WO2003025227A1 - Detection of rpob sequences of mycobacterium tuberculosis - Google Patents
Detection of rpob sequences of mycobacterium tuberculosis Download PDFInfo
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- WO2003025227A1 WO2003025227A1 PCT/US2001/029361 US0129361W WO03025227A1 WO 2003025227 A1 WO2003025227 A1 WO 2003025227A1 US 0129361 W US0129361 W US 0129361W WO 03025227 A1 WO03025227 A1 WO 03025227A1
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- 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
Definitions
- This invention relates to in vitro diagnostic detection of pathogenic bacteria, and specifically relates to compositions and assays for detecting nucleic acid sequences associated with rifampin resistance of Mycobacterium Tuberculosis by using in vitro nucleic acid amplification of the rpoB gene and detection of amplified products.
- Rifampin an antibiotic synthesized from rifamycin B, is a key component of drug therapy against Mycobacterium tuberculosis.
- Rifampin has a unique site of action on the beta subunit of prokaryotic RNA polymerase. In Escherichia coll, missense mutations and short deletions in the central region of the RNA polymerase subunit gene ⁇ rpoB) result in strains resistant to rifampin (Lisityn et al., 1984, Mol.Gen.Genet. 196 : 173-174).
- tuberculosis a wide variety of mutations in the rpoB gene have been identified that confer rifampin resistance (Telenti et al., 1993, Lancet 341 : 647-650). More than 90% of rifampin- resistant M. tuberculosis isolates are also resistant to isoniazid, and, therefore, rifampin resistance is a valuable surrogate marker for multiple drug resistance. Thus, there is a need for tests that can detect rapidly the genetic basis for rifampin resistance for diagnosis that leads to appropriate treatment of infected individuals.
- PCR polymerase chain reaction
- Patent Nos. 5,643,723 Persing et al.
- 5,851 ,763 Heym et al.
- 6,228,575 Gafas et al.
- 6,242,584 Korean et al.
- the present invention provides compositions and simple diagnostic methods to detect rifampin resistance in M. tuberculosis that may be present in a clinical sample. Summary of the Invention
- a method of detecting rpoB sequences of Mycobacterium tuberculosis present in a biological sample includes the steps of providing a biological sample containing nucleic acid from M. tuberculosis comprising a rpoB DNA sequence; amplifying the M. tuberculosis rpoB DNA sequence in an in vitro nucleic acid amplification mixture comprising at least one polymerase activity, and at least two primers having sequences selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 3, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:11 to produce amplified M.
- the method also includes the steps of adding to the biological sample at least one capture oligonucleotide that specifically hybridizes to a M. tuberculosis DNA sequence and an immobilized nucleic acid that hybridizes to the capture oligonucleotide under hybridizing conditions to produce a hybridization complex comprising the M. tuberculosis DNA sequence, the capture oligonucleotide and the immobilized nucleic acid; and separating the hybridization complex from other components of the biological sample before the amplifying step.
- the detecting step uses at least one detection probe that hybridizes specifically to the amplified M. tuberculosis nucleic acid. In one embodiment, the detecting step uses at least one labeled detection probe that hybridizes specifically to the amplified M. tuberculosis nucleic acid, whereas another embodiment uses a plurality of detection probes that hybridize specifically to the amplified M. tuberculosis nucleic acid.
- the amplifying step uses a combination of at least a first primer and a second primer wherein the first primer has the sequence of SEQ ID NO:2 and the second primer has the sequence of SEQ ID NO:3; the first primer has the sequence of SEQ ID NO:2 and the second primer has the sequence of SEQ ID NO:8; or the first primer has the sequence of SEQ ID NO:10 and the second primer has the sequence of SEQ ID NO:11.
- Another aspect of the invention provides a composition for detecting a rpoB sequence of M. tuberculosis, comprising one or more oligonucleotides having a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 6 and SEQ ID NO:8 to SEQ ID NO:12.
- the composition comprises at least one first oligonucleotide containing the sequence of any one of SEQ ID NO:1 to SEQ ID NO:3 and SEQ ID NO:8, and at least one second oligonucleotide containing the sequence of any one of SEQ ID NO:4 to SEQ ID NO:6 and SEQ ID NO:9.
- the composition comprises at least one first oligonucleotide containing the sequence of any one of SEQ ID NO:1 to SEQ ID NO:3 and SEQ ID NO:8, at least one second oligonucleotide containing the sequence of SEQ ID NO:4, and at least one third oligonucleotide containing the sequence of any one of SEQ ID N0:5, SEQ ID N0:6 and SEQ ID N0:9.
- One embodiment of the composition comprises at least one first oligonucleotide containing the sequence of any one of SEQ ID NO:10 and SEQ ID N0:11 , and at least one second oligonucleotide containing the sequence of SEQ ID N0:12.
- Another embodiment of the invention is a kit containing any of the aforementioned oligonucleotides and combinations of oligonucleotides.
- the present invention includes methods of detecting rpoB sequences for Mycobacterium tuberculosis present in biological samples derived from humans, preferably in processed sputum samples.
- the present invention also includes compositions that include nucleic acid capture oligomers that specifically hybridize to M. tuberculosis sequences present in a biological sample, thereby providing a means for capturing the target sequence from the sample components, nucleic acid amplification oligomers (or primers) that specifically amplify selected portions of the rpoB DNA sequences, and nucleic acid probe oligomers (or detection probes) for detecting such amplified sequences.
- the nucleic acid sequences of this invention are useful for capturing, amplifying and detecting mutations of rpoB gene for M. tuberculosis present in a biological sample.
