WO2000043545A2 - Detection d'organismes pharmacoresistants - Google Patents

Detection d'organismes pharmacoresistants Download PDF

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WO2000043545A2
WO2000043545A2 PCT/US1999/029517 US9929517W WO0043545A2 WO 2000043545 A2 WO2000043545 A2 WO 2000043545A2 US 9929517 W US9929517 W US 9929517W WO 0043545 A2 WO0043545 A2 WO 0043545A2
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sequence
seq
primers
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primer
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PCT/US1999/029517
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WO2000043545A3 (fr
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Yen Ping Liu
Nurith Kurn
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Dade Behring Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research.
  • ssDNA single stranded deoxyribonucleic acid
  • RNA ribonucleic acid
  • Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
  • One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.
  • a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane.
  • PCR polymerase chain reaction
  • the branch point between two duplex DNA's that have exchanged a pair of strands is thought to be an important intermediate in homologous recombination. This branch point is otherwise referred to as the Holliday junction. Movement of the Holliday junction by branch migration can increase or decrease the amount of genetic information exchanged between homologues. In vivo strand exchange is protein mediated, unlike the spontaneous migration that occurs in vitro.
  • mycobacteria are causative agents of disease. Cases of mycobacterial infections are increasing in the United States. Of particular concern is tuberculosis, the etiological agent of which is M. tuberculosis. Many of these new cases are related to the AIDS epidemic, which provides an immune compromised population that is particularly susceptible to infection by Mycobacteria. Other mycobacterial infections are also increasing as a result of the increase in available immune compromised patients. Mycobacte um avium, Mycobacterium kansasii and other non-tuberculosis mycobacteria are found as opportunistic pathogens in HIV infected and other immune compromised patients.
  • Multidrug resistance is one factor in the tuberculosis problem.
  • An increase in the frequency of Mycobacterium tuberculosis strains resistant to one or more anti- mycobacterial agents has been reported.
  • Immunocompromised patients such as those infected with human immunodeficiency virus (HIV-1), who are not infected with M. tuberculosis, are frequently infected with other mycobacterial strains, which are often resistant to the drugs used to treat M. tuberculosis. Consequently, it is important to accurately determine drug sensitivities and identification of the mycobacteria species. Additionally, many new cases of M.
  • HIV-1 human immunodeficiency virus
  • tuberculosis are resistant to one or more of the primary anti-tuberculosis drugs, namely, isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide.
  • the determination of drug resistance has become central concern during the diagnosis of mycobacterial diseases.
  • Methods used to determine drug sensitivity information include culture methods. Mycobacteria are judged to be resistant to particular drugs by use of either the standard proportional plate method or minimal inhibitory concentration method. More recently, approaches to determine drug sensitivity based on molecular genetics have been developed.
  • M. tuberculosis Most drug resistance phenotypes in M. tuberculosis appear to arise as a consequence of chromosomal mutations. Multidrug resistance appears to be due to stepwise accumulation of mutations conferring resistance to individual therapeutic agents.
  • Rifampin resistance in M. tuberculosis is largely associated with point mutations localized in a small core region of 81 base pairs (bp) in the rpoB gene, which encodes for the RNA polymerase beta subunit (The rpoB Gene of M. tuberculosis, Miller, et al., Antimicrobial Agents and Chemotherapy, 38, 805, 1994).
  • Mutations in this 81-bp region of the gene account for rifampin resistance in 96% of M. tuberculosis strains and many other Mycobacterium species.
  • the molecular mechanism of rifampin activity involves inhibition of DNA-dependent RNA polymerase.
  • PCR primers specific for rpoB are used to amplify the portion of the rpoB gene that contains the most common mutations associated with rifampin resistance.
  • the amplification products are then analyzed for the presence of rifampin-associated mutations.
  • the most common methods used for mutation detection are DNA sequencing (Hunt, infra), DNA conformation-dependent methods such as single-strand conformation polymorphism (SSCP) (Talent et al. infra), or dideoxy fingerprinting (Felmee, et al., infra), as well as detection of specific PCR amplification products (Whelan, et al., infra).
  • Pyrazinamide is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in M. tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance (Konno K. et al., Am. Rev. Respir. Pis. 1967, 98:461-469). The gene (pncA) encoding the M. tuberculosis PZase has recently been cloned and sequenced (Angelo S.
  • Mutations in pncA gene are the major mechanism of pyrazinamide resistance in M. tuberculosis and these were identified in both acquired PZA-resistant M. tuberculosis and naturally resistant M. bovis strain (Angelo S. and Ying Z., supra; Angelo S. er a/., Antimicrobial Agents and Chemotherapy, 1997, 41:540-543; Srinand S. et al, Antimicrobial Agents and Chemotherapy, 1997, 41:636-640). Transformation of these strains with a functional pncA gene restored PZase activity and PZA susceptibility. This demonstrates that mutations identified in the pncA gene are the cause for PZA resistance in both types of resistant bacilli.
  • tuberculosis susceptibility to PZA utilizes determination of PZase activity (Wayne L. G., Am. Rev. Respir. Pis., 1974, 109:147-151 ; Trivedl S. S. and Desai S. G., Tubercle, 1987, 88:221-224).
  • the automated and relatively faster Bactec method is preferred in the clinical laboratory; however, the growth based method requires at least 5 days for susceptibility testing following initial growth of the isolate.
  • a rapid test method based on detection of pncA mutations, i.e., genotypic drug resistance determination will not only avoid the problems of the current PZA susceptibility testing, but it will also open a new way for PZA susceptibility testing.
  • tuberculosis which are based on either automated sequencing or PCR-SSCP (single strand conformation polymorphism), were recently reported (Angelo S et al., Antimicrobial Agents and Chemotherapy, 1997, 41 , 540-543; U.S. Patent Application Serial No. 846,718 (Zhang Ying and Scorpio 1998).
  • U.S. Patent No. 5,643,723 discloses detection of genetic locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens.
  • U.S. Patent No. 5,470,723 discloses detection of mycobacteria by multiplex nucleic acid amplification.
  • U.S. Patent No. 5,667,994 discloses amplification and detection of Mycobacterium avium complex species. Miller, et al., describe the rpoB gene of M. tuberculosis in Antimicrobial Agents and Chemotherapy (1994) 38:805.
  • U.S. Patent No. 5,652,106 discloses rapid amplification-based subtyping of mycobacterium tuberculosis.
  • Cockerill, et al. disclose detection of isoniazid resistant strains of M. tuberculosis in U.S. Patent Nos. 5,688,639 (Cockerill 1) and 5,658,733 (Cockerill 2).
  • Kapur, et al. disclose characterization by automated PNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase ⁇ subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas in J. Clinical Microbiol. (1994) 32(4): 1095-1098.
  • Whelen, et al. disclose direct genotypic detection of rifampin resistance in M. tuberculosis in clinical specimens by using single-tube heminested PCR in J. Clinical Microbiology (1995) 33:556.
  • Telenti et al., describe the direct automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis in Antimicrobial Agents and Chemotherapy (1993) 37(10):2054- 2058.
  • the characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis is discussed by Scorpio, et al., Antimicrobial Agents and Chemotherapy (1997) 41:540-543.
  • Scorpio et al., Journal of Clinical Microbiology (1997) 35(1): 106-110 discloses rapid differentiation of bovine and human tubercle bacilli based on a characteristic mutation in the bovine pyrazinamidase gene.
  • U.S. Patent No. 5,786,149 discloses materials and methods for the detection of mycobacterium tuberculosis.
  • U.S. Patent No. 5,554,503 discusses a sample processing method for Mycobacterium tuberculosis.
  • U.S. Patent No. 5,376,527 discloses a process for lysing mycobacteria.
  • a displacement polynucleotide assay method and polynucleotide complex reagent therefor is discussed in U.S. Patent No. 4, 766,062 (Diamond, et al.).
  • a strand displacement assay and complex useful therefor is discussed in PCT application WO 94/06937 (Eadie, et al.).
  • One method in accordance with the present invention is directed to the detection of drug resistance in a strain of an organism.
  • the presence of at least one mutation in a predetermined region within the gene of the strain is detected.
  • the predetermined region has a multiplicity of mutations among strains of the organism that differ from a corresponding region of the wild type strain of the organism.
  • a complex is formed comprising a first sequence representing the predetermined region of the gene of the organism and a second sequence representing the corresponding region of the gene of the wild type organism in double stranded form.
  • Each member of at least one pair of non-complementary strands within the complex has a label.
  • the association of the labels within the complex is detected wherein the association of the labels in the complex is related to the presence of the mutation.
  • the presence of the mutation is related to the drug resistance of the strain.
  • Another aspect of the present invention is a method for detecting drug resistance in a strain of M. tuberculosis.
  • the presence of at least one mutation in a first sequence representing a predetermined region within the gene of the strain is detected.
  • a tailed target partial duplex A' is formed from the first sequence comprised of a duplex of the first sequence, a label at one end of the duplex, two non-complementary oligonucleotides, one linked to each strand.
  • the tailed target partial duplex A' is provided in combination with a tailed reference partial duplex B' from a second sequence representing a corresponding region of a wild type strain having a label as a part thereof.
  • the labels are present in non- complementary strands of the tailed target and tailed reference partial duplexes, respectively.
  • the formation of a complex between the tailed partial duplexes is detected by means of the labels.
  • the formation of the complex is directly related to the presence of the mutation.
  • the presence of the mutation is related to the drug resistance of the strain of M. tuberculosis.
  • Another aspect of the present invention is a method for detecting drug resistance in a strain of M. tuberculosis.
  • the presence of at least one mutation in a first sequence representing a predetermined region within a gene of the strain is detected.
  • An amplification of the first sequence is carried out by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA.
  • the primer P1 is comprised of a 3'-end portion Pa that can hybridize with the first sequence and 5'-end portion B1 that cannot hybridize with the first sequence.
  • a primer P3 is extended by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A'.
  • the primer P3 is comprised of the 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to the first sequence or its complement.
