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  • 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
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6862—Ligase chain reaction [LCR]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• the invention relates to oligonucleotides useful for detecting bacteria of the genus Mycobacterium. This invention also relates to methods useful in amplifying and/or detecting bacteria of the genus Mycobacterium. 5 BACKGROUND OF THE INVENTION
• tuberculosis the causative agent of tuberculosis, most commonly occurs by inhalation of droplets containing only a few live bacilli.
• the mycobacteria replicate in lung tissue to form a primary focus of infection and o from there enter the local lymphatic system.
• the infection then disseminates widely through the body via the blood and lymphatic system.
• the initial lesions usually heal to form tiny granulomas that may harbor viable tubercle bacilli indefinitely.
• Post-primary tuberculosis is the most common form of clinical tuberculosis and is usually pulmonary.
• the disease can occur many years after initial infection and is thought to be due to a 5 temporary loss or diminishing of cell-mediated immunity (due to, for example, increasing age, illness, malnutrition, or alcoholism) leading to reactivation of dormant tubercle bacilli in lesions.
• Acquired Immunodeficiency Syndrome AIDS
• a tentative diagnosis of tuberculosis or other mycobacterial diseases may be made on a basis of clinical grounds, radiology, and on the finding of acid-fast bacilli in smears of sputum, blood bronchial-lavage, gastric-washing, urine, or cerebral spinal fluid.
• Smears of these specimens typically are stained by the Ziehl-Neelsen technique or by fluorescent rhodamine-aurarnine dye and examined by microscopy.
• 5 microscopically positive sputum samples are found in only about 30 to 50 percent of pulmonary tuberculosis patients and thus, cultures must always be performed. Culturing for the presence of M. tuberculosis and other mycobacterial species is a time-consuming and difficult process because some mycobacterial species are very slow-growing and have fastidious nutritional requirements. While some samples may be inoculated directly onto o culture medium, most specimens require decontamination with, for example, strong alkali.
• Specimens are then typically inoculated onto egg-based media such as Lowenstein- Jensen medium and/or defined media such as Middlebrook 7H9 broth in the presence of antibiotics such as penicillin to inhibit growth of other bacteria. Cultures are then incubated at 35 C to 37 C for 1-7 weeks. However, even culture techniques are only about 70% effective.
• Hogan, et al. a method is described for the detection of non-viral organisms including M. tuberculosis and M. intracellulare using nucleic acid hybridization techniques.
• the method comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a region of ribosomal RNA (rRNA) selected to be unique to a particular non- o viral organism or a group of non-viral organisms sought to be detected.
• the target rRNA is selected by comparing one or more variable region rRNA sequences of the non-viral organisms of interest with one or more variable region rRNA sequences from one or more non-viral organisms sought to be distinguished.
• Probe sequences which are specific for 16S rRNA variable subsequences of Mycobacterium avium, Mycobacterium intracellulare, 5 and the Mycobacterium tuberculosis-complex bacteria and which do not cross-react with nucleic acids from each other or any other bacterial species under proper stringency are identified.
• a probe specific to three 23S rRNA variable region subsequences from the Mycobacterium tuberculosis-complex bacteria is also disclosed as are rRNA variable region probes useful in hybridization assays for bacteria of the genus Mycobacterium o (15S, 23S rRNA specific); for Mycobacterium avium in the region corresponding to bases
• the patent describes the cloning and sequencing of a repetitive element found in M. tuberculosis chromosomal DNA and further describes the use of these cloned repetitive elements as probes and primers for the detection of representative strains of the M. tuberculosis complex.
• U.S. Patent No. 5,183,737 to Crawford et al. (divisional of U.S. Patent No. 5,168,039 described above) teaches the use of the aforementioned repetitive elements as primers for the detection of bacteria of M. tuberculosis complex using the polymerase chain reaction.
• the patent also teaches the use of the repetitive elements as probes for the detection of bacteria of the M. tuberculosis complex using a membrane based nucleic acid hybridization assay.
• PCT Application WO90/15,157 by Lane etal. published December 13, 1990 addresses "universal" nucleic acid probes for eubacteria and methods for the detection of bacteria.
• the application describes nucleic acid probes and probe sets which hybridize under specific conditions to the ribosomal RNA molecules (rRNA), rRNA genes (rDNA), and certain amplification and in vitro transcription products thereof but which do not hybridize under the same conditions to rRNA or rDNA of eukaryotic cells which may be present in test samples. More specifically, the probes described in this application are specifically complementary to certain highly conserved bacterial 23 S or 16S rRNA sequences.
• the probes were selected using a computer algorithm operating on aligned sets of 16S and 23S rRNA sequences to identify regions of greatest similarity among the eubacteria. Nucleic acid probes so derived hybridize most widely among diverse bacterial species. Probes found homologous among the bacteria species were also assessed for differences with non-bacterial rRNA sequences using a computer algorithm. Ultimately, 41 probes were selected based on these analysis; 22 targeting 23S rRNA and 19 targeting 16S rRNA.
• the 16S amplification primers described in that application include primers which detect most eubacteria, Borrelia and spirochetes, the enterics, Deinococcus, Campylobacter, and the Fusobacteria and Bacillus species.
• probes are assertedly capable of detecting Mycobacteria kansasii and M. bovis.