- the methods of the present invention are important for diagnosis of a drug resistance phenotype of M. tuberculosis by giving the clinician information useful in determining appropriate treatment of the M. tuberculosis infected patient.
- biological sample is meant any tissue or material derived from a living or dead human which may contain M. tuberculosis nucleic acid.
- Samples include, for example, sputum, respiratory tissue or exudates, peripheral blood, plasma or serum, cervical swab samples, biopsy tissue, gastrointestinal tissue, urine, feces, semen or other body fluids, tissues or materials. Samples also include bacterial cultures (from liquid or solid media) and environmental samples. A biological sample may be treated to physically disrupt tissue or cell structure, thus releasing intracellular components into a solution which may contain enzymes, buffers, salts, detergents and the like which are used to prepare the sample for analysis.
- nucleic acid is meant a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases, or base analogs, where the nucleosides are covalently linked via a backbone structure to form a polynucleotide.
- a nucleic acid backbone may comprise a variety of known linkages known, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds ("peptide nucleic acids"; PCT No.
- WO 95/32305 (Hydig-Hielsen ef al.)), phosphorothioate linkages, methylphosphonate linkages or combinations of known linkages.
- Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2 1 methoxy and/or 2' halide substitutions.
- Nitrogenous bases may be conventional bases (A, G, C, T, U), known base analogs ⁇ e.g., inosine; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11 th ed., 1992), or known derivatives of purine or pyrimidine bases (PCT No. WO
- a nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions ⁇ e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more analogs).
- oligonucleotide or "oligomer” is meant a nucleic acid having generally less than
- Oligomers may be in a size range having a lower limit of about 5 to about 15 residues and an upper limit of about 50 to 600 residues; and preferably, in a size range having a lower limit of about 10 residues and an upper limit of about 100 residues. Oligomers can be purified from natural sources, but generally are synthesized in vitro using well-known methods.
- amplification oligonucleotide or “amplification oligomer” is meant an oligonucleotide or oligomer that hybridizes to a target nucleic acid, or its complement, and participates in an in vitro nucleic acid amplification reaction. These may be called “primers” because they initiate polymerization from a template by enzymatic activity that adds nucleotide monomers at their 3' ends.
- An amplification oligonucleotide generally contains at least 10 to 12 contiguous bases that are complementary to a region of the target nucleic acid sequence (or its complementary strand).
- the contiguous bases are preferably at least 80%, more preferably at least 90% complementary to the sequence to which the amplification oligonucleotide binds.
- An amplification oligonucleotide is preferably about 10 to about 60 bases long and may include modified nucleotides, base analogs or additional functional sequences, such as a 5' promoter sequence recognized by an RNA polymerase (such amplification oligonucleotides may be called "promoter primers").
- any oligomer that can function as a primer can be modified to include a 5' promoter sequence, and thus function as a promoter primer.
- any promoter primer can serve as a primer, independent of its promoter sequence.
- amplification is meant an in vitro procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or fragments thereof.
- In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement.
- Known amplification methods include, for example, transcription-mediated amplification (TMA), replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification and strand- displacement amplification (SDA).
- TMA transcription-mediated amplification
- PCR polymerase chain reaction
- LCR ligase chain reaction
- SDA strand- displacement amplification
- Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as Q Beta-replicase (U.S. Pat. Nos.
- PCR amplification uses thermal cycling with a DNA polymerase and, usually, two or more primers to synthesize multiple copies of two complementary DNA strands (Mullis et al., 1987, Methods in Enzymology 155: 335-350; U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 (Mullis et al.)).
- LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (EP Pat. No.
- SDA uses a primer that contains a recognition site for a restriction endonuclease which will nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by a series of primer extension and strand displacement steps to amplify DNA (U.S. Pat. No. 5,422,252 (Walker et al.)).
- Transcription-mediated amplification (TMA) is used in preferred embodiments of the present invention.
- TMA Transcription-mediated amplification
- transcription-mediated amplification or “transcription-associated amplification” is meant nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template.
- TMA generally uses an RNA polymerase activity, a DNA polymerase activity, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter primer and a second primer, and optionally may include one or more additional oligonucleotides (sometimes referred to as "helper” or “displacer” oligonucleotides).
- helper or "displacer” oligonucleotides
- WO 94/03472 (McDonough et al.); and PCT No. WO 95/03430 (Ryder et al.)).
- Preferred TMA methods have been disclosed in U.S. Pat. Nos. 5,399,491 , 5,554,516 and 5,786,183, and PCT No. WO 93/22461.
- probe is meant a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid or its complement, preferably in an amplified nucleic acid, under conditions that promote hybridization, thereby allowing detection of the target or amplified nucleic acid.
- Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target sequence or amplified nucleic acid) or indirect ⁇ i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target sequence or amplified nucleic acid).
- a probe's "target” generally refers to a sequence in (i.e., a subset) a larger nucleic acid sequence that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding (i.e., base pairing). Sequences that are "sufficiently complementary" allow stable hybridization of a probe oligomer to a target sequence, even if the two sequences are not completely complementary.
- a probe may be labeled or unlabeled, depending on the detection method used, which methods are well known in the art.
- “sufficiently complementary” is meant a contiguous nucleic acid base sequence that hybridizes to another base sequence by hydrogen bonding between a series of complementary bases under hybridization conditions. Sequences may be complementary at each position in a sequence using standard base pairing (i.e., G:C, A:T or A:U pairing) or may contain one or more residues that are not complementary by standard hydrogen bonding (including abasic residues), but in which the entire base sequence is capable of specifically hybridizing with another base sequence in appropriate hybridization conditions.