  • a second sequence representing a region in wild type M. tuberculosis that corresponds to the predetermined region is amplified, using the primer P2 and the primer P3, by polymerase chain reaction to produce amplicon BB.
  • the primer P2 comprises a label when the primer P2 above comprises a label and the primer P3 comprises a label when the primer P1 above comprises a label.
  • the primer P1 is extended by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex B'.
  • the tailed target partial duplex A' is allowed to bind to the tailed reference partial duplex B'.
  • the binding of one of the labels to another of the labels as a result of the formation of a complex between the tailed partial duplexes is detected.
  • the binding is directly related to the presence of the mutation.
  • the presence of the mutation is related to the drug resistance of the strain of M. tuberculosis.
  • Another aspect of the present invention is a method for detecting rifampin resistance in a strain of M. tuberculosis.
  • a first sequence representing a predetermined region within the rpoB gene of the strain and a second sequence representing a sequence within wild type strain that corresponds to the first sequence are subjected to polymerase chain reaction.
  • a 5'-labeled primer P2 selected from the group consisting of 5'-L-GAGCGGATGACCACCCAGGAC-3' (SEQ ID NO:1) and
  • 5'-L-CCACCCAGGACGTGGAGGC-3' SEQ ID NO:2
  • 5'-tailed primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of the P1 and P3.
  • the common nucleotide sequence is selected from the group consisting of
  • the product of the polymerase chain reactions is a tailed partial duplex A' produced from the first sequence and a tailed partial duplex B' produced from the second sequence.
  • the tailed target partial duplex A' is allowed to bind to the tailed reference partial duplex B'.
  • the binding of one of the labels to another of the labels as a result of the formation of a complex between the tailed partial duplexes is detected.
  • the binding thereof is directly related to the presence of the mutation.
  • the presence of the mutation is related to the rifampin resistance of the strain of M. tuberculosis.
  • kits for carrying out a method for detecting rifampin resistance in a strain of M. tuberculosis comprises (a) a second sequence representing a sequence within wild type strain that corresponds to the first sequence, (b) a 5'-labeled primer P2 selected from the group consisting of
  • 5'-L-CCACCCAGGACGTGGAGGC-3' (SEQ ID NO:2), wherein two different labels are employed for each labeled primer P2, and (c) 5'-tailed primers P1 and P3 comprising a common nucleotide sequence and a different oligonucleotide tail for each of the P1 and P3, wherein the common nucleotide sequence is selected from the group consisting of
  • Another embodiment of the present invention is a method for detecting pyrazinamide resistance in a strain of M. tuberculosis.
  • a medium is subjected to conditions for polymerase chain reaction.
  • the medium comprises (i) a first sequence representing a predetermined region within the pncA gene of the strain, (ii) a 5'-labeled primer P2, which is 5'- L-tggtgccgcgtcggtagg-3'
  • kits for detecting the presence of a difference between two related nucleic acid sequences in an organism comprises in packaged combination (a) two oligonucleotide primers each comprising a sequence that is hybridizable to the nucleic acid sequences in a region adjacent the site of potential difference and each comprising a label wherein the labels are different, and (b) a lysing agent.
  • Figs. 1 and 1A are schematic diagrams depicting the formation and non-formation, respectively, of a quadramolecular complex in accordance with the present invention.
  • Fig. 2 is a schematic diagram depicting an embodiment in accordance with the present invention.
  • Fig. 3 is a schematic diagram depicting an initial amplification in accordance with the present invention.
  • Fig. 4 is a depiction of the sequence of the M. tuberculosis rpoB gene (GenBank Ace. No. U12205).
  • Fig. 5 is a graph of results obtained using a method in accordance with the present invention for the detection of an rpoB mutation in a M. tuberculosis strain versus a wild type M. tuberculosis.
  • Fig. 6 is a graph of results obtained using a method in accordance with the present invention for the detection of a an rpoB mutation in a M. tuberculosis strain versus a wild type M. tuberculosis employing cell disruption.
  • Fig. 7 is a graph of results obtained using a method in accordance with the present invention for the detection of a pncA mutation in a M. tuberculosis strain employing two different enzymes.
  • Fig. 8 is a graph of results obtained using a method in accordance with the present invention for the detection of a pncA mutation in a M. tuberculosis strain.
  • Fig. 9 is a graph of results obtained using a method in accordance with the present invention for the detection of a pncA mutation in a M. tuberculosis strain and four mycobacteria other than tuberculosis strains. 11
  • Fig 10 is a graph of results obtained using a method in accordance with the present invention for the detection of a pncA mutation in a M tuberculosis strain versus a wild type M tuberculosis
  • the present invention provides a method and reagents for the determination of drug resistance in an organism such as a mycobacterium
  • the drug is one that is effective against the organism, usually, one that is employed in the treatment of an individual infected with the organism
  • the method may be applied to any situation where drug resistance of an organism resides in the genome i e , where drug resistance is genetically determined
  • the method usually involves PCR amplification for the production of substrates capable of formation of four-stranded DNA structures that will undergo spontaneous branch migration
  • the formation of a four-stranded DNA structure or complex from DNA involves producing two partial duplexes by amplification by using three different primers in the polymerase chain reaction and allowing the amplified products to anneal
  • the complex dissociates into normal duplex structures by strand exchange by means of branch migration when the hybridized portions of each partial duplex are identical However, where there is a difference between the two hybridized portions, the complex does not dissociate and can be detected as an indication of the presence of a mutation, which
  • Mycobacteria - a genus of bacteria that are acid-fast non-motile gram-positive rods
  • the genus comprises several species that include, but are not limited to Mycobactenum afncanum, M avium, M bovis, M bovis-BCG, M cnelonae, M fortuitum, M gordonae, M mtracellulare, M kansasu, M microti, M scorfulaceum, M paratuberculosis and M tuberculosis
  • Nucleic acid -- a compound or composition that is a polymeric nucleotide or polynucleotide
  • nucleic acids include both nucleic acids and fragments thereof from any source in purified or unpu ⁇ fied form including DNA (dsDNA and ssDNA) and RNA, t-RNA, m-RNA, r-RNA, and the like
  • nucleic acids include both nucleic acids and fragments thereof from any source in
  • Sample the material containing the nucleic acid
  • biological fluids such as blood, serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus, feces, urine, spinal fluid, and the like
  • Other samples include clinical isolates, cell cultures and the like
  • biological material may be pretreated with reagents to liquefy it and/or release nucleic acids from binding substances to thereby produce the sample to be tested or the biological may be used directly without release of nucleic acids
  • the biological material such as cells from an organism may be disrupted by mechanical or chemical treatment either prior to or during the present method including amplification of the nucleic acid
  • Mechanical treatment includes, for example, treatment with heat, sonication, treatment with microwaves and the like
  • Chemical treatment includes, for example, treatment with a lysing agent
  • Amplification of nucleic acids any method that results in the formation of one or more copies of a nucleic acid (exponential amplification)
  • Amplification of nucleic acids any method that results in
  • Primer length can vary from about 10 to 50 or more nucleotides and are usually selected to be at least about 15 nucleotides to ensure high specificity.
  • the double stranded fragment that is produced is called an "amplicon" and may vary in length form as few as about 30 nucleotides to 10,000 or more.
  • Chain extension of nucleic acids extension of the 3'-end of a polynucleotide in which additional nucleotides or bases are appended.
  • Chain extension relevant to the present invention is template dependent, that is, the appended nucleotides are determined by the sequence of a template nucleic acid to which the extending chain is hybridized.
  • the chain extension product sequence that is produced is complementary to the template sequence.
  • chain extension is enzyme catalyzed, preferably, in the present invention, by a thermophilic DNA polymerase.
  • First sequence a sequence of nucleotides within a gene to be studied; a target nucleic acid sequence.
  • the first sequence represents a predetermined region within the gene of a strain of an organism and may be double stranded or single stranded.
  • the method of the present invention produces a nucleic acid duplex comprising the single stranded first sequence.
  • the predetermined region exists within a portion of a gene of a strain of a microorganism to be studied.
  • the predetermined region has a multiplicity of mutations among strains of the organism that differ from a corresponding region of the wild type strain of the microorganism.
  • the identity of the predetermined region is known at least to an extent sufficient so that such mutations are known.
  • the identity of the predetermined region is generally known to allow preparation of various primers necessary for introducing one or more priming sites and/or conducting an amplification of the predetermined region in accordance with the present invention. Accordingly, other than the above, the identity of the gene may or may not be known. Examples of genes in conjunction with M. tuberculosis, by way of illustration and not limitation, include the rpoB gene and the pncA gene.
  • primers hybridize to, and are extended along (chain extended), at least a portion of a target nucleic acid sequence such as the predetermined region, and, thus, the predetermined region acts as a template.
  • the minimum number of nucleotides in the target sequence is selected to assure that a determination of a mutation in accordance with the present invention can be achieved.
