known techniques. In a preferred embodiment, the amplified product is separated on the basis of molecular weight by gel electrophoresis, and the separated products are than visualized by the use of nucleic acid specific stains which allow one to observe the discreet species of resolved amplified product present in the gel. The presence of a particular amplified product, that is one having a specific molecular weight when resolved by gel electrophoresis, indicates that the polynucleotides were present in the DNA duplex and acted as primers in the PCR reaction to produce multiple copies of a nucleic acid having a discreet nucleotide sequence that includes the target nucleotide sequence. Although numerous nucleic acid specific stains exist and would be suitable to visualize the electrophoretically separated nucleic acids, ethidium bromide is preferred and used as exemplary herein.

Alternative methods suitable to detect the amplified nucleic acid product include hybridization- based detection means that use a labeled polynucleotide probe capable of hybridizing to the amplified product. Exemplary of such detection means include the Southern blot analysis, described above, ribonuclease protection analysis using in vitro labeled polyribonuσleotide probes, and the like methods for detecting nucleic acids having specific nucleotide sequences. See, for example, Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, 1987.

5. Probes, Compositions and Kits In another embodiment the present invention contemplates specific polynucleotide probes, and compositions and diagnostic kits containing those polynucleotide probes. A contemplated polynucleotide probe is less than about 100 nucleotides in length, preferably less than about 50 and more preferably is in the range of about 20 to about 30 nucleotides in legnth. Preferably, the entire nucleotide sequence of a subject polynucleotide probe is identical to the P. carinii RNA sequence reported in Figure 1 of Edman et al., Nature, 334:519-522 (1988).

A polynucleotide probe of the present invention is further characterized as including one of the sequences shown in Table 1.

A composition containing, in about equal molar amounts, a plurality of the subject polynucleotide probes that do not substantially hybridize with each other is also contemplated. Preferably the probes present are of about equal length. Preferably the composition contains two probes, the first including the sequence of PN03 and the second including the sequence of PN40. A diagnostic system, in kit form, for assay for P. carinii in a vascular fluid sample, containing a subject probe and/or composition is also contemplated. The probe or composition is present in an amount sufficent to perform at least one assay.

Many of the compounds and groups involved in the instant specification (e.g., nucleic acids) have a number of forms, particularly variably protonated forms, in equilibrium with each other. As the skilled will understand, representation herein of one form of a compound or group is intended to include all forms thereof that are in equilibrium with each other.

In the present specification, "uM" means micromolar, "ul" means microliter, and "ug" means microgram.

Examples

The following examples are given for illustrative purposes only and do not in any way limit the scope of the invention.

1. Polynucleotide Synthesis Polynucleotides PN03 and PN40, having the deoxyribonucleotide sequences shown in Table 1 were prepared by chemical synthesis by Synthetic Genetics Inc. (San Diego, CA)

After chemical synthesis, each polynucleotide was purified by polyaer 1amide gel electrophoresis on 15% gels, deprotected in ammonium hydroxide for 16 hours at 65C, and then stored until used at -70C at a concentration of about 250 ug/ l of water.

2. Preparation of P. carinii cyst antigen.

Male Sprague Dawley rats that were latently infected with P. carinii were immunosuppressed to develop P. carinii pneumonia. The immunosuppression was achieved by the following regimen of medication added to their drinking water: 2 mg/liter prednisolone (9-alpha-flouro-16- alpha-methyl-prednisolone) for 18 days, no prednisolone for 10 days, and 1-1.5 mg/liter prednisolone for 2.5 months. Tetracycline-HCl was also added at a concentration of 500 mg/liter to the drinking water intermittently to prevent deaths from bacterial infections. Six to eight weeks after immunosuppression the pneumonic rats were sacrificed and the P. carinii infected lung tissues were removed. Touch imprints made from the lungs of each animal were examined for the presence of P. carinii cysts by staining with toluidine blue 0 according to the methods of Chalvavdjian and Grawe, J. Clin. Pathol. , 16:383, 1963. Lungs infected with P. carinii were homogenized in a Waring blender. The homogenate was passed through cotton gauze to remove any large clumps of tissue, and then passed through a 12MM filter (Nuclepore, Pleasanton, CA) . The filtered homogenate was centrifuged at 400 g for 30 minutes at room temperature. The resulting supernatant was discarded and the pellet was resuspended in about 5 volumes of phosphate buffered saline (PBS, pH 7.2) supplemented with 200 U of penicillin/ml, 200 mg of streptomycin/ml (GIBCO Laboratories, Grand Island, NY) and 4 mg of amphotericin B/ml to inhibit any bacterial and fungal growth. The number of P. carinii cysts present was determined according to Pifer et al., Pediatr. Res. 11:305-316, 1977. The isolated P. carinii cysts were stored in small aliquotes at -70°C.