- Contiguous bases are preferably at least about 80%, more preferably at least about 90% complementary to a sequence to which an oligomer specifically hybridizes.
- Appropriate hybridization conditions are well known to those skilled in the art, can be predicted readily based on sequence composition and conditions, or can be determined empirically by using routine testing (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at ⁇ 1.90-1.91 , 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly at ⁇ 9.50-9.51 , 11.12-11.13, 11.45-11.47 and 11.55-11.57).
- capture oligonucleotide or “capture oligomer” or “capture probe” is meant at least one nucleic acid oligomer that provides means for specifically joining a target sequence and an immobilized oligomer based on base pair hybridization (U.S. Patent No. 6,110,678 (Weisburg et al.)).
- a capture oligomer includes two binding regions: a target-specific binding region and an immobilized probe-specific binding region.
- immobilized probe or “immobilized oligomer” is meant a nucleic acid that joins, directly or indirectly, a capture oligomer to a solid support.
- An immobilized probe is an oligonucleotide joined to a solid support provides a means for separating a bound target sequence from other sample components.
- Suitable solid supports include matrices and particles in solution, made of any known material (e.g., nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene and metal particles, preferably paramagnetic particles).
- Preferred supports are monodisperse paramagnetic spheres (i.e., uniform in size ⁇ about 5%)), to which an immobilized probe is stably joined directly (e.g., via direct covalent linkage, chelation, or ionic interaction), or indirectly (e.g., via hybridization with one or more linkers), thus permitting hybridization to another nucleic acid in solution.
- separating or “purifying” is meant that one or more components of the biological sample are removed from other sample components.
- Sample components generally are an aqueous solution that includes nucleic acids and other materials (e.g., proteins, carbohydrates, lipids and/or nucleic acids).
- a separating or purifying step removes at least about 70%, preferably at least about 90%, and more preferably at least about 95% of the other sample components.
- label is meant a molecular moiety or compound that can be detected or can lead to a detectable response.
- a label is joined, directly or indirectly, to a nucleic acid probe or to a nucleic acid to be detected (e.g., an amplified nucleic acid).
- Direct labeling can occur through bonds or interactions that link the label to the probe (e.g., via covalent bonds or non-covalent interactions).
- Indirect labeling can occur through use of a bridging moiety or linker, such as additional oligonucleotide(s), which is either directly or indirectly labeled.
- Bridging moieties can be used to amplify detectable signal.
- Labels can be any known detectable moiety (e.g., radionuclide, ligand, such as biotin or avidin, enzyme, enzyme substrate, reactive group, chromophore, e.g., dye or colored particle, luminescent compound, including bioluminescent, phosphorescent, chemiluminescent and fluorescent compounds).
- the label on a labeled probe is detectable in a homogeneous assay system (i.e., in a mixture, bound labeled probe exhibits a detectable change compared to unbound labeled probe).
- chemiluminsecent labels and their use in homogenous detection assays have been described in detail (U.S. Pat. Nos. 5,283,174 (Arnold Jr., et al.), 5,656,207 (Woodhead et al.), 5,658,737 (Nelson et al.) and 5,639,604 (Arnold Jr., et al.)).
- Such labels include acridinium ester ("AE") compounds, e.g., standard AE or its derivatives.
- AE acridinium ester
- a homogeneous detectable label has the advantage of being detectable without physically separating hybridized from unhybridized label or labeled probe.
- DNA probe array is meant a solid support on which are immobilized at least 2, and preferably 10 or more, different capture oligonucleotide.
- DNA probe arrays are well known in the art (Ramsay, 1998, Nature BiotechAQ : 40-44; Cheng et al., 1996, Molec. diagnosis 1 (3) :183-200; Livache et al., 1994, Nucl. Acids Res. 22(15) :2915-2921; Cheng et al., 1998, Nature Biotech. 16 :541-546 ; U.S. Pat. Nos. 4,981,783 (Augenlicht), 5,700,637 (Southern), 5,445,934 and 5,744,305 (Fodor), and 5,807,522 (Brown)).
- compositions, kits or methods of the present invention include additional component(s), composition (s) or method step(s) that do not materially change the basic and novel characteristics of the invention. Such characteristics include the ability to detect rpoB sequences of M. tuberculosis in a biological sample at about 20 to 200 or more copies per sample. Any component, composition, or method step that has a material effect on the invention's basic characteristics would fall outside of this term.
- rpoB sequences from different Mycobacterium isolates including known mutations, available from publicly accessible databases (e.g., GenBank) were aligned by matching regions of the same or similar sequences and compared using well known molecular biology techniques.
- GenBank publicly accessible databases
- use of algorithms may facilitate sequence comparisons, those skilled in the art can readily perform such comparisons manually and visually.
- portions of sequences containing relatively few variants between the compared sequences were chosen as a basis for designing synthetic oligomers suitable for use in capture and amplification of . tuberculosis nucleic acid sequences in the rpoB region and detection of amplified sequences.
- oligomers included the relative GC content which affects T m of the sequence and the relative absence of predicted secondary structure within a sequence, all well known in the art. Based on these analyses, the oligomers having sequences of SEQ ID NO:1 to SEQ ID NO:6 and SEQ ID NO:8 to SEQ ID NO:12 were designed and synthesized.
- Target capture may be included to increase the concentration or purity of the target nucleic acid before in vitro amplification.
- target capture involves a relatively simple method of hybridizing and isolating the target nucleic acid, as described in detail elsewhere (U.S. Pat. No. 6,110,678 and PCT No. WO 98/50583 (Weisburg et al.)).