  • Second sequence a nucleic acid sequence that is related to the first sequence, a reference nucleic acid sequence
  • the second sequence represents a region of a wild type strain that corresponds to the predetermined region of the first sequence in that the two sequences are identical except for the presence of a mutation
  • the region of the wild type strain may be part of the sample
  • the identity of the second sequence need be known so that the presence of mutations in the first sequence is known
  • the identity of the corresponding region is generally known to allow preparation of various primers necessary for introducing one or more priming sites and/or conducting an amplification of the corresponding region in accordance with the present invention Accordingly, other than the above, the identity of the wild type strain may or may not be known
  • the corresponding region of the wild type strain may be a reagent employed in the methods in accordance with the present invention In that regard the corresponding region may be obtained from a natural source or prepared by known methods such as those described
  • Holliday junction the branch point in a four-way junction in a complex of two identical nucleic acid sequences and their complementary sequences The junction is capable of undergoing branch migration resulting in dissociation into two double stranded sequences where sequence identity and complementarity extend to the ends of the strands
  • Complex a complex of four nucleic acid strands containing a Holliday junction, which is inhibited from dissociation into two double stranded sequences because of a mutation in one of the sequences and/or their complements Accordingly, the complex is quadramolecular
  • Mutation a change in the sequence of nucleotides of a normally conserved nucleic acid sequence resulting in the formation of a mutant as differentiated from the normal (unaltered) or wild type sequence Mutations can generally be divided into two general classes, namely, base-pair substitutions and frameshift mutations The latter entail the insertion or deletion of one to several nucleotide pairs A difference of one nucleotide can be significant as to phenotypic normality or abnormality as in the case of, for example, sickle cell anemia
  • Partial duplex a fully complementary double stranded nucleic acid sequence wherein one end thereof has non-complementary oligonucleotide sequences, one linked to each strand of the double stranded molecule, each non-complementary sequence having 8 to 60, preferably, 10 to 50, more preferably, 15 to 40, nucleotides
  • the partial duplex is said to be "tailed" because each strand of the duplex has a single stranded oligonucleotide chain linked thereto
  • Duplex a double stranded nucleic acid sequence wherein all of the nucleotides therein are complementary
  • Oligonucleotide a single stranded polynucleotide, usually a synthetic polynucleotide
  • the ol ⁇ gonucleot ⁇ de(s) are usually comprised of a sequence of 10 to 100 nucleotides, preferably, 20 to 80 nucleotides, and more preferably, 30 to 60 nucleotides in length
  • oligonucleotide utilized in the present invention
  • Such oligonucleotide can be obtained by biological synthesis or by chemical synthesis
  • chemical synthesis will frequently be more economical as compared to the biological synthesis
  • chemical synthesis provides a convenient way of incorporating low molecular weight compounds and/or modified bases during the synthesis step
  • chemical synthesis is very flexible in the choice of length and region of the target polynucleotide binding sequence
  • the oligonucleotide can be synthesized by standard methods such as those used in commercial automated nucleic acid synthesizers
  • Chemical synthesis of DNA on a suitably modified glass or resin can result in DNA covalently attached to the surface This may offer advantages in washing and sample handling
  • standard replication methods employed in molecular biology can be used such as the use of M 3 for single stranded DNA as described by J Messing (1983) Methods Enzvmol, 101.
  • oligonucleotide synthesis examples include phosphot ⁇ ester and phosphodiester methods (Narang, et al (1979) Meth Enzymol 68 90) and synthesis on a support (Beaucage, et al (1981) Tetrahedron Letters 22 1859-1862) as well as phosphoramidate technique, Caruthers, M H , et al_, “Methods in Enzymology,” Vol 154, pp 287-314 (1988), and others described in “Synthesis and Applications of DNA and RNA,” S A Narang, editor, Academic Press, New York, 1987, and the references contained therein
  • the oligonucleotide primer is usually a synthetic oligonucleotide that is single stranded, containing a hybridizable sequence at its 3'-end that is capable of hybridizing with a defined sequence of the target or reference polynucleotide Normally, the hybridizable sequence of the oligonucleotide primer has at least 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence or primer binding site
  • the number of nucleotides in the hybridizable sequence of an oligonucleotide primer should be such that stringency conditions used to hybridize the oligonucleotide primer will prevent excessive random non-specific hybridization Usually, the number of nucleotides in the hybridizable
  • Nucleoside tnphosphates nucleosides having a 5'-tr ⁇ phosphate substituent
  • the nucleosides are pentose sugar derivatives of nitrogenous bases of either pu ⁇ ne or pynmidine derivation, covalently bonded to the 1 '-carbon of the pentose sugar, which is usually a deoxy ⁇ bose or a ⁇ bose
  • the pu ⁇ ne bases comprise aden ⁇ ne(A), guanine (G), inosine (I), and derivatives and analogs thereof
  • the pynmidine bases comprise cyto- sine (C), thymine (T), uracil (U), and derivatives and analogs thereof
  • Nucleoside tnphosphates include deoxy ⁇ bonucleoside tnphosphates such as the four common tnphosphates dATP, dCTP, dGTP and dTTP and nbonucleoside tnphosphates
  • nucleoside tnphosphates also includes derivatives and analogs thereof, which are exemplified by those derivatives that are recognized and polymerized in a similar manner to the unde ⁇ vatized nucleoside tnphosphates
  • derivatives or analogs by way of illustration and not limitation, are those which are biotinylated, amme modified, alkylated, and the like and also include phosphorothioate, phosphite, ring atom modified derivatives, and the like
  • Nucleotide - a base-sugar-phosphate combination that is the monomenc unit of nucleic acid polymers, i e , DNA and RNA Modified nucleotide - is the unit in a nucleic acid polymer that results from the incorporation of a modified nucleoside t ⁇ phosphate during an amplification reaction and therefore becomes part of the nucleic acid polymer
  • Nucleoside -- is a base-sugar combination or a nucleotide lacking a phosphate moiety
  • the nucleotide polymerase is a template dependent polynucleotide polymerase and utilizes nucleoside tnphosphates as building blocks for extending the 3'-end of a polynucleotide to provide a sequence complementary with the polynucleotide template.
  • the catalysts are enzymes, such as DNA polymerases, for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase, T7 PNA polymerase, Klenow fragment, and reverse transcriptase, and are preferably thermally stable PNA polymerases such as Vent DNA polymerase, VentR DNA polymerase, Pfu DNA polymerase, Tag DNA polymerase, and the like, derived from any source such as cells, bacteria, such as E. coli, plants, animals, virus, thermophilic bacteria, and so forth.
  • DNA polymerases for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase, T7 PNA polymerase, Klenow fragment, and reverse transcriptase
  • thermally stable PNA polymerases such as Vent DNA polymerase, VentR DNA polymerase, Pfu DNA polymerase, Tag DNA polymerase, and the like, derived from any source such as cells, bacteria, such as E. coli, plants,
  • one or more may be combined with one or more of the remaining agents to form a subcombination. Subcombination and remaining agents can then be combined and can be subjected to the present method.
  • Hybridization and binding—in the context of nucleotide sequences these terms are used interchangeably herein.
  • the ability of two nucleotide sequences to hybridize with each other is based on the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs.
  • the more nucleotides in a given sequence that are complementary to another sequence the more stringent the conditions can be for hybridization and the more specific will be the binding of the two sequences.
  • Increased stringency is achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration, and the like.
  • Complementary -- two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3'-end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence.
  • Copy - means a sequence that is a direct identical copy of a single stranded polynucleotide sequence as differentiated from a sequence that is complementary to the sequence of such single stranded polynucleotide.
  • Conditions for extending a primer - includes a nucleotide polymerase, nucleoside tnphosphates or analogs thereof capable of acting as substrates for the polymerase and other materials and conditions required for enzyme activity such as a divalent metal ion (usually magnesium), pH, ionic strength, organic solvent (such as formamide), and the like.
  • Hgand and receptor Hgand and receptor
  • Ligand any compound for which a receptor naturally exists or can be prepared.
  • Receptor any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e.g., epitopic or determinant site.
  • Illustrative receptors include naturally occurring and synthetic receptors, e.g., thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, repressors, oligonucleotides, protein A, complement component C1q, or DNA binding proteins and the like.
  • Small organic molecule a compound of molecular weight less than about 1500, preferably 100 to 1000, more preferably 300 to 600 such as biotin, digoxin, fluorescein, rhodamine and other dyes, tetracycline and other protein binding molecules, and haptens, etc.
  • the small organic molecule can provide a means for attachment of a nucleotide sequence to a label or to a support.
  • the support can be hydrophihc or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), etc.; either used by themselves or in conjunction with other materials; glass available as Bioglass, ceramics, metals, and the like.