• Detection of P. carinii Specific Nucleic Acids in the Blood of a P. carinii Infected Rat The following samples were each separately admixed with 100 ul acetone to begin preparation of a denatured sample: (i) 5 ul blood from a rat having P. carinii infected lungs, (ii) 5 ul blood from a normal (negative control) rat, (iii) 1 ul isolated P. carinii rRNA containing 0.224 ug rRNA (positive control) , (iv) 1 ul sterile water (negative control) , and (v) 1 ul of a suspension of isolated P. carinii cysts. The admixtures were then maintained at 100C for 5 minutes, further admixed with 86 ul of sterile water and maintained at 100C for 10 min to form denatured samples. The denatured samples were centrifuged at

12,000 x g in a microfuge to pellet any debris present. Each clarified sample was then admixed with 10 ul of 10X TAQ buffer (100 mM Tris-HCl, pH 8.5, 500 mM KC1, 25 mM MgC12, 1.8 mM each of dGTP, dATP, dTTP and dCTP, and 100 ug/ml BSA) , 2 ul of a solution containing 200 picomoles (pmol) of polynucleotide PN03 and 2 ul of a solution containing 200 pmol of polynucleotide PN40, each oligonucleotide having been prepared in Example 1. The admixture was first maintained at 100C for 2 in, then maintained at RT in a water bath for 1 min to form a TAQ buffer mixture.

One ul of a solution containing 27.5 U of avian myeloblastosis virus reverse transcriptase (AMV-RT) was admixed with the TAQ buffer admixture and further maintained at 45C for 2 min to form a cDNA copy of any RNA present in the TAQ buffer admixture. Two ul of a solution containing 5 U of Thermus aquaticus (TAQ) DNA polymerase were admixed with the cDNA containing admixture to form a first polymerase chain reaction (PCR) admixture. The resulting PCR admixture was subjected to numerous cycles of PCR as described below to form amplified nucleic acid product. One cycle of PCR consists of maintaining the admixture first at 95C for 1 min, then at 30C for 1 min, and finally at 65C for 2 min. After the admixture was subjected to 12 PCR cycles, 8 ul of cycled admixture was removed and retained, 1 ul of TAQ DNA polymerase was admixed with the remaining PCR admixture and the admixture was subjected to 8 more PCR cycles. Eight ul of cycled admixture was removed and retained, and the retained samples after 12 cycles or 20 cycles were both analyzed by agarose gel electrophoresis to detect the PCR amplified DNA duplex nucleic acid molecules contained in the retained samples.

The PCR treated and retained samples were each admixed with 2 ul of agarose gel loading buffer and the admixture was electrophoresed for 1 hr at 120 V on a 4% Nu-Seive agarose gel containing 0.5 ug/ul ethidium bromide (EtBr) in 1 x TBE buffer. TBE buffer was prepared by diluting 10 x TBE buffer to 1 X using water. EDTA buffer was prepared by admixing 2 gm NaOH and 18.6 gm disodium EDTA into 100 ml water. 10 x TBE was prepared by admixing 108 gm Tris-base, 55 gm powdered boric acid, 40 ml EDTA buffer, and EtBr to a final concentration of 0.5 ug/ml, all in a volume of 1 liter water. Loading buffer containing 0.1% bromophenol blue dye, 33% sucrose, 0.5 ug/ml ETBr and 1 x TBE. After gel electrophoresis, the electrophoresed DNA duplexes were visualized by exposing the gel to a U.V. light source and photographing the illuminated nucleic acids. Results obtained by the above process show detection of an illuminated nucleic acid band of approximately 220 nucleotide base pairs (bp) in apparent molecular weight. The 220 bp band was observed in samples processed after 20 PCR cycles from P. carinii infected rats, from isolated P. carinii rRNA, or from isolated P. carinii cysts. Low levels of the 220 bp band were also observed in samples processed after 20 PCR cycles from control normal rats, but the observed faint band is believed to reflect the process's sensitivity at detecting low amounts of P. carinii indigenous in normal rats. Low levels of the 200 bp band were observed in samples processed from the negative control of water, but that result is believed to result during electrophoresis by cross contamination from the adjacent gel lanes that contained isolated P. carinii rRNA as positive controls.