• the application describes the use of sandwich type hybridization assays for the detection of hybridization between probes and rRNAs in samples suspected of containing bacteria and also describes the use of the polymerase chain reaction (PCR) directed to 16S rRNAs 5 using probes as derived above.
• PCR polymerase chain reaction
• the probes described in that application both 16S, and 23S rRNA probes were assertedly able to detect a wide variety of bacterial species.
• PCT application WO90/12,875 by Hance et al. published November 1, 1990 discloses nucleotide sequences of actinomycetales and their application to the synthesis or detection of nucleic acids found in actinomycetales.
• the application discloses 0 a 383 base pair polynucleotide coding for the 65 kD (kilodalton) mycobacterial antigen which has homologs in 8 species of mycobacteria including M. tuberculosis, M. avium, M.fortuitum, M. paratuberculosis, BCG, M. kansasii, M. malmoense, and M. marinum.
• bovis bovis bovis bovis were obtained from a variable intergenic region intermediate the 16S ribosomal RNA gene and the 23S ribosomal rRNA gene and from the V6 variable region of the gene coding for 16S . ribosomal RNA.
• a DNA probe for M. tuberculosis was obtained from the o variable intergenic region intermediate the 16S and 23S ribosomal RNA genes.
• Protein Antigen B gene of Mycobacterium tuberculosis was described by Anderson and Hanson, Inf. and Immun. 57:2481-2488 (1989).
• the pab gene is 1993 nucleotides in length.
• the deduced amino acid sequence of the pab gene reveals 30% homology to a phosphate-binding protein 5 (Ps+S) from E. coli.
• Protein antigen b was selected for analysis because of its association with virulent strains of M. tuberculosis and, to a lesser extent, with M. bovis BCG and because BCG has been used with some success as a vaccine against tuberculosis. See, e.g., Hart, P. D. and Sutherland, I., British Medical Tuberculosis Journal 2:293-295 (1977).
• Baird et al. J. Gen. Microbiol. 135: 931-939 (1989) have cloned and sequenced the 10 kD antigen gene of Mycobacterium tuberculosis.
• the 10 kD antigen has been implicated (by analogy to the 10 kD protein of M. bovis) in the induction of T-cell mediated delayed type hypersensitivity.
• the 10 kD antigen gene was isolated using a DNA probe corresponding to the N-terminal amino acid sequence of the M. bovis 10 kD antigen. This probe was selected because of the demonstrated immunological cross- reactivity between the 10 kD M. bovis antigen and the lOkD antigen of M. tuberculosis. Cloning and sequencing of the M.
• tuberculosis lOkD antigen gene revealed a coding sequence of 300 nucleotides which encode 99 amino acids having an aggregate molecular weight of 10.7 kD. The sequence was shown to be homologous to two procaryotic heat- shock proteins and additionally to have heat-shock like promoter sequences upstream from the initiation codon. Rogall et al. Int. J. Syst. Bact. 40:323-330 sequenced and analyzed the
• oligonucleotide probes are used for the detection of various microorganisms by means of nucleic acid hybridization using techniques such as dot blot, slot blot, Southern blots, solution hybridization, in situ hybridization and others.
• the polymerase chain reaction may be used which amplifies the target DNA.
• U.S. Patent No. 4,683,195 to Mullis et al. describes the details of the polymerase chain 5 reaction.
• LCR ligase chain reaction
• probe pairs are used which include two primary probe partners (first and second probes) and two secondary probe partners (third and fourth) all of which are employed in excess.
• the first o probe of a probe pair hybridizes to a first segment of the target strand and the second probe of a probe pair hybridizes to a second segment of the same target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3' hydroxyl relationship and so that a ligase can covalently fuse or ligate the two probes of the pair into a fused product.
• a third (secondary) probe can 5 hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion.
• the secondary probes will also hybridize to the target complement in the first instance.
• the third and fourth probes which can be o ligated to form a complementary, secondary fused product. It is important to realize that the fused products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in K. Backman, et al. EP-A-320 308 published June 14, 1989 and K. Backman, et al., EP-A-439 182 published July 31, 5 1991.
• a potential problem associated with ligase chain reaction is background signal caused by target independent ligation of the probes. Since the third probe hybridizes to the first probe and the fourth probe hybridizes to the second probe, the probes, which are added in excess, can easily form duplexes among themselves. These duplexes can become ligated independently of the presence of target to form a fused product which is then indistinguishable from the desired amplified target, yet which is still capable of supporting further amplification. Although target independent blunt-end ligation of these duplexes is a relatively rare event, it is sufficiently common to cause undesirable high background signals in diagnostic assays.
• WO 90/01069 (Segev Diagnostics) and GB 2 225 112 A (Imperial Chemical Industries Pic.) describe versions of a ligation-based amplification scheme which includes a polymerase-mediated gap-filling step prior to ligation.
• EP-A-439 182 to K. Backman et al. published July 31, 1991, teaches variations of LCR that reduce background. One such variation involves gap filling and ligation.
• At least one probe of a probe pair initially includes a "modified” end which renders the resultant duplex "non-blunt" and/or not a substrate for the ligase catalyzed fusion of the probe pair.
• a "modified” end is defined with respect to the point of ligation rather than with respect to its complementary probe.
• a "modified end” has omitted bases to create a "gap" between the terrriinus of one probe of a probe pair and the terminus of the other probe of a probe pair.