  • inorganic powders such as silica, magnesium sulfate, and alumina
  • natural polymeric materials particularly cellulosic materials and materials derived from
  • Natural or synthetic assemblies such as liposomes, phospholipid vesicles, and cells can also be employed. Binding of sbp members to a support or surface may be accomplished by well-known techniques, commonly available in the literature See, for example, "Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J Biol Chem , 245 3059 (1970)
  • the surface can have any one of a number of shapes, such as strip, rod, particle, including bead, and the like
  • Labels include reporter molecules or reporter groups that can be detected directly by virtue of generating a signal, and specific binding pair members that may be detected indirectly by subsequent binding to a cognate that contains a reporter molecule such as oligonucleotide sequences that can serve to bind a complementary sequence or a specific DNA binding protein, organic molecules such as biotin or digoxigenin that can bind respectively to streptavidin and antidigoxin antibodies, respectively, polypeptides, polysacchandes, and the like
  • a reporter molecule such as oligonucleotide sequences that can serve to bind a complementary sequence or a specific DNA binding protein, organic molecules such as biotin or digoxigenin that can bind respectively to streptavidin and antidigoxin antibodies, respectively, polypeptides, polysacchandes, and the like
  • any reporter molecule that is detectable can be used
  • the reporter molecule can be isotopic or nonisotopic, usually non-isotopic, and can be a catalyst, such as an enzyme
  • the label is a member of a signal producing system and can generate a detectable signal either alone or together with other members of the signal producing system
  • a reporter molecule can serve as a label and can be bound directly to a nucleotide sequence
  • the reporter molecule can bind to a nucleotide sequence by being bound to an sbp member complementary to an sbp member that comprises a label bound to a nucleotide sequence
  • Examples of particular labels or reporter molecules and their detection can be found in U S Patent No 5,508,178, the relevant disclosure of which is incorporated herein by reference
  • Signal producing system the signal producing system may have one or more components, at least one component being the label
  • the signal producing system generates a signal that relates to the presence of a mutation between the first sequence and the second sequence
  • the signal producing system includes all of the reagents required to produce a measurable signal
  • the reporter molecule is normally bound to an sbp member complementary to an sbp member complementary to an sbp member complementary
  • buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components Frequently, in addition to these additives, proteins may be included, such as albumins, organic solvents such as formamide, quaternary ammonium salts, polycations such as dextran sulfate, surfactants, particularly non-ionic surfactants, binding enhancers, e g , polyalkylene glycols, or the like
  • one aspect of the present invention concerns a method for detecting drug resistance in a strain of an organism
  • the presence of at least one mutation in a first sequence representing a predetermined region within the gene of the strain is detected
  • the predetermined region has a multiplicity of mutations among strains of the organism that differ from a corresponding region of the wild type strain of the organism
  • This may be most readily understood with regard to drug resistant phenotypes that appear to arise as a consequence of chromosomal mutations In this situation multidrug resistance appears to be due to stepwise accumulation of mutations conferring resistance to individual therapeutic agents
  • the predetermined region is analyzed to detect a mutation
  • a quadramolecular complex is formed comprising the first sequence and a second sequence that represents the corresponding region of the gene of the wild type organism in double stranded form
  • the complex comprises a Holliday junction
  • Each member of at least one pair of non-complementary strands within the complex has a label The association of the labels within the complex is detected
  • a mutation M is present in a first sequence A
  • the method comprises forming from the first sequence a tailed target partial duplex A' comprised of a duplex of the first sequence, a label L1 and at one end of the duplex, two non-complementary ohgonucleotides A1 and A2, one linked to each strand of duplex A'
  • Ohgonucleotides A1 and A2 have from 8 to 60 nucleotides, preferably, 15 to 40 nucleotides
  • the tailed target partial duplex is provided in combination with a labeled tailed reference partial duplex B' lacking mutation M, which is a second sequence of the wild type strain of the organism Accordingly, one terminus of the tailed reference partial duplex B' has, as the end part of each strand, a sequence of nucleotides B1 and B2, respectively, that are complementary to A2 and A1 , respectively, of A' and are not complementary to each other Labels L1 and L2
  • a complex C is formed Oligonucleotide tail A1 of A' is hybridized to corresponding oligonucleotide tail B2 of B' and, similarly, oligonucleotide tail A2 of A' is hybridized to oligonucleotide tail B1 of B' Because oligonucleotide tails A1 and B1 are different, branch migration can only proceed away from these tails and then only until mutation M is reached, at which point branch migration stops Thus, when a mutation is present, complex C is stable and can be detected by determining whether both labels L1 and L2 have become associated The association of the labels indicates the presence of complex C The formation of complex C is directly related to the presence of the mutation If mutation M is not present in the nucleic acid (see Fig 1A), branch migration continues until complete strand exchange has occurred and only the separate duplexes D and E are present In this event no complex C is detected As mentioned above, the presence of the mutation is related to the drug resistance of the gene that is being tested
  • Fig 2 depicts, by way of example and not limitation, the method of the present invention in step-wise fashion
  • A is amplified by the polymerase chain reaction using primers P1 and P2 to produce an amplicon AA
  • Primer P2 contains a label L1
  • primer P1 is comprised of a 3'-end portion Pa that can hybridize with the first sequence and 5'-end portion B1 that cannot hybridize with the first sequence
  • the amplification is carried out in the presence of a nucleotide polymerase and nucleoside tnphosphates using temperature cycling Amphcon AA has two strands, a labeled strand derived from primer P2 and an unlabeled strand derived from primer P1
  • the above amplification is carried out by polymerase chain reaction (PCR) utilizing temperature cycling to achieve denaturation of duplexes, oligonucleotide primer annealing, and primer extension by thermophilic template dependent nucleotide polymerase
  • PCR polymerase chain reaction
  • the temperatures for the present method for the amplification by PCR generally range from about 50°C to about 100°C, more usually, from about 60°C to about 95°C Relatively low temperatures of from about 50°C to about 80°C are employed for the hybridization steps, while denaturation is carried out at a temperature of from about 80°C to about 100°C and extension is carried out at a temperature of from about 70°C to about 80°C, usually about 72°C to about 74°C
  • the amplification is conducted for a time sufficient to achieve a desired number of copies for an accurate determination of whether or not the first sequence has a mutation Generally, the time period for conducting the method is from about 10 seconds to
  • the medium is heated to a temperature of about 90°C to about 100°C for a period of about 5 to about 500 seconds and then cooled to about 20°C to about 80°C for a period of about 5 to about 2000 seconds followed by heating to about 40°C to about 80°C for a period of about 5 to about 2000 seconds
  • the medium is subjected to heating at about 90°C to about 100°C for a period of about 10 seconds to about 3 minutes, cooling to about 50°C to about 75°C for a period of about 10 seconds to about 2 minutes and heating to about 70°C to about 80°C for a period of about 30 seconds to about 5 minutes
  • an aqueous medium is employed
  • the sample to be analyzed is present in the medium
  • the sample may be cells from the organism that have been disrupted as discussed above or the medium may be treated mechanically or chemically to disrupt the cells
  • Other polar cosolvents may also be employed, usually oxygenated organic solvents of from 1-6 more usually from 1-4, carbon atoms, including alcohols, ethers and the like Usually these cosolvents, if used, are present in less than about 70 weight percent, more usually in less than about 30 weight percent
  • the pH for the medium is usually in the range of about 4 5 to about 9 5, more usually in the range of about 5 5 to about 8 5, and preferably in the range of about 6 to about 8, usually about 8
  • the pH and temperature are chosen and varied, as the case may be, so as to cause, either simultaneously or sequentially, dissociation of any internally hybridized sequences, hybridization of the oligonucleotide primer with the target nucleic acid sequence, extension of the primer, and dissociation of the extended primer
  • Various buffers may be used to achieve the desired pH and maintain the pH during the determination
  • Illustrative buffers include borate, phosphate, carbonate, T ⁇ s, barbital and the like
  • the particular buffer employed is not critical to this invention but in individual methods one buffer may be preferred over another
  • the buffer employed in the present methods normally contains magnesium ion (Mg 2+ ) at a concentration of from about 1 to about 10 mM, which is commonly used with many known polymerases, although other metal ions such as manganese have also been
  • magnesium ion is used at a concentration of from about 1 to about 20mM, preferably, from about 1 to about 10mM
  • the magnesium can be provided as a salt, for example, magnesium chloride and the like
  • the primary consideration is that the metal ion permits the determination of a mutation in accordance with the present invention
  • the concentration of the nucleotide polymerase is usually determined empirically
  • a concentration is used that is sufficient such that further increase in the concentration does not decrease the time for the amplification by over about 5-fold, preferably about 2-fold
  • the primary limiting factor generally is the cost of the reagent
  • the amount of the nucleic acid that is to be examined in accordance with the present invention can be as low as one or two molecules in a sample
  • the priming specificity of the primers used for the detection of a mutation and other factors will be considered with regard to the need to conduct an initial amplification of the first sequence It is within the purview of the present invention for detection of a mutation to carry out a preliminary amplification reaction to increase, by a factor of about 10 2 or more, the number of molecules of the first sequence
  • the amplification can be by any convenient method such as PCR, amplification by single primer, NASBA, and so forth, but will preferably be by PCR
  • the amount of the first sequence to be subjected to subsequent amplification using primers in accordance with the present invention may vary from about 1 to about 10 3 8 2 1
  • -10 -19 -1 in the medium may be about 10 to about 10 M, more usually about 10 to about
  • primer P1 should be removed prior to the extension of primer P3
  • an enzyme that can digest only single stranded DNA for example, an enzyme may be employed that has both 5' to 3' and 3' to 5' exonuclease activities, such as, e g , exo VII
  • the medium is incubated at a temperature and for a period of time sufficient to digest the primers Usually, incubation at about 20°C to about 40°C for a period of 10 to 60 minutes is
  • the amount of the oligonucleotide p ⁇ mer(s) used in the amplification reaction in the present invention will be at least as great as the number of copies desired and will usually be about 10-9 to about 10-3 M, preferably, about 10-7 to about 10-4 M
  • the concentration of the oligonucleotide ⁇ mer(s) is substantially in excess over, preferably at least about 100 times greater than, more preferably, at least about 1000 times greater than, the concentration of the first sequence
  • concentration of the nucleoside tnphosphates in the medium can vary widely, preferably, these reagents are present in an excess amount for both amplification and chain extension
  • the nucleoside tnphosphates are usually present in about 10-6 to about 10-2M, preferably about 10-5 to about 10-3M
  • the order of combining the various reagents may vary
  • the first sequence may be combined with a pre-prepared combination of primers nucleoside tnphosphates and nucleotide polymerase
  • the target nucleic acid for example, can be combined with certain primers together with the nucleoside tnphosphates and polymerase
  • the reaction mixture can be combined with the remaining primers As depicted in Fig 2, second sequence B, in the presence of primer P2 and primer
  • primer P3 is amplified in a polymerase chain reaction to produce amplicon BB
  • the amplification occurs using temperature cycling under the conditions described above in the presence of a nucleotide polymerase and nucleoside tnphosphates B is comprised of a sequence identical to A except for mutation M
  • primer P2 used for this amplification contains a label L2 that may be the same as or different than L1
  • Amplicon BB has two strands, a labeled strand derived from primer P2 and an unlabeled strand derived from primer P3