A molecular weight of 220 bp for the illuminated band is in agreement with the approximate size predicted of an amplified nucleic acid product produced using polynucleotides PN03 and PN40 by PCR. Therefore, the visualization of the 220 bp band represents the detection of P^_ carinii specific amplified nucleic acid products, and therefore represents the detection of a P. carinii infection in the rats.

Polynucleotides PN20 and PR02, prepared as described in Example 1, were also used in the above described detection process in place of the polynucleotides PN40 and PN03. Results obtained by conducting the detection process with PN20 and PR02 on the same samples as before produced an amplified nucleic acid product having a molecular weight of about 59 bp. The molecular weight of about 59 bp is in agreement with the size predicted of an amplified nucleic acid product produced by PCR using the polynucleotides PN20 and PR02. The 59 bp amplified product was observed when the process was conducted on samples of blood from P. carinii infected rats, isolated P. carinii isolated rRNA, or isolated P. carinii cysts, but was not observed when the control samples were processed. Therefore, the visualization of the 59 bp amplified product represents detection of P. carinii infection when using the PN20 and PR02 polynucleotide probes.

4. Detection of P. carinii Specific Nucleic Acids in the Blood of a P. carinii Infected Human Serum samples were obtained from human patients clinically diagnosed as having either HIV positive serum and P. carinii pneumonia or HIV negative serum and no pneumonia. The serum samples were then treated as described in Example 3 with the following exceptions as noted. After the denatured samples were admixed with 10 x TAQ buffer and polynucleotides, 1 ul of TAQ DNA polymerase was further admixed and the resulting admixture was subjected to 35 cycles of PCR. Thereafter, 8 ul of cycled admixture was removed and analyzed by agarose gel electrophoresis as before.

Results obtained by the above process show the detection of the same 220 bp band as that described in Example 3. The HIV positive patient antisera obtained from patients having known P. carinii pneumonia and the isolated P. carinii cyst sample both exhibited a 220 bp band after being subjected to the above detection process. Neither a HIV negative-P. carinii negative patient's antisera, nor a negative control of water exhibited any detectable 220 bp band, showing the specificity of the contemplated process. The above detection of amplified P. carinii specific nucleic acid products was conducted without the use of reverse transcriptase (RT) in contrast to the procedure used in Example 3. Detection of amplified nucleic acid products without using RT indicate that P. carinii infected patient's antisera contains DNA-DNA duplexes that are detectable by the described procedure. Therefore visualization of a P. carinii specific amplified nucleic acid product, and therefore detection of P. carinii infection in humans, can be carried out following the teachings of the above process.

The foregoing description and the examples illustrate the present invention, but are not intended to limit the scope of the invention. Those skilled in the art will recognize modifications and variations of the exemplified embodiments that are within the spirit and scope of the invention described and claimed herein.