- a sample that potentially contains M. tuberculosis DNA is contacted with a capture oligomer containing a sequence specific for hybridization to M. tuberculosis DNA, and an immobilized oligonucleotide attached to a solid support that can hybridize to another portion of the capture oligomer under appropriate hybridization conditions.
- the hybridization complex attached to the solid support is separated from other sample components. Then, the M. tuberculosis target nucleic acid linked to the solid support is washed and rpoB sequences are amplified in an in vitro amplification reaction.
- the capture oligomer sequence includes a 5' target-binding sequence that binds specifically to the M. tuberculosis DNA target sequence, and a 3' tail sequence (e.g., poly-dA) that binds to the complementary immobilized sequence (e.g., poly-dT) on the solid support.
- a capture oligomer may use any backbone to link the base sequence, including standard deoxyribose-phosphate linkages and O-methoxy linkages.
- Preferred capture oligomers have sequences of: GGCCACCATCGAATATCTGGTCCGCTTGCACTTT(A) o (SEQ ID NO:5), CATGTCGCGGATGGAGCGGGTGGTC(A) 30 (SEQ ID NO:6 ), and CATCGAATATCTGGTCCGCTTGCAC(A) 3 o (SEQ ID NOS).
- Amplifying the captured rpoB region can be accomplished using a variety of known nucleic acid amplification reactions, but preferably uses a transcription-mediated amplification (TMA).
- TMA transcription-mediated amplification
- many strands of nucleic acid are produced from a single copy of target nucleic acid, thus permitting detection of the target by specifically binding the amplified rpoB sequences to one or more detecting probes.
- TMA has been described in detail previously (U.S. Pat. Nos. 5,399,491 and 5,554,516 (Kacian et al.)).
- this amplification method uses two types of primers (one being a "promoter primer” that contains a promoter sequence for an RNA polymerase), two enzymes (a reverse transcriptase and an RNA polymerase), substrates (deoxyribonucleoside triphosphates, ribonucleoside triphosphates) and appropriate salts and buffers in solution to produce multiple RNA transcripts from a nucleic acid template.
- a promoter primer hybridizes specifically to its target nucleic acid sequence and reverse transcriptase creates a first strand cDNA by extension from the 3' end of the promoter primer.
- the cDNA becomes available for hybridization with the second primer by enzymatic activity that degrades the complementary strand (e.g., RNase H activity of the reverse transcriptase) or by localized or complete denaturaton of the duplex.
- a second primer binds to the cDNA and a new strand of DNA is synthesized from the 3' end of the second primer using reverse transcriptase to create a double-stranded DNA having a functional promoter sequence at one end.
- RNA polymerase binds to the double-stranded promoter sequence and transcription produces multiple transcripts or "amplicons.” Amplicons are used in further steps in the process, each serving as a template for a new round of replication as described above, thus generating large amounts of single-stranded amplified nucleic acid in a substantially isothermal process. For example, about 100 to about 3,000 copies of RNA transcripts are synthesized from a single template.
- Primer sequences e.g., SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:11
- promoter primers may include a T7 RNA polymerase promoter sequence (SEQ ID NO:7) as a 5' portion of the sequence.
- SEQ ID NO:7 T7 RNA polymerase promoter sequence
- the methods include the steps of providing a biological sample that potentially contains the target M. tuberculosis rpoB gene, target capture of DNA containing an rpoB sequence, in vitro nucleic acid amplification and detection of the amplified nucleic acid products to determine if an rpoB mutation is present in the amplified nucleic acid.
- the amplification mixture includes the captured target DNA, at least one T7 promoter primer that includes a target-specific sequence and a T7 promoter sequence, at least one second primer that hybridizes specifically to a first strand cDNA made from the target using the T7 promoter primer, and substrates and cofactors for enzymatic polymerization by reverse transcriptase and T7 RNA polymerase.
- the captured target does not have to be separated from the solid support for use in the TMA reaction.
- the functional T7 promoter sequence combined with T7 RNA polymerase produces multiple transcripts which can be detected using any of a variety of known methods, including hybridizing specifically the amplified products, or portions thereof, to one or more complementary probe sequences.
- a labeled probe is used to detect the amplified products
- the amplified products are labeled and hybridized to immobilized probes, preferably to an array of many probes.
- the hybridization complex of the probe and amplified product is detected.
- the pattern of hybridization on the array indicates the sequence of the amplified rpoB gene, which provides information on whether an rpoB mutation of M. tuberculosis is present in the sample assayed. Typical assay conditions described below.
- Sample preparation A sample (e.g., 0.5 ml of sputum sediment or bacterial culture) was mixed with an equal volume of a 2X lysis buffer (e.g., 20 mM HEPES, 0.5 % (w/v) lithium lauryl sulfate (LLS), pH 8). To release nucleic acids from the bacteria, the mixture was vortexed in the presence of glass beads, or sonicated for 15 min, and then the mixture was heated at 95° C for 15 min. For positive control reactions, an equal volume of water or buffer containing a known amount of M. tuberculosis genomic DNA (gDNA) was used in place of the sputum sediment or bacterial culture.
- gDNA M. tuberculosis genomic DNA
- the gDNA was prepared using a combination of standard methods that have been described in detail previously. Briefly, cells were grown in broth to late log phase and treated 18 hr with 1 mg/ml ampicillin and 0.1 mg/ml D-Cycloserine (Crawford et al., 1979, Infect. Immun. 24: 979-81). Then, cells were collected and lysed with SDS and treated with Proteinase K to release DNA into an aqueous solution which was extracted twice with sodium perchlorate and a phenol/chloroform mixture, and DNA was spooled out of the solution after adding about two volumes of ethanol (Maniatis et al.,
- Target capture Generally, lysate prepared from a sample was used in the target capture step (U.S. Patent No. 6,110,678 (Weisburg et al.)). To capture the target M. tuberculosis DNA, the mixture included 250 ⁇ l of prepared sample lysate, 250 ⁇ l of a target capture solution containing 3 pmole of SEQ ID NO:5 and 3 pmole of SEQ ID NO:6 or 3 pmol of SEQ ID NO:9, and 40 ⁇ g of paramagnetic particles (0.7-1.05 ⁇ , Seradyn, Indianapolis, IN) with attached immobilized poly-dT ⁇ 4 oligomers (Lund, etal., 1988, Nuc.