  • the unlabeled strand has end portion A1 of primer P3 and the labeled strand has corresponding end portion B2, which is the complement of A1
  • a chain extension of p ⁇ mer P1 along the labeled strand of amphcon BB occurs, under the conditions mentioned above for the chain extension of primer P3 along the labeled strand in duplex AA, to produce tailed reference partial duplex B' comprising the second sequence
  • primer P1 is comprised of portion Pa, which binds to the labeled strand of BB and portion B1 that does not bind to amphcon BB
  • the chain extension is carried out in the presence of a nucleotide polymerase and nucleoside tnphosphates under appropriate temperature conditions so that only the complement of the labeled strand is produced and not a copy
  • the extended primer P1 has a 5'-end portion B1 , which is not complementary to end portion B2 of the labeled strand of B'
  • A' and B' are related in that each of their labeled strands is complementary, except for mutation M, to the unlabeled strand of the other
  • labels L1 and L2 are incorporated into the partial duplexes that comprise complex C and provide a means for detection of complex C
  • L1 or L2 which comprises an sbp member or a reporter molecule
  • a receptor for the sbp member and a receptor that can bind to complex C by virtue of a feature other than L1 or L2 can both bind to complex C and provide a means for detection
  • a combination is provided in a single medium
  • the combination comprises (i) a sample containing a first sequence of within the gene of the strain of a microorganism where the region is suspected of having a mutation, (n) a second sequence of the wild type strain of the microorganism, which may be added separately if it is not known to be present in the sample, (in) a nucleotide polymerase, (iv) nucleoside tnphosphates, and (v) primers P1 , P2 and P3, wherein P2 may include primer P2 labeled with L1 and primer P2 labeled with L2, or P2 may be unlabeled and primers P1 and P3 may be labeled respectively with L1 and L2
  • the medium is then subjected to multiple temperature cycles of heating and cooling to simultaneously achieve all of the amplification and chain extension reactions as depicted in
  • PCR amplification of first sequence A and second sequence B each using primers P1 , P2 and P3, can be conducted in separate solutions
  • the solution can then be combined, heated to about 90°C to about 100°C to denature strands and then incubated as before at about 40°C to about 80°C to permit formation of duplexes and complex C when a mutation is present
  • Detection of complex C can then be carried out directly in the combined solutions or by adding reagents required for detection or by separating the complex C, for example, on a solid surface, and detecting its presence on the surface
  • primers PX1 , PX2, P1 , P2 and P3 may all be combined with the target and reference sequences prior to temperature cycling This is more readily seen in Fig 3, which depicts the initial amplification for a first sequence TS Two primers PX1 and PX2 are employed and bind to sites on TS that are upstream of the sites to which primers P1 and P2, respectively, bind These sites are indicated by
  • primers PX1 and PX2 when an initial amplification using primers PX1 and PX2 is carried out, these primers will be designed to anneal to the first sequence and the second sequence nucleic acids at a higher temperature than that for primers P1 , P2 and P3, respectively This is usually achieved by selecting PX1 and PX2 sequences that are longer or more GC rich than P2 and the Pa binding sequence in P1 and P3 The initial amplification is then carried out at temperatures that exceed the temperature required for binding P1 , P2 and P3 and the subsequent amplifications to form AA and BB are carried out at lower temperatures that permit P1 P2 and P3 to bind It is then possible to detect a mutation by combining the sequences, primers PX1 , PX2, P1 , P2 and P3 wherein P2 or P1 and P3 are labeled, polynucleotide polymerase, nucleotides tnphosphates, and optionally the reagents needed to detect complex C,
  • one means of detecting the quadramolecular complex involves the use of two labels on non-complementary strands
  • the labels become associated by virtue of both being present in the quadramolecular complex if a mutation is present
  • Detection of the two labels in the complex provides for detection of the complex
  • the association of the labels within the complex is detected
  • one of the labels can be an sbp member and a complementary sbp member is provided attached to a support
  • the complex becomes bound to the support and is separated from the reaction medium
  • the other label employed is a reporter molecule that is then detected on the support
  • the presence of the reporter molecule on the support indicates the presence of the complex on the support, which in turn indicates the presence of the mutation in the target nucleic acid sequence
  • ELISA enzyme-linked immunosorbent assay
  • the product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotomet ⁇ cally, or a product that can be detected by other spectromet ⁇ c or electromet ⁇ c means If the reporter molecule is a fluorescent molecule, the medium can be irradiated and the fluorescence determined Where the label is a radioactive group, the medium can be counted to determine the radioactive count
  • the association of the labels within the complex may also be determined by using labels that provide a signal only if the labels become part of the complex This approach is particularly attractive when it is desired to conduct the present invention in a homogeneous manner
  • labels include enzyme channeling immunoassay, fluorescence energy transfer immunoassay, electrochemilummescence assay, induced luminescence assay, latex agglutination and the like
  • detection of the complex is accomplished by employing at least one suspendable particle as a support, which may be bound directly to a nucleic acid strand or may be bound to an sbp member that is complementary to an sbp member attached to a nucleic acid strand
  • a particle serves as a means of segregating the bound first sequence from the bulk solution, for example, by settling, electrophoretic separation or magnetic separation
  • a second label which becomes part of the complex if a mutation is present, is a part of the signal producing system that is separated or concentrated in a small region of the solution to facilitate detection
  • Typical labels that may be used in this particular embodiment are fluo- rescent labels, particles containing a sensitizer and a chemiluminescent olefin (see U S Serial No 07/923,069 filed July 31 , 1992, the disclosure of which is incorporated herein by reference), chemiluminescent and
  • the particle itself can serve as part of a signal producing system that can function without separation or segregation
  • the second label is also part of the signal producing system and can produce a signal in concert with the particle to provide a homogeneous assay detection method
  • a variety of combinations of labels can be used for this purpose
  • the labels are limited to those that are stable to the elevated temperatures used for amplification, chain extension, and branch migration
  • polynucleotide or polynucleotide analogs having about 5 to about 20 or more nucleotides depending on the nucleotides used and the nature of the analog
  • Polynucleotide analogs include structures such as poly ⁇ bonucleotides, polynucleoside phosphonates, peptido-nucleic acids, polynucleoside phosphorothioates, homo DNA and the like
  • unchanged nucleic acid analogs provide stronger binding and shorter sequences can be used Included in the reaction
  • an oligonucleotide or polynucleotide analog attached to a reporter molecule or particle can bind to its complementary polynucleotide analog or oligonucleotide separated by an abasic site that has become incorporated into partial duplexes A' and B' as labels during amplification If the partial duplexes become part of a quadramolecular complex, the reporter molecule or particle becomes part of the complex
  • L1 and L2 two different reporter molecules or particles can become part of the complex Various combinations of particles and reporter molecules can be used
  • the particles may be simple latex particles or may be particles comprising a sensitizer, chemiluminescer, fluorescer, dye, and the like
  • Typical parti- cle/reporter molecule pairs include a dye crystallite and a fluorescent label where binding causes fluorescence quenching or a t ⁇ tiated reporter molecule and a particle containing a scintillator
  • Typical reporter molecule pairs include a fluorescent energy donor and a fluorescent acceptor dye
  • Typical particle pairs include (1) two latex particles, the association of which is detected by light scattering or turbidimetry, (2) one particle capable of absorbing light and a second label particle which fluoresces upon accepting energy from the first, and (3) one particle incorporating a sensitizer and a second particle incorporating a chemiluminescer as described for the induced luminescence immunoassay referred to in U S Serial No 07/704,569, filed May 22, 1991 , entitled "Assay Method Utilizing Induced Luminescence" (and corresponding European Patent Application
  • detection of the quadramolecular complex using the induced luminescence assay as applied in the present invention involves employing a photosensitizer as part of one label and a chemiluminescent compound as part of the other label If the complex is present the photosensitizer and the chemiluminescent compound come into close proximity The photosensitizer generates singlet oxygen and activates the chemiluminescent compound when the two labels are in close proximity The activated chemiluminescent compound subsequently produces light The amount of light produced is related to the amount of the complex formed
  • a particle which comprises the chemiluminescent compound associated therewith such as by incorporation therein or attachment thereto
  • the particles have a recognition sequence, usually an oligonucleotide or polynucleotide analog, attached thereto with a complementary sequence incorporated into one of the nucleic acid strands as a label, L1
  • Another particle is employed that has the photosensitizer associated therewith
  • These particles have a recognition sequence attached thereto, which is different than that attached to the chemiluminescent particles
  • a complementary sequence is incorporated as a label L2 in the nucleic acid strand in complex C that is not complementary to the nucleic acid strand carrying label L1
  • An amplification of the first sequence is carried out by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA
  • the nature of the primers is determined by the nature of the first sequence and in particular the nature of the predetermined region
  • Exemplary P1 (and P3) primers include, by way of illustration and not limitation, the following
  • each of the above sequences may further comprise at the 3'-end one or more nucleotides and/or modified nucleotides such as, for example, the group NNA wherein each N is independently a modified nucleotide.
  • One of the primers P1 , P2 and P3 comprises a label.
  • the primer P1 is comprised of a 3'-end portion Pa that can hybridize with the first sequence and 5'-end portion B1 that cannot hybridize with the first sequence.
  • B1 does not hybridize with the first sequence and is referred to above as an oligonucleotide tail.
  • the oligonucleotide tail B1 may be selected from the following:
  • oligonucleotide tails may be used.
  • the main criteria for a tail useful in the present invention are that the tail does not hybridize with the genome under the conditions of the interrogation and that the tail has a high Tm for hybridization at the temperature of branch migration.
  • a primer P3 is extended by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A'.
  • the primer P3 is comprised of the 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to the first sequence or its complement.
  • the nature of Pa and A1 is determined by the nature of the first sequence and of B1. In general, A1 and B1 are selected to have minimal homology to the target sequence and to each other.
  • oligonucleotide tail A1 does not hybridize with the first sequence, or with B1 , and is referred to above as an oligonucleotide tail.
  • the oligonucleotide tail A1 may be selected from the following: 5'- ACCATGCTCGAGATTACGGAG-3' (SEQ ID NO:9) and
  • a second sequence representing a region in wild type M tuberculosis that corresponds to the predetermined region is amplified, using the primer P2 and the primer P3, by polymerase chain reaction to produce amplicon BB Obviously, the nature of the second sequence is directly related to the nature of the first sequence
  • the primer P2 used for the second sequence comprises a label when the primer P2 referred to above with respect to the first sequence comprises a label and the primer P3 comprises a label when the primer P1 above comprises a label
  • the primer P1 is extended by chain extension along one strand of amp con BB to produce a tailed reference partial duplex B'
  • the tailed target partial duplex A' binds to the tailed reference partial duplex B'
  • the binding of one of the labels to another of the labels as a result of the formation of a complex between the tailed partial duplexes is detected The binding is directly related to the presence
  • sequence alteration in a sample polynucleotide that is representative of a mutation cluster is determined relative to a sequence that corresponds to the polynucleotide except for the mutation
  • This alteration is then related to the drug resistance characteristics of the organism from which the sample polynucleotide was obtained.