Claims

What is Claimed is:
1. A process for detecting the presence of a Pneumocystis carinii infection in a mammal which process comprises: (a) admixing a denatured vascular fluid sample from said mammal with a Pneumocystis carinii polynucleotide probe capable of hybridizing with Pneumocystis carinii genomic DNA or a RNA transcript thereof, (b) maintaining said admixture under hybridizing conditions for a time period sufficient for said probe to hybridize to any complementary nucleic acid sequence present in said sample to form a hybridized duplex, and (c) detecting the presence of any duplex formed in step (b) and thereby the presence of Pneumocystis carinii infection in said mammal.
2. The process of claim 1 wherein said polynucleotide probe has a sequence that is complementary to a Pneumocystis carinii specific portion of the nucleotide sequence of Pneumocystis carinii ribosomal RNA.
3. The process of claim 1 wherein said step (c) comprises detecting an amplified nucleic acid product.
4. The process of claim 3 wherein first and second polynucleotide probes are admixed in step (a) having nucleic acid sequences represented by the formulas: CAG AGC CAG CAA GTT CAT TT, and
AAT AAC CCA TCA CCA GTC CGA, respectively.
5. The process of claim 3 wherein first and second polynucleotide probes are admixed in step (a) having nucleic acid sequences represented by the formulas:
CAG AGC CAG CAA GTT CAT TT, and AAT AAC CCA TCA CCA GTC CGA, respectively.
6. The process of claim 1 wherein said mammal is a human.
7. A method for detecting the presence of a Pneumocystis carinii infection in a patient, which method comprises: (a) deproteinating double stranded nucleic acids from a vascular fluid sample from said patient;
(b) separating the strands of said deproteinated nucleic acids to form complementary single stranded templates;
(c) treating said complementary single stranded templates, under condition suitable for polymerase chain reaction amplification, with first and second polynucleotides selected so as to be capable of amplifying a Pneumocystis carinii- specific nucleic acid; and
(d) detecting the presence of any Pneumocystis carinii-specific nucleic acid amplification product formed in step (c) , and thereby the presence of a Pneumocystis carinii infection in said patient.
8. The process of claim 7 wherein first and second polynucleotides are admixed in step (c) having nucleic acid sequences represented by the formulas:
CAG AGC CAG CAA GTT CAT TT, and AAT AAC CCA TCA CCA GTC CGA, respectively.
9. The process of claim 7 wherein first and second polynucleotides are admixed in step (c) having nucleic acid sequences represented by the formulas:
ATT TAG ATA CCT TA, and TTA CCG CGG CTG GCA C.
10. A polynucleotide of less than about 100 nucleotides in length and including a sequence represented by the formula:
CAG AGC CAG CAA GTT CAT TT, or AAT AAC CCA TCA CCA GTC CGA.
11. The polynucleotide of claim 10 consisting essentially of the sequence
CAG AGC CAG CAA GTT CAT TT, or AAT AAC CCA TCA CCA GTC CGA.
12. A composition comprising, in about equal molar amounts, first and second polynucleotides that do not hybridize with each other and each having less than about 100 nucleotides, said first polynucleotide including the nucleotide sequence:
CAG AGC CAG CAA GTT CAT TT, and said second polynucleotide including the nucleotide sequence:
ATT AAC CCA TCA CCA GTC CGA.
13. The composition of claim 12 wherein said first and second polynucleotides are represented by the formulas, respectively:
CAG AGC CAG CAA GTT CAT TT, and ATT AAC CCA TCA CCA GTC CGA.
PCT/US1990/002633 1989-05-11 1990-05-11 Nucleic acid based diagnostic system and process for the detection of pneumocystis carinii in blood products WO1990013669A1 (en)

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WO1991002092A1 (en) * 1989-08-11 1991-02-21 Gene-Trak Systems NUCLEIC ACID PROBES FOR THE DETECTION OF $i(PNEUMOCYSTIS CARINII)
WO1991019005A1 (en) * 1990-06-01 1991-12-12 Isis Innovation Limited Dna for diagnosing pneumocystis carinii
US5593836A (en) * 1993-05-14 1997-01-14 Niemiec; John T. Primers and probes for detecting Pneumocystis carinii
US5958693A (en) * 1995-01-13 1999-09-28 Bayer Corporation Extraction of DNA by boiling cells in an alkaline phenol/guanidine thiocyanate solution
US6180339B1 (en) 1995-01-13 2001-01-30 Bayer Corporation Nucleic acid probes for the detection and identification of fungi

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GB8802667D0 (en) * 1988-02-05 1988-03-02 Chancellor Masters & Scholars Probe for pneumoxystis carinii
AT143700T (en) * 1991-09-23 1996-10-15 Pfizer A method for the detection of specific mRNA and DNA in cells

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991002092A1 (en) * 1989-08-11 1991-02-21 Gene-Trak Systems NUCLEIC ACID PROBES FOR THE DETECTION OF $i(PNEUMOCYSTIS CARINII)
WO1991019005A1 (en) * 1990-06-01 1991-12-12 Isis Innovation Limited Dna for diagnosing pneumocystis carinii
US5593836A (en) * 1993-05-14 1997-01-14 Niemiec; John T. Primers and probes for detecting Pneumocystis carinii
US5958693A (en) * 1995-01-13 1999-09-28 Bayer Corporation Extraction of DNA by boiling cells in an alkaline phenol/guanidine thiocyanate solution
US6180339B1 (en) 1995-01-13 2001-01-30 Bayer Corporation Nucleic acid probes for the detection and identification of fungi

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JPH04505257A (en) 1992-09-17
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EP0471789A4 (en) 1992-04-22
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