• the present invention is directed to oligonucleotide probes useful for detection of target DNA from a plurality of Mycobacterium species.
• Such an oligonucleotide probe is from 10 to about 50 nucleotides long and possesses sufficient complementarity or homology to the sequences shown in SEQ ID NOS. 34, 39, 44, 49, 5 54, 59 and 64 to hybridize with such sequence or its complement under hybridizing conditions, as defined herein.
• Sufficient complementarity or homology generally requires about 80% to 100% complementarity or homology preferably 90% or more. Shorter probes typically require higher percentage ranges, while longer probes typically are useful with lower percentage ranges.
• Such an oligonucleotide probe detects at lest two species of Mycobacterium, preferably three or more, and ideally all species, while not cross reacting substantially with other related organisms including closely related organisms.
• Preferred examples of such oligonucleotide probes of the invention are the probes of SEQ ID NOS. 35-38, 40-43, 45-48, 50-53, 55-58, 60-63, 65-68, 69-72, and 73-76.
• Another aspect of the present invention includes probe compositions useful in detecting target DNA from Mycobacterium species including: probe set 8 (SEQ ID NOS.
• probe set 9 SEQ ID NOS. 40-43
• probe set 10 SEQ ID NOS. 45-48
• probe set 11 SEQ ID NOS. 50-53
• probe set 12 SEQ ID NOS. 55-58
• probe set 13 SEQ ID NOS. 60-63
• probe set 14 SEQ JD NOS. 65-68
• probe set 15 SEQ ID NOS. o 69-72
• probe set 16 SEQ ID NOS. 73-76
• Tne methods of the present invention generally comprise providing a sample suspected of containing said target DNA; providing one or more probe sets according to the compositions of the present invention wherein at least one probe of said probe set has a label capable of detection; and providing one to three deoxynucleotide triphosphates, a polymerase, and a ligase.
• the following cycle is then performed at least once: mixing o said probe set with said sample suspected of containing said target DNA; denaturing said mixture of said probe set and said sample suspected of containing said target DNA; hybridizing said denatured probe set to said denatured target DNA thereby creating hybridized probes; correcting said hybridized probes in a template dependent manner thereby creating adjacent probes; ligating said extended "corrected” adjacent probes using 5 said ligase to form reorganized probes; detecting said label in said reorganized probes.
• "Correction" as used herein is used in the same sense as in EP 439,182. For the above mentioned oligonucleotides, probe compositions and methods, bacteria from non-mycobacterium genera are substantially not detected, while multiple species and/or strains within the Mycobacterium genus are detected.
• kits useful for the detection of Mycobacterium comprising suitable containers containing one or more probe sets according to the present invention, and a ligase reagent.
• Additional kits of the present invention include a ligase reagent, a polymerase reagent, one or more deoxynucleotide triphosphates, one or more probe sets and wherein at least one probe from said probe set has a label.
• Figure 1A, IB and 1C illustrate M. tuberculosis species-specific target DNAs and oligonucleotide probes aligned with their respective targets.
• Figure 2A, 2B and 2C illustrate Mycobacterium genus-specific target DNAs and oligonucleotide probes aligned with their respective targets.
• the bases shown in lower case are mismatches with respect to target.
• Target DNAs are double stranded, though are shown as only a single strand.
• the oligonucleotide sequences of the present invention are derived from the gene coding for protein antigen B from M. tuberculosis, Anderson et al., Infection and Immunity 57:2481-2488 (1989); from the direct repeats reported along with insertion element IS987 from M. bovis, Hermans et al., Infection and Immunity 59:2695-2705; 5 from the insertion-like element IS 6110 from M. tuberculosis, Thierry et al. , Nucleic Acids Res. 18:188 (1990), from the 16S ribosomal RNA gene of M. tuberculosis according to Rogall et al. (Int. J. Syst. Bact.
• the modified ligase chain reaction (LCR) utilized in the present invention uses two pairs of probes herein designated A, B (primary probes), and A', B' (secondary probes).
• Probe pairs as used herein refers to two probes which are directed to the same target strand and which will ultimately be ligated to one another after annealing to the 5 target.
• At least one probe of one of the probe pairs initially includes a "modified” end which renders the resultant duplex "nonblunt" and/or not a suitable substrate for a ligase catalyzed fusion of the two probe duplexes.
• a "modified end” is defined with respect to the point of ligation rather than with respect to its complementary probe.
• a “modified end” has omitted bases to create a "gap" between one probe te ⁇ ninus and the next probe o terminus when the probe pair is annealed to a target sequence.
• Other modified ends include a base mismatched with the target sequence.
• a modified end is referred to as a "recess", the recess being the gap between two primary or two secondary probes after hybridizing to the target.
• the presence of these modified ends reduces the falsely 5 positive signal created by blunt-end ligation of complementary probe duplexes to one another in the absence of target.
• “Correction” of the modification is subsequently carried out to render the probes ligatable.
• correction refers to the process of rendering, in a target dependent manner, the two primary probes or the two secondary probes ligatable to their o partners. Thus, only those probes hybridized to target, target complement or polynucleotide sequences generated therefrom are “corrected.”
• “Correction” can be accomplished by several procedures, depending on the type of modified end used. Gap- filling and nick translation activity are two correction methods described further in the examples. 5
• point of ligation or “intended point of ligation” refers to a specific location between probe pairs that are to be ligated in a template-dependent manner.