- Acids fies.16:10861-10880 The target capture mixture was heated at 60°C for about 20 min and then cooled to room temperature. A magnetic field was applied for 5 min to attract the magnetic particles with the attached complex containing the target DNA to a location on the reaction container (substantially as described in U.S. Pat. No. 4,895,650 (Wang)). Particles with attached hybridization complexes were washed twice with 1 ml of a washing buffer (10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 150 mM NaCI, 0.1% (w/v) sodium lauryl sulfate) by resuspending the particles in the washing buffer and then repeating the magnetic separation step. Amplification.
- a washing buffer (10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 150 mM NaCI, 0.1% (w/v) sodium lauryl sulfate
- Transcription mediated amplification was performed substantially as described previously (U.S. Pat. Nos. 5,399,491 and 5,554,516 (Kacian et al.)). Washed particles from the target capture step were suspended in 75 ⁇ I of amplification reagent solution (0.08 mM rUTP, 1.3 mM rATP, 4 mM rCTP, 6 mM rGTP, 1.3 mM each dNTP, 66 mM Tris, 17.3 mM MgCI 2 ). The relatively low concentration of rUTP is important for amplification efficiency of the rpoB target DNA.
- At least two amplification oligomers were included in the amplification reaction, i.e., at least one promoter primer and a second primer, usually at 0.08 ⁇ M final concentration.
- Amplification oligomers may also include helper or displacer oligomers and may be hybridized to the target before other amplification reagents are added to the mixture.
- the reaction mixture was covered with a layer (200 ⁇ I) of inert oil to prevent evaporation and incubated at 42°C for 5 min.
- enzyme reagent 25 ⁇ l was added (about 1750 U of MMLV reverse transcriptase and 400 U of T7 RNA polymerase per reaction, in a buffer containing 50 mM HEPES, 1 mM EDTA, 10% (v/v) t-octylphenoxypolyethoxyethanol (TRITON TM X-100), 120 mM KCI, 20% (v/v) glycerol).
- MMLV reverse transcriptase incorporates 1 nmol of dTTP in 10 min at 37 °C using a polyA template primed with 200-400 ⁇ M oligo(dT); and one unit of T7 RNA polymerase incorporates 1 nmol of ATP into RNA in 1 hr at 37 °C using a DNA template containing a T7 promoter sequence.
- Negative controls consisted of all of the same reagents but substituting an equal volume of water or buffer that contained no target nucleic acid for the sample. Detection. In some cases, amplified M.
- tuberculosis sequences were detected using an acridinium ester (AE)-labeled probe (e.g., 5'-GTTGTTCTGGTCCATGAA (SEQ ID NO:4)) which was detected by chemiluminescence in a luminometer (e.g., LEADERTM luminometer, Gen-Probe Incorporated, San Diego, CA) and signal is expressed in relative light units (RLU) substantially as described previously (U.S. Pat. No. 5,658,737 (Nelson et al.) at column 25, lines 27-46; Nelson et al., 1996, Biochem. 35: 8429-8438, at 8432). Generally, the average (mean) of detected RLU for replicate assays are reported.
- the labeled detection probe has the base sequence of SEQ ID NO:4 linked by a 2'-0-methoxy backbone.
- the amplified sequences were detected on an immobilized array of DNA probes specific for detection of M. tuberculosis rpoB sequences, as described in detail previously (Troesch et al., 1999, J. Clin. MicrobioL 37: 49-55).
- the amplicons generated by the amplification reaction were labeled with a fluorescent label before hybridization to the array using methods substantially as described in detail elsewhere (PCT Nos. WO 99/65926 and WO 01/44507 (Laayoun et al.)).
- Hybridization of the probe arrays was performed with the GENECHIPTM Fluidics Station (Affymetrix, Santa Clara, CA) substantially as previously described (Troesch et al., 1999, J. Clin. MicrobioL 37: 49-55). An additional step, antibody staining, allows signal amplification as described elsewhere (PCT No. WO 01/44506 (Laayoun et al.)).
- the DNACHIPTM was flushed and a second step of staining was performed using staining solution containing 300 ⁇ I of 2 M MES, 2.4 ⁇ I of bovine serum albumin (BSA), 6 ⁇ I of normal goat IgG, 1.2 ⁇ I of anti-fluorescein antibody, and water (600 ⁇ I final volume).
- staining solution containing 300 ⁇ I of 2 M MES, 2.4 ⁇ I of bovine serum albumin (BSA), 6 ⁇ I of normal goat IgG, 1.2 ⁇ I of anti-fluorescein antibody, and water (600 ⁇ I final volume).
- Anti-fluorescein, rabbit IgG fraction, biotin-XX conjugate, were supplied by Molecular Probes (Eugene, OR); acetylated BSA solution was supplied by GibcoBRL Life Technologies, (Rockville, MD); and goat IgG (Reagent Grade) was supplied by Sigma Chemical, (St. Louis, MO).