  • low signals obtained in the above process where induced luminescence is employed in detection indicate the identity of the sample polynucleotide relative to the reference sequence
  • Another embodiment of a method in accordance with the present invention is a method for detecting pyrazinamide resistance in a strain of M tuberculosis
  • the presence of at least one mutation in a first sequence representing a predetermined region within the pncA gene (see Example 4) of the strain is detected
  • An amplification of the first sequence is carried out by polymerase chain reaction as described above for the detection of rifampin resistance, using the following as primers P1 and P3
  • each of the above sequences may further comprise at the 3'-end one or more nucleotides and/or modified nucleotides such as, for example, the group nna wherein each "n" is independently a modified nucleotide
  • one of the primers P1 , P2 and P3 comprises a label
  • P2 comprises a label
  • the primer P1 is comprised of a 3'-end portion Pa that can hybridize with the first sequence and 5'-end portion B1 that cannot hybridize with the first sequence B1 does not hybridize with the first sequence and is referred to above as an oligonucleotide tail
  • the oligonucleotide tail B1 may be selected from the following 5'-accatgctcgagattacgag-3' (SEQ ID NO 9) 5'-gatcctaggcctcacgtatt-3' (SEQ ID NO 10)
  • a primer P3 is extended by chain extension along one strand of amphcon AA to produce a tailed target partial duplex A'
  • the primer P3 is comprised of the 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to the first sequence or its complement
  • the nature of Pa and A1 is determined by the nature of the first sequence and of B1
  • A1 and B1 are selected to have minimal homology to the target sequence and to each other A1 does not hybridize with the first sequence, or with B1 , and is referred to above as an oligonucleotide tail
  • the oligonucleotide tail A1 may be selected from the following 5'-accatgctcgagattacgag-3' (SEQ ID NO 9) and 5'-gatcctaggcctcacgtatt-3' (SEQ ID NO 10), which are set forth above
  • a second sequence representing a region in wild type M tuberculosis that corresponds to the predetermined region is amplified, using the primer P2 and the primer P3, by polymerase chain reaction to produce amphcon BB
  • the primer P2 used for the second sequence comprises a label when the primer P2 referred to above with respect to the first sequence comprises a label and the primer P3 comprises a label when the primer P1 above comprises a label
  • the primer P1 is extended by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex B'
  • the tailed target partial duplex A' binds to the tailed reference partial duplex B'
  • the binding of one of the labels to another of the labels as a result of the formation of a complex between the tailed partial duplexes is detected The binding is directly related to the presence of the mutation
  • the presence of the mutation is related to the pyrazinamide resistance of the strain of M tuberculosis As a
  • primer P2 can be labeled, but primers P1 and P3 alternatively may be labeled
  • the kit can also include reagents for conducting an amplification of the first sequence prior to subjecting the first sequence to the methods of the present invention
  • the kit can also include nucleoside tnphosphates and a nucleotide polymerase
  • the kit can further include two additional oligonucleotide primers PX1 and PX2 where the primers are related in that a product of the extension of one along the first sequence serves as a template for the extension of the other
  • the kit can further include particles as described above capable of binding to the label on at least one of the primers
  • the kit can further include members of a signal producing system and also various buffered media, some of which may contain one or more of the above reagents
  • primers PX1 , PX2, P1 , P2 and P3 are packaged in a single container More preferably, at least all of the above components other than buffer are packaged in a single
  • kits for this embodiment can comprise in packaged combination (a) a second sequence representing a sequence within a wild type strain that corresponds to the first sequence, (b) a 5'-labeled primer P2, which is 5'- L-tggtgccgcgtcggtagg-3' (SEQ ID NO 21) wherein two different labels are employed for each labeled primer P2 as discussed above and wherein the above sequence may further comprise at the 3 -end one or more nucleotides and/or modified nucleotides such as, for example, the group nnt wherein each "n" is independently a modified nucleotide, and (c) 5'-ta ⁇ led primers P1 and P3 5'-accatgctcgacattacgagtcaggagctgcaaaccaactc-3' (SEQ ID NO 22) 5'-gatcctag
  • kits for detecting the presence of a difference between two related nucleic acid sequences in an organism
  • the kit comprises in packaged combination (a) two oligonucleotide primers each comprising a sequence that is hybridizable to the nucleic acid sequences in a region adjacent the site of potential difference and each comprising a label wherein the labels are different, and (b) a lysing agent
  • kits can be varied widely to provide for concentrations of the reagents that substantially optimize the reactions that need to occur during the present method and to further substantially optimize the sensitivity of the method in detecting a mutation
  • one or more of the reagents in the kit can be provided as a dry powder, usually lyophilized, including ex pients, which on dissolution will provide for a reagent solution having the appropriate concentrations for performing a method in accordance with the present invention
  • Each reagent can be packaged in separate containers or some or all of the reagents can be combined in one container where cross-reactivity and shelf life permit
  • the reagents are packaged in a single container
  • the kits may also include a written description of a method in accordance with the present invention as described above
  • T ⁇ s - T ⁇ s(hydroxymethyl)am ⁇ nomethane-HCI (a 10X solution) from BioWhittaker, Walkersville, MD BSA - bovine serum albumin from Gibco BRL, Gaithersburg MD bp - base pairs wt (+) - wild type allele mut (-) - mutant allele sec - seconds hr - hours mm - minutes
  • Buffer A 10mM T ⁇ s-HCI (pH8 3), 50mM KCI, 1 5 ⁇ M MgCI 2 200 ⁇ g/ml BSA
  • Buffer B 100mM T ⁇ s-HCI (pH8 3), 500mM KCI, 15mM MgCI 2 200 ⁇ g/ml BSA
  • Buffer C - 0 1 M T ⁇ s, 0 3M NaCI, 25 mM EDTA, 0 1 % BSA, 0 1 % dextran T-500, a 1 320 dilution of mouse IgG (HBR-1 from Scantibodies Laboratory Inc , Los Angeles, CA), 0 05% Kathon (Rohm and Haas, Philadelphia, PA), and 0 01 % gentamycin sulfate Buffer D - 10 mM T ⁇ s-HCI, pH 8 3, 50 mM KCI, 1 5 mM MgCI 2 , and 200 ⁇ g/ml
  • oligodeoxy ⁇ bonucleotides PCR primers
  • modified oligodeoxy ⁇ bonucleotides were obtained from Oligo Etc , Inc (Wilsonville, OR)
  • Molecular weight size markers 50 to 2 kb) were purchased from Bio-Rad (Hercules, CA) Cloned Pfu polymerase and Turbo Pfu were purchased from Stratagene ( La Jolla, CA) 2'- deoxynucleoside 5'-t ⁇ phosphates (dNTPs) was purchased as 100 mM solution from Pharmacia Biotech (Piscataway, NJ)
  • Bovine Serum Albumin (BSA) was purchased from Gibco LifeTech (Gaithersburg, MD)
  • PCR tubes were purchased from Corning and ISC Bioexpress (Kaysville, UT) Native 4- 10% precast gradient polyacrylamide gels were purchased from Novex and used to analyze the amplification products (L
  • Monoclonal antibodies were produced by standard hybrid cell technology Briefly, the appropriated immunogen was injected into a host, usually a mouse or other suitable animal, and after a suitable period of time the spleen cells from the host were obtained Alternatively, unsensitized cells from the host were isolated and directly sensitized with the immunogen in vitro Hybrid cells were formed by fusing the above cells with an appropriate myeloma cell line and cultu ⁇ ng the fused cells The antibodies produced by the cultured hybrid cells were screened for their binding affinity to the particular antigen, dig-BSA conjugate A number of screening techniques were employed such as, for example, ELISA screens Selected fusions were then recloned
  • Hydroxypropylaminodextran (1 NH 2 / 7 glucose) was prepared by dissolving Dextran T-500 (Pharmacia, Uppsala, Sweden) (50 g) in 150 L of H 2 0 in a 3-neck round- bottom flask equipped with mechanical stirrer and dropping funnel To the above solution was added 18 8 g of Zn(BF 4 ) 2 and the temperature was brought to 87°C with a hot water bath Epichlorohyd ⁇ n (350 mL) was added dropwise with stirring over about 30 mm while the temperature was maintained at 87-88°C The mixture was stirred for 4 hr while the temperature was maintained between 80°C and 95°C, then the mixture was cooled to room temperature Chlorodextran product was precipitated by pouring slowly into 3 L of methanol with vigorous stirring, recovered by filtration and dried overnight in a vacuum oven
  • the chlorodextran product was dissolved in 200 mL of water and added to 2 L of concentrated aqueous ammonia (36%) This solution was stirred for 4 days at room temperature, then concentrated to about 190 mL on a rotary evaporator The concentrate was divided into two equal batches, and each batch was precipitated by pouring slowly into 2 L of rapidly stirring methanol The final product was recovered by filtration and dried under vacuum
  • Hydroxypropylaminodextran (1 NH 2 / 7 glucose), prepared above, was dissolved in 50 mM MOPS, pH 7 2, at 12 5 mg/mL The solution was stirred for 8 hr at room temperature, stored under refrigeration and centrifuged for 45 mm at 15,000 rpm in a Sorvall RC-5B centrifuge immediately before use to remove a trace of solid material To 10 mL of this solution was added 23 1mg of Sulfo-SMCC in 1 mL of water This mixture was incubated for 1 hr at room temperature and used without further purification C-28 thioxene was prepared as follows
  • the following dye composition was employed 20% C-28 thioxene (prepared as described above), 1 6% 1-chloro-9,10-b ⁇ s(phenylethynyl)anthracene (1-CI-BPEA) (from Ald ⁇ ch Chemical Company) and 2 7% rubrene (from Ald ⁇ ch Chemical Company)
  • the particles were latex particles (Seradyn Particle Technology, Indianapolis IN)
  • the dye composition (240-250 mM C-28 thioxene, 8-16 mM 1-CI-BPEA, and 20-30 mM rubrene) was incorporated into the latex beads in a manner similar to that described in U S Patent 5,340,716 issued August 23, 1994 (the 716 patent), at column 48, lines 24-45, which is incorporated herein by reference
  • the dyeing process involved the addition of the latex beads (10% solids) into a mixture of ethylene glycol (65 4%), 2-ethoxyethanol (32 2%) and 0 1 N NaOH (2 3%) The beads
  • Silicon tetra-t-butyl phthalocyanine was prepared as follows
  • the sensitizer beads were prepared placing 600 mL of carboxylate modified beads (Seradyn) in a three-necked, round-bottom flask equipped with a mechanical stirrer, a glass stopper with a thermometer attached to it in one neck, and a funnel in the opposite neck
  • the flask had been immersed in an oil bath maintained at 94+ /-1°C
  • the beads were added to the flask through the funnel in the neck and the bead container was rinsed with 830 mL of ethoxyethanol, 1700 mL of ethylene glycol and 60 mL of 0 1 N NaOH and the rinse was added to the flask through the funnel
  • the funnel was replaced with a 24- 40 rubber septum
  • the beads were stirred at 765 rpm at a temperature of 94+ /-1°C for 40 mm
  • Silicon tetra-t-butyl phthalocyanine (10 0 g) was dissolved in 300 mL of benzyl alcohol
  • Streptavidin was bound to the above beads using 25 mg streptavidin for 100 mg of beads 25 mg streptavidin (50 mg Aaston solid from Aaston, Wellesley, MA) was dissolved in 1 mL of 1 mM EDTA, pH 7 5, and 77 ⁇ L of 2 5 mg/mL SATA in ethanol was added thereto The mixture was incubated for 30 mm at room temperature A deacetylation solution was prepared containing 1M hydroxylamine-HCI, 50 mM Na 2 P0 4 , 25 mM EDTA, pH 7 0 0 1 mL of this deacetylation solution was added to the above solution and incubated for 1 hr at room temperature The resulting thiolated streptavidin was purified on a Pharmacia PD10 column and washed with a column buffer containing 50 mM MOPS, 50 mM EDTA, pH 7 2 The volume of the sample was brought to 2 5 mL by adding 1 5 mL of the above column buffer
  • N etheno dA modification
  • the position of hybridization of the primers to the rpoB gene sequence is indicated by the primer number The number indicates the target nucleotide complementary to the 5'-end of the primer (shown in bold) In the case of the reverse primers the position is related to the complementary sequence only not including tail 1 or tail 2
  • the positions of the forward and reverse PCR primers are denoted in Fig 4, detailing the full sequence of the rpoB gene (GenBank Accession No 012205) (SEQ ID NO 11)
  • the forward PCR primers are 5'-labeled with biotin or digoxigenm (Dig )
  • the reverse PCR primers are composed of two parts The 3'-port ⁇ on of the primers is identical 3 1
  • tail 1 and tail 2 are designed to form the tails of the heteroduplexes, which upon annealing result in the formation of a four-stranded DNA structure or quadramolecular complex
  • PCR amplification of the rpoB gene sequence PCR amplification of the rpoB gene sequence was carried out using a hot start procedure referred to as the wax bead-based method, in which commercially available PCR gems (AmpliWax from Perkin Elmer) were utilized.