• Each of the probes comprise deoxyribonucleic acid (DNA) which may be o routinely synthesized using conventional nucleotide phosphoramidite chemistry and the instruments available from Applied Biosystems, Inc, (Foster City, CA); DuPont, (Wilmington, DE); or Milligen, (Bedford, MA). Phosphorylation of the 5' ends of the appropriate probes, is necessary for ligation by ligase, and may be accomplished by a kinase or by commercial synthesis reagents, as is known in the art.
• DNA deoxyribonucleic acid
• the LCR methods useful in the practice of the present invention comprise repeated steps of denaturation of the target DNA and (a) hybridizing the modified probes to the target (and, if double stranded so that target complement is present, to the target complement); (b) correcting the modification in a target dependent manner (e.g. filling the gap) to render the probes ligatable; (c) ligating the corrected probe to its o partner to form a fused or ligated product; and (d) dissociating the fused product from the target and repeating the hybridization, correction and ligation steps to amplify the desired target sequence. Steps (a), (c) and (d) are essentially the same for all of the embodiments and can be discussed together.
• Step (b) varies depending on the type of modification employed, but only gap filling and "exo" variations are discussed herein.
• Hybridization or “hybridizing” conditions is defined generally as conditions which promote nucleation and annealing. It is well known in the art, however, that such annealing is dependent in a rather predictable manner on several parameters, including temperature, ionic strength, probe length and G:C content of the probes. For example, lowering the temperature of the reaction promotes annealing. For any given set 0 of probes, melt temperature, or Tm, can be estimated by any of several known methods. Typically, diagnostic applications utilize hybridization temperatures which are slightly below the melt temperature.
• the G:C content and length will be known and can be accounted for in determining precisely what 5 "hybridization conditions" will encompass. Since ionic strength is typically optimized for enzymatic activity, the only parameter left to vary is the temperature. For improved specificity, the hybridization temperature is selected slightly below the Tm of the probe; typically 2-10 °C below the Tm. Thus, obtaining suitable "hybridization conditions" for a particular probe set and system is well within ordinary skill of one practicing this art. o For LCR, the probes are added in approximately equimolar concentration since they are expected to react stoichiometrically.
• Each probe is generally present in a concentration ranging from about 5 nanomolar (nM) to about 90 nM; preferably from about 10 nM to about 35 nM. For a typical reaction volume of 50 ⁇ L, this is equivalent to adding from about 3 x 10 11 to about 1 x 10 ⁇ molecules of each probe; and around 5 x 5 1011 molecules per 50 ⁇ L has been a good starting point.
• the optimum quantity of probe used for each reaction also varies depending on the number of cycles which must be performed and the reaction volume. Probe concentrations can readily be determined by one of ordinary skill in this art to provide optimum signal for a given number of cycles.
• each corrected primary probe is ligated to its associated primary partner and each corrected secondary probe is ligated to its associated secondary partner.
• An "adjacent" probe is either one of two probes hybridizable with the target in a contiguous orientation, one of which lies with its phosphorylated 5' 5 end in abutment with the 3' hydroxyl end of the partner probe. "Adjacent" probes are created upon correction of the modified end(s) in a target dependent manner.
• Ligating reagents useful in the present invention include T4 ligase, and prokaryotic ligases such as E. coli DNA ligase, and Thermus aquaticus DNA ligase available from Molecular Biological Resources (Catalog Nos. 107001 and 107002, Milwaukee, WI).
• thermostable ligase is 5 presently preferred for its ability to maintain activity during the thermal cycling of LCR. Absent a thermally stable ligase, the ligase must be added again each time the cycle is repeated. Also useful are eukaryotic ligases, including DNA ligase of Drosophila, reported by Rabin, et al., J. Biol. Chem. 261:10637-10647 (1986). Once ligated, the fused (reorganized) probe is dissociated (e.g. melted) from the target and, as with conventional LCR, the process is repeated for several cycles. The number of repeat cycles may vary from 1 to about 100, although from about 15 to about 70 are preferred presently.
• ligation of the "wrong" outside ends of the probes can be prevented by blocking the end of at least one of the probes with a "hook” or marker moiety as will be 5 described in detail below.
• the outside ends of the probes can be staggered so that if they are joined, they will not serve as template for exponential amplification.
• the amplified sequences can be detected by a number of conventional ways known in the art. Typically, detection is performed after o separation, by determining the amount of label in the separated fraction. Of course, label in the separated fraction can also be determined subtractively by knowing the total amount of label added to the system and measuring the amount present in the unseparated fraction. Separation may be accomplished by electrophoresis, by chromatography or by the preferred method described below. 5 In a particularly preferred configuration, haptens, or "hooks" (also referred to as labels), are attached at the available outside ends of at least two probes (opposite ends of fused product), and preferably to the outside ends of all four probes. A "hook” is any moiety having an affinity to a binding partner.
• the hook(s) at one end of the fused product comprises an antigen or o hapten capable of being immobilized by a specific binding reagent (such as antibody or avidin) coated onto a solid phase.
• the hook(s) at the other end e.g. the 3' end of B and the 5 ' end of B '
• Exemplary hooks include but are not limited to chromogens, catalysts such as enzymes, luminescent compounds, 5 chemiluminescent compounds, radioactive elements such as 32p ?