- the chip was flushed, washed with a washing buffer containing 6 X SSPE, 0.01% polyoxyethylenesorbitan (TWEENTM 20), and a third hybridization step was performed, using second staining solution of 300 ⁇ I of 2M MES, 6 ⁇ I of BSA and 6 ⁇ I of streptavidin, R- phycoerythrin conjugate, and water (600 ⁇ I final volume). Streptavidin and R-phycoerythrin conjugate were supplied by Molecular Probes (Eugene, OR). After a 10 min hybridization, the chip was flushed and washed using the washing buffer described above.
- the analysis to detect the intensity and pattern of fluorescent signals (expressed as relative fluorescence units or RFU) on the hybridized array was performed on the GENECHIPTM instrumentation system (Affymetrix, Santa Clara, CA).
- This system comprises a GENECHIPTM fluidics station, a GENEARRAYTM scanner (Hewlett-Packard, Palo Alto, CA) and GENECHIPTM analysis software, an algorithm to determine nucleotide base calling and determine the nucleic acid sequence present in the amplified nucleic acid.
- This system generates a report of the rpoB mutations present in the amplified nucleic acid sequences applied to the chip.
- the following examples demonstrate embodiments of the present invention.
- Oligonucleotides This example shows the sensitivity of the amplification oligonucleotides of the present invention when used in a TMA reaction. Primers were designed to amplify M. tuberculosis specifically and not other Mycobacterium species.
- amplification oligonucleotides SEQ ID NO:1 (GACCACCCAGGACGTG) as a helper oligomer
- SEQ ID NO: 2 AATTTAATACGACTCACTATAGGGAGACGATCACACCGCAGACGTTG
- SEQ ID NO: 3 GCTCGCGCTCACGTG
- Target sequences for this assay were purified gDNA extracted from a lysed bacterial culture of M. tuberculosis and provided at 20, 200 or 10 3 copies per in vitro amplification reaction.
- the AE-labeled detection probe of SEQ ID NO:4 (GTTGTTCTGGTCCATGAA) was mixed with unlabeled probe of the same sequence (ratio of labeled probe/unlabeled probe was 1/5000) to provide a signal within the linear range detectable by the LEADERTM luminometer (Gen-Probe Incorporated, San Diego, CA). Signals of 2 x 10 4 or greater RLU were considered positive.
- the RLU results mean of 10 assays for each assay condition) are shown in Table 1.
- the results shown in Table 1 demonstrate that the amplification reaction was sensitive at as few as 20 copies of gDNA, a level more sensitive than the desired sensitivity of a clinical smear positive specimen which usually contains at least 1000 bacteria.
- the rpoB region was amplified similarly but using a combination of amplification oligonucleotides of SEQ ID NO:1 as a helper oligomer, SEQ ID NO: 2 as a promoter primer, and SEQ ID NO:8 (CGGCACGCTCACGTG) as primer.
- the sensitivity of the assay in these experiments, as detected by hybridization with an AE-labeled probe, was at least about 200 copies of target per reaction.
- the target was provided at 800, 500 and 200 copies per reaction (5 reactions for each condition). All reactions gave positive results (7.88 x 10 5 to 2.66 x 10 6 mean RLU) compared to the negative controls with no M. tuberculosis DNA in the reaction (which produced 1.93 x 10 3 mean RLU for two reactions).
- Example 2 Specificity of Amplification This example shows the specificity of the amplification oligomers of the present invention as demonstrated using a TMA reaction performed using amplification oligomers and procedures substantially as described in Example 1 and above.
- the target sequence for this assay was purified Mycobacterium gDNA extracted from bacteria obtained from the American Type Culture Collection ("ATCC", Manassas, VA) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH culture collection ("DSM”, Braunschweig, Germany) and grown in vitro using standard microbiology procedures.
- the target DNA were provided at 200, 10 3 , 10 4 , 10 5 , and 10 6 copies per assay.
- the negative control reaction contained no Mycobacterium DNA (i.e., an equal volume of water was substituted for the sample volume).
- the homogeneous detection assay using a labeled probe as described in Example 1 was used except that undiluted labeled probe was used.
- the average RLU results (four assays per species DNA) obtained from these assays are shown in Table 2. For these, a signal of 5 x 10 4 RLU or greater was considered positive.
- the assay amplified and detected 200 copies of M. tuberculosis DNA in the reactions.
- the results were negative even when much more target DNA was used (10 3 to 10 5 copies per reaction).
- the specificity for M. tuberculosis using these amplification oligomers and probe was demonstrated by the minimal detectable signal obtained for the other Mycobacterium species even when more DNA was provided.
- the amplicons were also detected on a DNA probe array.
- Amplicons are chemically fragmented and fluorescentiy labeled using procedures substantially as previously described (PCT No. WO 01/44507 (Laayoun et al.)).
- the labeled fragments were detected on the DNA probe array using the GENECHIPTM System for detecting M. tuberculosis sequences as described above.
- Table 3 present the percentage of correct base calling that is used to identify a predetermined sequence diagnostic of M. tuberculosis. That is, for amplicons obtained from sample DNA from each of the Mycobacterium species listed in Table 3, the relative amount of correct base calling on the M. fabercu os/s-specific DNA probe chip is shown, with the average signal intensity for the detected signal (mean relative fluorescence units or RFU). A result of greater than 85% base calling is considered positive identification of the M. tuberculosis sequence.
- Example 3 Detection of Mutant Clones This example shows the detection of rpoB sequences after target capture, amplification and detection. Detection was done by using labeled probe binding in a homogeneous detection assay and by binding labeled amplicons to a DNA probe array, substantially as described in Example 2. For detection, the same amplification reaction for each clone was divided into two parts.