  • the choice of primers and conditions for PCR amplification provided for specific and efficient production of PCR derived substrates for subsequent analysis
  • the high GC content of the specific sequence of M tuberculosis rpoB gene also influences the effectiveness of amplification
  • the amplification conditions described in the following section were selected for maximum specificity of the present detection method
  • test target was carried out using 5'-b ⁇ ot ⁇ n-labeled forward primer and two related reverse primers
  • PCR amplification with wax bead-based hot start was carried as follows A master mixture (Mix 1) containing 10 mM T ⁇ s-HCI pH 8 3 50 mM KCI, 1 5 mM MgCI 2 , 0 2 mg/ml BSA, 200 ⁇ M of each of the four dNTPs, and 250 nM of each of the primers was prepared 25 ⁇ l of Mix 1 was added to PCR tubes containing a wax gem, and the tubes were incubated at 80°C for 2 minutes to melt the wax gems The reaction tubes were then cooled to room temperature to form the wax barrier on top of the liquid reaction mixture A second reaction mixture containing 10 mM T ⁇ s-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI 2 , 0 2 mg/ml BSA and 2 5 U/25 ⁇ l of Pfu DNA polymerase was also prepared 20 ⁇ l of Mix 2 and 5 ⁇ l of test or reference target were added to each of the reaction tubes prepared as above PCR
  • Example 2 Direct genotypic detection of M tuberculosis rifampin resistance was carried out using cells grown in culture M tuberculosis clinical isolates grown in culture were suspended in 1X or 10X buffer (100 mM T ⁇ s-HCI pH 8 3, 500 mM KCI, 15 mM MgCI 2 and 2 mg/ml BSA) (IHBB) and heat inactivated by boiling for 30 mm at 95°C Direct analysis was achieved following sonication (12-15 pulse on Branson Son ⁇ f ⁇ er 450 ), heat-treatment (98°C for 15-m ⁇ n , using thermocycler) or treatment in a microwave (25 sec to 1-m ⁇ n at high power setting) The direct analysis of 10 M tuberculosis clinical isolates, obtained using cell suspensions pretreated as detailed above, are shown in Fig 6, demonstrating the feasibility of direct genotypic detection procedure The procedure is suitable for use in a clinical microbiology laboratory
  • RLU-1 * relative luminescence unit for M. tuberculosis cells heat killed in Buffer A
  • RLU-2* * relative luminescence unit for M. tuberculosis cells heat killed in Buffer B
  • Detection of genotype of M. tuberculosis associated with rifampin resistance As shown in previous examples, the branch migration method was effective in the detection of rpoB gene alteration. Signals were indicative of sequence alteration of test sequence relative to reference sequence. Low signals indicated identity of the test sequence to reference sequence. However, since the signal was related also to the amount of test target relative to the reference target, low input test target may also result in low signal, which may lead to false determination of mutant genotype as a wild type genotype.
  • the following experiments demonstrate a method for detection of genotype associated with rifampin resistance using a normalization of the signals relative to input test target.
  • the normalization method is based on formation of stable four stranded DNA structures when test target amplification products are mixed with similarly produced products of amplification of non-relevant reference sequence. In so far as the two sequences are not related, signals are produced from all test samples, regardless of the specific genotype. The ratio of signals produced with relevant sequence to those produced with non-relevant sequence are the normalized signals and represent test genotype regardless of input target sequence.
  • test DNA amplification products were tested against relevant wild type target genomic DNA and non-relevant reference sequence, in this case amplification products of the human cystic fibrosis gene. Amplification of the non-relevant reference sequence was carried out with reverse primers designed for branch migration analysis and composed of a 3' target specific portion and 5' tails, which were the same as those used for the M. tuberculosis rpoB analysis. This feature is important for the ability of forming four stranded structures between the test and non- relevant amplification products.
  • PCR amplification of the rpoB gene sequence was carried out using one of two hot start procedures: one such procedure was the wax bead-based method using commercially available PCR gems (AmpliWax from Perkin Elmer), the other such procedure involved the use of an anti-etheno A monoclonal antibody, which binds to the primers, which comprise etheno-dA moieties at their respective 3-'ends, until the temperature of the reaction medium is raised whereupon the antibody dissociates from the primers and is denatured.
  • the choice of primers and conditions for PCR amplification are chosen for specific and efficient production of PCR derived substrates for subsequent branch migration analysis in accordance with the present invention.
  • the high GC content of the specific sequence of M. tuberculosis rpoB gene also influenced the effectiveness of amplification.
  • the amplification conditions described in the following were selected for maximum specificity of the present mutation detection method.
  • test target was carried out using 5'-biotin labeled forward primer and two related reverse primers.
  • the reference target wild type, was amplified using the corresponding 5'-dig labeled forward primer and the same set of reverse primers as for the test target amplification.
  • PCR amplification with wax bead based hot start was carried out as follows A master mixture (Mix 1) containing 10 mM T ⁇ s-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI 2 , 0 2 mg/ml BSA, 0 5 ⁇ M of each of the four dNTPs, and 0 5 ⁇ M of each of the primers, was prepared 25 ⁇ l of Mix 1 was added to PCR tubes containing a Wax gem, and the tubes were incubated at 80°C, for 2 minutes, to melt the wax gems The reaction tubes were then cooled to room temperature, to form the wax barrier on top of the liquid reaction mixture A second reaction mixture (Mix 2) containing 10 mM T ⁇ s-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI2, 2 mg/ml BSA and 2 5 U/25 ⁇ l of Pfu DNA polymerase, was also prepared 20 ⁇ l of Mix 2 and 5 ⁇ l of test or reference target were added
  • PCR amplification using the antibody-based hot start procedure was carried out as follows a master reaction mixture containing 10 mM T ⁇ s-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI2, 0 25 mM of each of four dNTPs, 0 2 mg/ml BSA, 0 5 ⁇ M anti etheno A monoclonal antibody (from Inst fur Zelibiologie, Dr Petra Lorenz), and 1 25 U/25 ⁇ l Pfu DNA polymerase, was prepared 2 5 ⁇ l of test or reference target was added to 22 5 ⁇ l of the master reaction mix, in PCR tubes PCR amplification was carried out using conditions similar to the above PCR amplification of non-relevant target was carried out using the antibody-based hot start procedure as described above All of the reagents were the same as stated above except the target was cystic fibrosis exonl 1 (wild type) and the primers were a mixture of 5'-b ⁇ ot ⁇ n and 5'-d
  • the f2/rl primer set flanks 173 bases of the CFTR Exon 11 sequence, resulting in an amplicon which includes 217 bases from Exon 11 and 20 bases from the reverse primer tails, for a total of 237 bp.
  • Genomic DNA preparations of M. tuberculosis pyrazinamide resistant isolates and 29 genomic DNA preparations of wild type M. tuberculosis isolates were obtained from Dr. Peter Small at Stanford Medical Center and used in this study. Genomic DNA preparation of other
  • Mycobacteria strains were also obtained from Stanford University (P. Small).
  • gagttggttt gcagctcctg a (SEQ ID NO:20) 1.
  • the nucleotides denoted in bold letters represent sites in the pncA gene sequence of known mutations, which result in PZA resistance.