• exemplary hooks include adamantane acetic acid as described in U.S. 07/808,508 (by Mattingly, P.G., entitled, "Haptens, Tracers, Immunogens and Antibodies for 3-phenyl-l-adamantaneacetic Acids") and carbazole and dibenzofuran derivatives as described in co-owned, co-pending U.S. Patent Application Serial No. 07/808,839 (by Fino, J.R., entitled, "Haptens, Tracers, 5 Immunogens and Antibodies for Carbazole and Dibenzofuran Derivatives", both filed December 17, 1991.
• a method for adding a hapten to the 3 '-end of an oligonucleotide is disclosed in co-owned, co-pending U.S. Patent Application Serial Number 07/630,908, filed December 20, 1990.
• Other methods e.g. Amino Modifier LI, Clontech, Palo Alto, o California
• the method for adding a hapten to the 5' end is through the use of a phosphoramidite reagent as described in Thuong, N.T. et al., Tet. Letters, 29(46): 6905-5908 (1988), or Cohen, J.S. et al., U.S.
• exemplary ligated oligonucleotides may have a carbazole at one 5 end and an adamantane at the other end for the detection by the IMx® instrument (Abbott Laboratories, Abbott Park, IL) using the microparticle enzyme immunoassay (MEIA) technology.
• IMx® instrument Abbott Laboratories, Abbott Park, IL
• MEIA microparticle enzyme immunoassay
• the assay protocol is similar to that used in the commercially available alpha- fetoprotein assay, with the following adaptions: (1) the anti-alpha-fetoprotein antibody coated microparticles are replaced with anti-carbazole antibody coated microparticles; and o (2) the conjugates of anti-alpha-fetoprotein antibodies:alkaline phosphatase are replaced with the conjugates of anti-3-phenyl-l-adamantaneacetic acid antibodies:alkaline phosphatase.
• the protocol for the IMx® MEIA assays is further described in K. Backman et al., EP-A-439, 182 published July 31, 1991.
• the protocol is as 5 follows. A 100 ⁇ L of the reaction mixture which has been amplified by LCR is pipetted into the sample well. 30 ⁇ L of this sample is then pipetted into the incubation well, the anticarbazole antibody coated microparticles are added to the well. An appropriate period of incubation follows which allows the formation of a complex consisting of anticarbazole • antibodies and nucleic acid sequences with the carbazole ends.
• the o mixture is pipetted onto the glass fiber capture matrix of the IMx® reaction cell, and antiadamantane antibodies conjugated to alkaline phosphatase are added.
• the glass-fiber capture matrix is treated with 4-methylumbelliferyl phosphate (MUP).
• MUP 4-methylumbelliferyl phosphate
• the surface-bound enzyme converts the nonfluorogenic MUP to 4-methylumbelliferone (MU), whose fluorescence can be measured.
• probe pairs are labeled with a "fluorescein” hapten and a “biotin” hapten or with a “carbazole” hapten and an adamantaneacetic acid (“adamantane”) hapten.
• adamantane adamantaneacetic acid
• “fluorescein” and “biotin” are used together and “adamantane” and “carbazole” are used together in accordance with the o description above, although any combination of virtually any haptens would be possible.
• each member of a probe pair has a different label.
• results were read in an IMx® instrument. This is commercially available from Abbott Laboratories (Abbott Park, Illinois) and is described in EP-A-288 793 and in Fiore, M. et al Clin. Chem., 34/9:1726-1732 (1988). It should 5 be noted that the IMx® instrument typically generates "machine" noise or background in the range of 5-12 counts/sec/sec.
• Quantities of polymerase are expressed in units, defined as follows: 1 unit of enzyme equals the amount of enzyme required to incorporate 10 nanomoles of total nucleotide into acid-insoluble material in 30 min at 70°C. Units of ligase enzyme are defined herein as: 1 mg of 95% purified Thermus thermophilus DNA ligase has a specific activity of about 1 x 10& units. While this is not precisely standardized and may vary by 5 as much as 20%, optimization is within the skill of the routine practitioner.
• sequences of specific length are listed. It should be understood that sequences covering the same map positions but having slightly fewer or greater numbers of bases are deemed 0 to be equivalents of these sequences and fall within the scope of the invention, provided they will hybridize to the same positions on the target as the listed sequences. It is also understood that sequences having homology to the target sequences of about 80% or more also fall within the scope of the present invention. Preferably any base substitutions in the sequences of the present invention lie 3 or more nucleotides away from the modified ends.
• Target sequences and probes were selected so as to include a "stop base" as taught in EP 439 182 by K. Backman et al. published July 21, 1991 to terminate gap filling extension precisely at the point of ligation so that the extended probe abuts its probe partner and can be ligated to it.
• line diluent is a standard IMx® buffer reagent used to detect machine noise in the absence of target DNA and probe. All data are expressed as IMx® rates of counts/second/second (c/s/s).
• Example 1 Detection of M. tuberculosis Using Probe Set 1 (SEQ ID NOS. 2, 3, 4, and 5)
• Probe set 1 (SEQ ID NOS. 2, 3, 4, and 5) was selected to detect a target sequence in M. tuberculosis corresponding to nucleotides 347-390 (SEQ ID NO. 1) of the protein antigen b (pab) gene of M. tuberculosis.