- the bacterial rpoB clones to be detected were generated from cloned rpoB sequences contained in a fragment of about 700 bp which was amplifed by the PCR and ligated into a plasmid vector (pGEMTM-T EASY, Promega, Madison, Wl) using the methods described by Troesch et al. (J. Clin. MicrobioL, 1999, 37: 49-55).
- the insert sequence was characterized by DNA sequencing.
- the percentage of base calling (BC%) and the signal intensity (RFU) observed for the probe array for each clone are shown in columns 3 and 4, respectively.
- the last column of Table 4 shows the relative amount of M. tuberculosis amplicons obtained for each clone in one assay, as determined by hybridization to an AE-labeled probe and detected as relative light units (RLU) as described above.
- RLU relative light units
- This example shows the sensitivity of primers of the present invention when used in TMA amplification of clinical samples containing wild type M. tuberculosis and detection of the amplified RNA.
- Amplification was done substantially as described in Example 1.
- Amplicons were detected substantially as described in Example 2 on a solid support having an array of immobilized probes (GENECHIPTM) and by using a labeled probe in a homogeneous detection assay.
- NALC acts as a mucolytic agent to ensure liquefaction of the specimen and sodium hydroxide is a decontaminating agent.
- the smear intensity was determined based on the usual clinical classification of mycobacteria culture where "1+" means a low positive and "4 +" means a high positive.
- results obtained for 12 specimens are summarized in Table 5.
- the results show the smear intensity for each of the specimens (column 2) compared to the probe detection results obtained with the homogeneous detection assay with labeled probe (single assay RLU results, in column 3) and the results obtained following hybridization to the DNA probe array (columns 4, BC%, and 5, signal intensity).
- Example 5 PCR Amplification and Detection of Amplicons
- PCR polymerase chain reaction
- the DNA obtained following amplification were detected on a solid support having an array of immobilized probes (i.e., a GENECHIPTM).
- wild type M. tuberculosis ATCC Accession No. 27177
- Bacterial stock suspensions were made in water and adjusted to a concentration of about 6 X10 8 bacteria per ml. Successive dilutions in water were made to produce a concentration of 10 4 bacteria per ⁇ l (equivalent to 10 4 copies of bacterial DNA per ⁇ l) which were then inactivated by heating for 15 min at 95° C.
- PCR amplification was carried out in a plastic tube using a thermostable DNA polymerase isolated from Thermus aquaticus (FAST STARTTM Taq DNA polymerase, Roche Molecular Biochemicals). Briefly, the amplification mixture (50 ⁇ I final volume) contained 5 ⁇ I of 10X amp buffer, 0.4 ⁇ I of dNTP mix (25mM each of ATP, CTP, UTP and GTP), 1.5 ⁇ I each of primers of SEQ ID NO:2 and SEQ ID NO:3 (1 O ⁇ M), 0.4 ⁇ I of DNA polymerase (2U), 5 ⁇ I of target (or water for the negative control).
- FAST STARTTM Taq DNA polymerase Roche Molecular Biochemicals
- Thermal cycling was performed using an automated thermal cycler (PERKIN-ELMER 9600TM) with an initial denaturation step at 95°C for 4 min, followed by 35 cycles each consisting of 95° C for 30 sec, 50° C for 30 sec and 72° C for 45 sec, and a final cycle of 72° C for 7 min.
- PERKIN-ELMER 9600TM automated thermal cycler
- the amplification products were analyzed by agarose gel electrophoresis and stained with ethidium bromide, to detect the presence or absence of a 168 nt band of amplified DNA. No band was visible on the gel for the negative control (i.e., no target DNA). A DNA band was seen when 10 4 copies of target or more were used in the reaction.
- amplification products were detected on a DNA probe array (GENECHIPTM), substantially as described previously (Troesch et al., 1999, J. Clin. MicrobioL 37(1):49-55).
- Promoter-tagged PCR amplicons were used to generate single-stranded RNA targets by in vitro transcription reactions (20 ⁇ I) that each contained approximately 50 ng of PCR product, 20 U of T7 RNA polymerase (Promega), 40 mM Tris acetate (pH 8.1), 100 mM Mg(acetate) 2 , 10 mM dithiothreitol, 1.25 mM each of ATP, CTP, UTP and GTP, and were incubated at 37°C for 1 hr.
- RNA was fluorescentiy labeled substantially as described (PCT No. WO 01/44507) and the labeled RNA was hybridized to the probe array and analyzed (Troesch et al., supra). For hybridization, 5 ⁇ l the labeled RNA was diluted in 700 ⁇ I of hybridization buffer (0.90 M NaCI, 60 mM NaH 2 P0 4 , 6 mM EDTA, pH 7.4, and 0.05% (v/v) TRITON® X-100), applied to the probe array and incubated at 45° C for 30 min.
- hybridization buffer 0.90 M NaCI, 60 mM NaH 2 P0 4 , 6 mM EDTA, pH 7.4, and 0.05% (v/v) TRITON® X-100
- the probe array was washed twice in 3X SSPE (0.45 M NaCI, 30 mM NaH 2 P0 4 , 3 mM EDTA, pH 7.4) and 0.005% (v/v) TRITON® X-100 at 30°C and the fluorescent signal bound to the array was detected.
- the detected signal intensities mean, median and maximum RFU
- nucleotide % base calling and sequence determinations were generated by using the system algorithm (GENECHIPTM software, Affymetrix).
- a candidate selection index was determined by the percentage of homology between the experimentally derived sequence and reference sequences present on the array. The results obtained from this experiment showed that for 10 4 copies of target the sequence determined on the probe array was that of wild type M.