  • Oligonucleotide Seguences (PCR primers 5' to 3') M tuberculosis FP/(B ⁇ o/D ⁇ g)39ue 5'- B ⁇ o(D ⁇ g)-tggtgccgcgtcggtaggnnt-3' (SEQ ID NO 25)
  • SEQ ID NO 27 The above sequences correspond to SEQ ID NO 21 , SEQ ID NO 22 and
  • PCR amplification PCR amplification with wax bead based hot start was carried out as follows A master mixture (Mix 1) containing 10 mM T ⁇ s-HCI pH 8 3, 50 mM KCI, 1 5 mM MgCI2, 0 2mg/ml BSA, 200 ⁇ M of each of the four dNTPs, and 250 nM of each of the primer was prepared 25 ⁇ l of Mix 1 was added to PCR tubes containing a Wax gem, and the tubes were incubated at 80°C for 2 minutes to melt the wax gems The reaction tubes were then cooled to room temperature to form the wax barrier on top of the liquid reaction mixture.
  • a second reaction mixture (Mix 2) containing 10 mM Tris-HCI pH 8.3, 50 mM KCI, 1.5 mM MgCI 2 , 0.2 mg/ml BSA and 2.5 U/25 ⁇ l of Pfu DNA polymerase was also prepared. 20 ⁇ l of Mix 2 and 5 ⁇ l of test DNA or reference DNA (10 4 molecules) were added to each of the reaction tubes prepared as above. PCR amplification was carried out in the T3 thermocycler (Biometra Inc.). The following thermocycling protocol was used: The reaction mixture was first heated to 95°C for 4 min. for denaturation of the genomic DNA, followed by 39 cycles of 30 sec at 95°C, 1 min. at 64°C and 1 min. at 72°C, followed by 5 min. at 72°C for final extension. The amplification product generated by the disclosed primers is 620 bp long.
  • PCR amplification of non-relevant target was carried out the same way as above. All the reagents are the same as stated above except the target is cystic fibrosis exonl 1 genomic DNA (wild type) and the primers are f2/5'-dig labeled forward primer and a mixture of reverse primers modified with the same tail-1 and tail-2 (r1t1 &r1t2) as for M. tuberculosis.
  • Mutation analysis and detection were performed as follows: 1 ⁇ l of post-PCR amplification reaction mixture of reference target and 1 ⁇ l of post-PCR amplification reaction mixture of test target were added to PCR tubes containing 4 ⁇ l of 10x buffer (100 mM Tris-HCI pH8.3, 500 mM KCI, 15 mM MgCI2 and 2 mg/ml BSA). The mixtures were incubated for 2 min at 95°C followed by 30 min at 65°C, in a thermocycler. 50 ⁇ l of bead mixture that contained 2.5 ⁇ g of streptavidin-coated sensitizer beads and 1.25 ⁇ g of anti- dig-coated acceptor beads were added to each tube and the tubes were incubated at 37°C for 30 min.
  • 10x buffer 100 mM Tris-HCI pH8.3, 500 mM KCI, 15 mM MgCI2 and 2 mg/ml BSA.
  • Signal was read as previously described (3 cycles of 1 sec. illumination and 1 sec. read). Normalization of the signal reading was carried out as follows: 1 ⁇ l of post-PCR amplification reaction mixture of non-relevant target (wild type Cystic Fibrosis genomic DNA, 1/10 dilution) and 1 ⁇ l of test post PCR amplification reaction mixture of test target were added to PCR tubes containing 4 ⁇ l of 10x buffer (100 mM Tris-HCI pH8.3, 500 mM KCI, 15 mM MgCI 2 and 2 mg/ml BSA). The mixtures were incubated for 2 min at 95°C followed by 30 min at 65°C, in a thermocycler.
  • 10x buffer 100 mM Tris-HCI pH8.3, 500 mM KCI, 15 mM MgCI 2 and 2 mg/ml BSA.
  • the ratios of signals obtained from the mixtures of test post-PCR amplification reaction products and reference (wt M tuberculosis) post-PCR amplification products (S1) to the signals, similarly obtained from mixtures of the test post-PCR amplification products and the non-relevant reference post amplification products (S2), represent the normalized signals PCR products were also detected by gel electrophoresis 5 ⁇ l reaction mixture and 5 ⁇ l dye solution were mixed and subjected to electrophoresis using native 4-20% gradient polyacrylamide gel in T ⁇ s-glycme native gel buffer followed by staining with ethidium bromide
  • the primers used included two forward primers having the same sequence and labeled at the 5' end with either biotin or digoxigenm, and two reverse primers each having a 3' portion that was complementary to the target and was the same for both reverse primers, and 5' tail sequences that are not complementary to the target (test or reference) and are not complementary to each other These primers generated 620 bp long amplification products
  • the amplification products were labeled with either biotin or digoxigenm and had the tail sequences at one end
  • the test target was amplified with biotin labeled forward primer, and the reference target with a digoxigenm labeled forward primer Amplification reaction of both test and reference targets was
  • a total of 37 M tuberculosis isolates were tested in accordance with the present invention for genotypic determination of PZA susceptibility using the disclosed primers Included were 29 phenotypically wild type strains (used in the previous demonstration of determination of rifampin resistance in M. tuberculosis) and 8 phenotypically PZA resistant isolates (all obtained from Stanford University, P. Small).
  • the genotypic determination of PZA resistance is shown in Fig. 8.
  • the results demonstrate perfect correlation of the phenotypic and genotypic determination, with very strong discrimination of susceptible and resistant genotypes.
  • the normalized signals of the analysis for the detection of PZA resistant M. tuberculosis are given in Table 5.

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Abstract

Cette invention se rapporte à un procédé permettant de détecter la pharmacorésistance d'une souche d'un organisme. Dans ce procédé, on détecte la présence d'au moins une mutation dans une région prédéterminée du gène de la souche. Cette région prédéterminée comporte une multitude de mutations parmi les souches de l'organisme qui diffèrent d'une région correspondante de la souche de type sauvage de l'organisme. Pour détecter cette mutation, on forme un complexe comprenant la région prédéterminée du gène de l'organisme et la région correspondante du gène de l'organisme de type sauvage sous forme de double brin. Chaque élément d'au moins une paire de brins non complémentaires du complexe possède une étiquette. L'association des étiquettes à l'intérieur du complexe est détectée, de telle sorte que l'association des étiquettes dans le complexe est mise en relation avec la présence de la mutation. La présence de la mutation est mise en relation avec la pharmacorésistance de la souche.
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WO2003025227A1 (fr) * 2001-09-18 2003-03-27 Gen-Probe Incorporated Detection de sequences de rpob de mycobacterium tuberculosis
US7094542B2 (en) 2001-09-18 2006-08-22 Gen-Probe Incorporated Detection of rpoB sequences of Mycobacterium tuberculosis
EP2140033A1 (fr) * 2007-03-28 2010-01-06 Signal Diagnostics Systeme et procede pour l'analyse de resolution elevee d'acides nucleiques pour detecter des variations de sequence
US10370707B2 (en) 2013-10-09 2019-08-06 Fluoresentric, Inc. Multiplex probes
US10669574B2 (en) 2008-11-18 2020-06-02 XCR Diagnostics, Inc. DNA amplification technology
CN114107300A (zh) * 2021-11-30 2022-03-01 宁夏医科大学 基于CRISPR-Cas技术检测结核分枝杆菌利福平耐药性点突变的crRNA

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WO1997023646A1 (fr) * 1995-12-22 1997-07-03 Behringwerke Aktiengesellschaft Detection de differences dans les acides nucleiques
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025227A1 (fr) * 2001-09-18 2003-03-27 Gen-Probe Incorporated Detection de sequences de rpob de mycobacterium tuberculosis
US7094542B2 (en) 2001-09-18 2006-08-22 Gen-Probe Incorporated Detection of rpoB sequences of Mycobacterium tuberculosis
AU2001294599B2 (en) * 2001-09-18 2007-06-28 Biomerieux S.A. Detection of rpoB sequences of Mycobacterium tuberculosis
US7326533B2 (en) 2001-09-18 2008-02-05 Gen-Probe Incorporated Detection rpoB sequences of Mycobacterium tuberculosis
US7326532B2 (en) 2001-09-18 2008-02-05 Gen-Probe Incorporated Detection of rpoB sequences of mycobacterium tuberculosis
US9139882B2 (en) 2007-03-28 2015-09-22 Fluoresentric, Inc. System and method for high resolution analysis of nucleic acids to detect sequence variations
EP2140033A4 (fr) * 2007-03-28 2010-08-04 Signal Diagnostics Systeme et procede pour l'analyse de resolution elevee d'acides nucleiques pour detecter des variations de sequence
US7838235B2 (en) 2007-03-28 2010-11-23 Signal Diagnostics System and method for high resolution analysis of nucleic acids to detect sequence variations
EP2140033A1 (fr) * 2007-03-28 2010-01-06 Signal Diagnostics Systeme et procede pour l'analyse de resolution elevee d'acides nucleiques pour detecter des variations de sequence
US9353408B2 (en) 2007-03-28 2016-05-31 Fluoresentric, Inc. Dynamic flux nucleic acid sequence amplification
US9670531B2 (en) 2007-03-28 2017-06-06 Fluoresentric, Inc. Dynamic flux nucleic acid sequence amplification
US10337056B2 (en) 2007-03-28 2019-07-02 Fluoresentric, Inc. Dynamic flux nucleic acid sequence amplification
US10669574B2 (en) 2008-11-18 2020-06-02 XCR Diagnostics, Inc. DNA amplification technology
US10370707B2 (en) 2013-10-09 2019-08-06 Fluoresentric, Inc. Multiplex probes
CN114107300A (zh) * 2021-11-30 2022-03-01 宁夏医科大学 基于CRISPR-Cas技术检测结核分枝杆菌利福平耐药性点突变的crRNA
CN114107300B (zh) * 2021-11-30 2023-10-27 宁夏医科大学 基于CRISPR-Cas技术检测结核分枝杆菌利福平耐药性点突变的crRNA

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