• LCR reaction mixtures contained 50 mM EPPS, 30 mM MgCl2, 20 mM K+ (from KOH and KCl) (LCR Buffer), 10 ⁇ M NAD,
• 1.7 ⁇ M dATP, and 1.7 ⁇ M dCTP (gap filling nucleotides), 1 x 1012 molecules of each oligonucleotide probe, 18,000 units of Thermus thermophilus DNA ligase, 2 units Thermus polymerase, (Molecular Biology Resources, Milwaukee, WI, cat no. 1070.01) 2 ⁇ g human placental DNA, and target DNA as set out in Table 1 in a final volume of 200 ⁇ l. Cycling was performed on a Perkin-Elmer model 480 thermocycler at the following settings: 93 C for 1 second; 65 C for 1 second; then 68 ° C for 1 minute 15 seconds for a total of 40 cycles.
• Probes were labelled with carbazole and adamantane as described above. Following amplification, ligation products were detected using a sandwich immunoassay using an Abbott automated IMx® analyzer as described above. Table 1 shows the results of LCR using probe set 1 with 10, 25, and 100 molecules of target DNA derived from M. tuberculosis Erdman. Table 2 also shows the results of LCR using target DNA, derived from M. avium, M. intracellulare, M. kansasii, and human placental DNA.
• probe set 1 (SEQ ID NOS. 2, 3, 4, and 5) was capable of detecting as few as 10 molecules of DNA derived from M. tuberculosis and showed no cross-reactivity with as many as 10 ⁇ genomes of DNA derived from M. avium, M. intracellulare, and M. kansasii.
• Probe set 2 (SEQ ID NOS. 7, 8, 9 and 10) was selected to detect a target sequence in M. tuberculosis corresponding to nucleotides 350-387 (SEQ ID NO. 6) of the pab gene of M. tuberculosis.
• LCR was performed as described in Example 1 except that human placental DNA was present at 330 ng/rxn, and cycling was performed on a Perkin- Elmer model 480 thermocycler at the following settings: 94 C, 1 second; 55 C, 1 second; 60 C for 55 seconds, for a total of 40 cycles.
• M. tuberculosis target DNA was present at 10 molecules/reaction, other target DNAs were present at 1 x 10 ⁇ molecules/reaction. Probes were labelled with carbazole and adamantane and detected as above. Results are shown in Table 2.
• probe set 2 (SEQ ID NOS. 7, 8, 9 and 10) was capable of detecting as few as 10 molecules/reaction of M. tuberculosis DNA while other target DNAs present at 1 x 10 ⁇ molecules/reaction gave little if any detectable signal.
• Probe set 3 (SEQ ID NOS. 12, 13, 14, and 15) was selected to detect a target sequence in M. tuberculosis corresponding to nucleotides 2544-2593 (SEQ ID NO. 11) of the direct repeat sequences around IS987. Reactions were performed as described in Example 1 except that the gap filling nucleotides were dCTP and dTTP. Target DNAs and their concentrations are shown in Table 3. In addition, LCR was performed using both 1 x 10 2 molecules and 2 x 10 2 molecules of each oligonucleotide probe. LCR cycling was performed on a Perkin-Elmer model 480 thermocycler at the following settings: 93 ° C, 1 sec; 67 ° C, 1 sec; 70 ° C, 1 min 15 sec.
• Probes were labelled with carbazole and adamantane as described above and data was obtained using an automated IMx® analyzer as described above.
• Target DNA LMx® rate (c/s/s)
• probe set 3 (SEQ ID NOS. 12, 13, 14, and 15) was capable of detecting 25 and 100 molecules of M. tuberculosis target DNA using either 1 x 10*2 molecules of probe/rxn or 2 x 10 ⁇ 2 molecules of probe/rxn.
• Oligonucleotide probe set 4 (SEQ ID NOS. 17, 18, 19, and 20) was selected to detect a target sequence in M. tuberculosis corresponding to nucleotides 535- 578 (SEQ ID NO.16) of the IS-like element IS6110 of M. tuberculosis.
• reactions were performed as described in Example 1 except that dCTP and dTTP were used for gap filling and cycling was performed in a Perkin-Elmer Model 480 thermocycler at the following settings: 94 C, 1 second; 65 C, 1 second; 70 C, 55 seconds for a total of 40 cycles.
• Target DNAs and their quantities are shown in Table 4. Reaction products were then cooled and analyzed as described in Example 1. Probes were labelled with carbazole and adamantane as described above. Table 4
• probe set 4 (SEQ ID NOS. 17, 18, 19, and 20) is capable of detecting as few as 10 molecules of M. tuberculosis target DNA.
• Probe set 4 (SEQ ID NOS. 17, 18, 19, and 20) gave no signal when tested against i molecules of target DNA derived from M. intracellulare 1419, M. avium CSU Ser #1 and M. kansasii 1203.
• Human placental DNA (2 ⁇ g) gave an average IMx® Rate of 25.92 c/s/s which is artifically high due to 3 outliers.
• Oligonucleotide probe set 5 (SEQ ID NOS. 22, 23, 24 and 25) was selected to detect a target DNA corresponding to nucleotides 585-627 (SEQ ID NO. 21) of the pab gene of M. tuberculosis.
• Each reaction was preformed in LCR buffer and contained 5 x 10 1 molecules of each probe/rxn, 3400 units of ligase, 0.5 units polymerase, 50 ng of human placental DNA, 2000 copies/reaction of target DNA, all in a final volume of 50 ⁇ l.