- Example 6 Amplification of rpoB (+) Strand Target This example shows that the assay can amplify and detect amplicons made from the rpoB region but from the (+) DNA strand for comparison to the (-) DNA strand amplification described in Example 1.
- the amplification oligonucleotides used in the (+) strand amplification reaction were: SEQ ID NO:10 (AATTTAATACGACTCACTATAGGGAGAACGCTCACGTGACAGAC) as a promoter primer and SEQ ID NO: 11 (GGTCGCCGCGATCAAG) as a primer.
- the target sequences for this assay were purified gDNA extracted from a lysed bacterial culture of M. tuberculosis and provided at 5 X 10 3 copies per amplification reaction or a crude lysate of sonicated M. tuberculosis cells grown in broth culture, provided at about 5 X 10 3 copies per amplification reaction.
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Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
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EP01975259A EP1436414B1 (en) | 2001-09-18 | 2001-09-18 | Detection of rpob sequences of mycobacterium tuberculosis |
JP2003529999A JP4455881B2 (en) | 2001-09-18 | 2001-09-18 | Detection of rpoB sequence of Mycobacterium tuberculosis |
CA002443534A CA2443534C (en) | 2001-09-18 | 2001-09-18 | Detection of rpob sequences of mycobacterium tuberculosis |
PCT/US2001/029361 WO2003025227A1 (en) | 2001-09-18 | 2001-09-18 | Detection of rpob sequences of mycobacterium tuberculosis |
DE60136572T DE60136572D1 (en) | 2001-09-18 | 2001-09-18 | DETECTION OF RPOB SEQUENCES OF MYCOBACTERIUM TUBERCULOSIS |
AU2001294599A AU2001294599B2 (en) | 2001-09-18 | 2001-09-18 | Detection of rpoB sequences of Mycobacterium tuberculosis |
AT01975259T ATE414175T1 (en) | 2001-09-18 | 2001-09-18 | DETECTION OF RPOB SEQUENCES FROM MYCOBACTERIUM TUBERCULOSIS |
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JP (1) | JP4455881B2 (en) |
AT (1) | ATE414175T1 (en) |
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EP2270202A1 (en) * | 2009-07-03 | 2011-01-05 | John Ikonomopoulos | Mycobacterial detection |
Citations (7)
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WO1995033074A1 (en) * | 1994-05-26 | 1995-12-07 | Mayo Foundation For Medical Education And Research | Detection of a genetic locus encoding resistance to rifampin |
WO1995033851A2 (en) * | 1994-06-09 | 1995-12-14 | Innogenetics N.V. | Method for the detection of the antibiotic resistance spectrum of mycobacterium species |
EP0962536A1 (en) * | 1998-06-04 | 1999-12-08 | Roche Diagnostics GmbH | DNA detection by a strand reassociation complex |
WO2000043545A2 (en) * | 1999-01-19 | 2000-07-27 | Dade Behring Inc. | Detection of drug resistant organisms |
EP1076099A2 (en) * | 1999-08-03 | 2001-02-14 | Nisshinbo Industries, Inc. | Kit for diagnosis of tubercle bacilli |
WO2001034842A2 (en) * | 1999-11-12 | 2001-05-17 | University Of Chicago | Pcr amplification on microarrays of gel immobilized oligonucleotides |
WO2001066797A2 (en) * | 2000-03-03 | 2001-09-13 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Multiplex hybridization system for identification of pathogenic mycobacterium and method of use |
-
2001
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- 2001-09-18 AU AU2001294599A patent/AU2001294599B2/en not_active Ceased
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Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1995033074A1 (en) * | 1994-05-26 | 1995-12-07 | Mayo Foundation For Medical Education And Research | Detection of a genetic locus encoding resistance to rifampin |
WO1995033851A2 (en) * | 1994-06-09 | 1995-12-14 | Innogenetics N.V. | Method for the detection of the antibiotic resistance spectrum of mycobacterium species |
EP0962536A1 (en) * | 1998-06-04 | 1999-12-08 | Roche Diagnostics GmbH | DNA detection by a strand reassociation complex |
WO2000043545A2 (en) * | 1999-01-19 | 2000-07-27 | Dade Behring Inc. | Detection of drug resistant organisms |
EP1076099A2 (en) * | 1999-08-03 | 2001-02-14 | Nisshinbo Industries, Inc. | Kit for diagnosis of tubercle bacilli |
WO2001034842A2 (en) * | 1999-11-12 | 2001-05-17 | University Of Chicago | Pcr amplification on microarrays of gel immobilized oligonucleotides |
WO2001066797A2 (en) * | 2000-03-03 | 2001-09-13 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Multiplex hybridization system for identification of pathogenic mycobacterium and method of use |
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COLE S T ET AL: "Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence", NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 393, 11 June 1998 (1998-06-11), pages 537 - 544, XP002087941, ISSN: 0028-0836 * |
JONAS VIVIAN ET AL: "Detection of Mycobacterium tuberculosis by molecular methods.", CLINICS IN LABORATORY MEDICINE, vol. 17, no. 1, 1997, pages 119 - 128, XP001064491, ISSN: 0272-2712 * |
Cited By (1)
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EP2270202A1 (en) * | 2009-07-03 | 2011-01-05 | John Ikonomopoulos | Mycobacterial detection |
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JP2005503165A (en) | 2005-02-03 |
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ATE414175T1 (en) | 2008-11-15 |
EP1436414B1 (en) | 2008-11-12 |
CA2443534C (en) | 2010-01-12 |
EP1436414A1 (en) | 2004-07-14 |
CA2443534A1 (en) | 2003-03-27 |
AU2001294599B2 (en) | 2007-06-28 |
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