• the gap-filling nucleotide was dCTP.
• Reactions were overlaid with mineral oil and cycling was performed in a COY thermocycler at the following settings: 85 C, 30 sec; 55 C, 20 sec; for 45 cycles.
• the 50 ⁇ l samples were then diluted to 200 ⁇ l with IMx® line diluent to provide adequate volume for IMx® operation. Labels were biotin and fluorescein.
• the IMx® rate for M. scrofulaceum LR130 in this experiment is suspect because of the wide spread in the data (1687.5 c/s/s and 416.1 c/s/s) and in light of the low average seen for M. scrofulaceum 1302 which gave an average of 8.1 c/s/s. This is possibly due to a contamination of the LR130 sample. Similarly a single, spuriously high reading accounts for the unusually high average IMx® rate seen for M. chelonae which gave IMx® rates of 317.8 c/s/s and 47.5 c/s/s while M.
• chelonae 1343 gave IMx® rates of only 8.3 c/s/s and 8.4 c/s/s.
• M. phlei gave an unusually high average IMx® 0 rate by virtue of the fact that one of the reactions gave an IMx® rate of 510.5 c/s/s opposed to a rate of 9.1 c/s/s for its duplicate and an average rate of 8.7 c/s/s for M. phlei 1516.
• a modification of the LCR method described above was utilized to detect a target sequence corresponding to nucleotides 585-627 (SEQ ID NO. 21) of the protein antigen b (pab) gene of M. tuberculosis using probe set 5 (SEQ ID NOS. 26, 27, 28, and 29).
• the LCR method used in this example differs from Example 1 in that it utilizes two o sets of blunt-ended probes wherein the 5' end of probe B has a mismatch with the target corrected prior to ligation by 5' to 3' exonuclease activity of the DNA polymerase used in the reaction.
• This method variously known as the "exo” or "nick translation” method, the basis for which is described in more detail in co-pending, co-owned U.S.
• Ligation products were detected using an automated IMx® analyzer as described above. Experiments using 2 units and 4 units of polymerase gave nearly the same average IMx® rates of 1745.7 c/s/s and 1734.3 c/s/s, respectively. Control experiments using only 2 ⁇ g of human placental DNA/rxn and 2 and 4 units of polymerase gave nearly the same IMx® rates of 11.32 c/s/s and 12.44 c/s/s, respectively.
• Oligonucleotide probe set 7 (SEQ ID NOS. 30, 31, 32 and 33) was selected to detect a target DNA corresponding to nucleotides 585-633 (SEQ ID NO. 21) of the pab gene of M. tuberculosis. LCR was carried out as described in Example 1 with the following exceptions: human placental DNA was present at 300 ng/rxn, dGTP and dTTP were used for gap filling, and cycling was performed in a Perkin-Elmer model 480 thermocycler at the following settings: 93 C, 1 sec; 62 C, 1 sec; 65 C, 1 min. 10 sec for 40 cycles. The probe set was labelled with adamantane and carbazole as described above. Ligation products were detected as described in Example 1. Table 7 shows the results of the assay.
• probe set 7 (SEQ ID NOS. 30, 31, 32, and 33) was capable of detecting as few as 10 molecules of target DNA from M. tuberculosis.
• Probe Set 9 (SEQ ID NO. 40, 41, 42, and 43) Oligonucleotides probe set 9 (SEQ ID NOS . 40, 41 , 42, and 43) was selected to detect a target sequence corresponding to nucleotides 721-760 of the 16S rRNA gene of M. tuberculosis (SEQ ID NO. 39). LCR was performed as described in Example 1 using a Perkin-Elmer Model 480 thermocycler except that cycling was performed at the following settings: 94 ° C, 1 sec; 55 C, 1 sec; 60 C, 40 sec. for a total of 40 cycles, human placental DNA was present at 330 ng/rxn, and each probe was present at 7.5 x l ⁇ l 1 molecules/reaction. Gap filling nucleotides were dCTP and dTTP. Probes were labelled with fluorescein and biotin respectively. Table 8 shows the results of these assays.
• Target DNA 100 mol. target 1000 mol. target
• probe set 16S 721-760 is capable of detecting numerous species and strains of the genus Mycobacterium.
• Oligonucleotide probe set 11 (SEQ ID NOS. 50, 51, 52, 53) was selected to detect a target sequence corresponding to nucleotides 244-286 of the 65 kD heat shock gene of M. tuberculosis (SEQ ID NO. 49) with the aim of using the probe set to detect bacteria of the genus Mycobacterium.
• LCR was performed as described in Example 5 except that the gap filling nucleotide was dTTP.
• mycobacterial target DNA was present at 10 pg/rxn which is the equivalent of about 2000 copies of a typical mycobacterial genome.
• the final reaction volume was 50 ⁇ l.
• Oligonucleotide probe set 12 (SEQ ID NOS. 55, 56, 57 and 58) was selected to detect a target sequence corresponding to nucleotides 405-452 of the 65 kD heat shock gene (SEQ ID NO. 54) of M. tuberculosis with the aim of using the probe set as a probe for bacteria of the genus Mycobacterium.
• LCR was performed as described in Example 1 except that the gap filling nucleotides were dATP and dGTP, human placental DNA was present at 330 ng/rxn, each probe was present at 6 x 1012 probes/rxn, and cycling was performed on a Perkin-Elmer model 480 thermocycler at the following settings: 94 C, 1 se ; 55 C, 1 sec; and 60 C, 55 sec, for a total of 40 cycles. Probes were labelled with biotin and fluorescein respectively.
• Target DNA (10 4 molecules/reaction)
• IMx Rate (c/s/s)
• probe set 12 (SEQ ID NOS. 55, 56, 57, and 58) was capable of detecting 10,000 molecules each of genomic DNA fromM. tuberculosis, M. avium, and M. intracellulare.
• Oligonucleotide probe set 13 (SEQ ID NOS. 60, 61, 62, and 63) was selected to detect a target DNA corresponding to nucleotides 477-524 (SEQ ID NO 59) of the lOkD heat- shock protein gene from M. tuberculosis. LCR was performed as described in Example 1 except that the gap filling nucleotides were dTTP and dGTP, probes were present at 2 x 10 11 molecules each, and cycling was performed on a Perkin-Elmer model
• thermocycler at the following settings: 94 C, 1 sec; 60 C, 1 sec; and 65 C, 55 sec, for 40 cycles. Probes were labelled with carbazole and adamantane respectively. Reaction products were then analyzed by the automated IMx® procedure described in Example 1. Target DNAs and their amounts are listed in Table 11. The results of these experiments are summarized in Table 11.
• Target DNA 1000 molecules 100 molecules
• probe set 13 SEQ LD NOS. 60, 61, 62, and 63
• probe set gave little or no signal when used in reactions containing human placental DNA.
• Oligonucleotide probe set 14 (SEQ ID NOS. 65, 66, 67 and 68) were selected to detect a target DNA corresponding to nucleotides 1059-1098 (SEQ ID NO. 64) of the M. tuberculosis 16S ribosomal RNA gene. LCR assays were performed as described in Example 1 except probes were present at 5 x 10* molecules each and the reaction contained 330 ng human placental DNA. Gap filling nucleotides were dATP and
• probe set 14 SEQ ID NOS 65, 66, 67 and 68
• the results indicate that the probe set 14 (SEQ ID NOS 65, 66, 67 and 68) set was capable of detecting 1,000 molecules of all of the target DNAs tested.
• Probe Set 15 (SEQ ID NOS. 70, 71, 72 and 73)
• Oligonucleotide probe set 15 (SEQ ID NOS. 70, 71, 72, and 73) was used to detect a target DNA corresponding to nucleotides 690-732 (SEQ ID NO. 69) of the 16S ribosomal RNA of M. tuberculosis. LCR was carried out as described in Example 5 except that the gap-filling nucleotide was dATP, and 2 x 10 12 molecules of each probe was used. Probes were labelled with biotin and fluorescein. Reactions were overlaid with mineral oil and reactions were cycled in a COY thermocycler at the following settings: 85 C for 30 sec; 40 C for 30 sec; for 45 cycles. Reaction products were analyzed as described in Example 1. Results are shown in Table 13.
• Target DNA (1 ng) LMx® Rate (c/s/s) M. tuberculosis 35801P 487.2
• probe set 15 was capable of detecting target DNA from at least four species from the genus Mycobacterium.
• Probe Set 8 (SEQ ID NOS. 35, 36, 37, and 38)
• Oligonucleotide probe set 8 (SEQ ID NOS. 35, 36, 37, and 38) was selected to detect a target DNA corresponding to nucleotides 690-732 (SEQ ID NO 34) of the 16S ribosomal RNA gene of M. tuberculosis (and others). LCR was carried out as described in Example 5 except that probes were present at 2 x l ⁇ !2 molecules/reaction, and cycling was performed at: 85 C, 30 sec; 55 ° C for 30 sec; for a total of 45 cycles. Reaction products were analyzed as described in Example 1. Probes were labelled with biotin and fluorescein, respectively. Results are shown in Table 14.
• Target DNA (5 pg) IMx ® Rate (c/s/s) M. intracellulare LR158 558.5
• probe set 8 was capable of detecting target DNA from several species of bacteria of the genus Mycobacterium. However, this probe set failed to detect target DNA from M. terrae.
• Probe Set 16 (SEQ ID NOS. 73, 74, 75 and 76)
• Oligonucleotide probe set 16 (SEQ ID NOS. 73, 74, 75 and 76) was selected to detect a target DNA of the 16S ribosomal RNA gene of various mycobacteria. LCR was performed as described in Example 1 using dATP as the gap-filling nucleotide, and 2 x 1012 molecules of each probe. Thermocycling was performed in a Sutter Dunker at the following settings: 85 C for 85 sec; 62 C for 120 sec; for a total of 40 cycles. Reaction products were analyzed as described in Example 1. Results are shown in Table 15. Table 15
• probe set 16 (SEQ ID NOS. 73, 74, 75 and 76) was capable of detecting a subset of target DNAs from bacteria of the genus Mycobacterium.
• Example 16 Detection of Bacteria of the Genus Mycobacterium with Probes from Probe Sets 8 (SEQ NOS. 35, 36, 37, and 38) and 16 (SEQ ID NOS. 73, 74, 75 and 76). Probes from oligonucleotide probe sets 8 (SEQ ID NOS. 35, 36, 37, and
• 16 SEQ ID NOS. 73, 74, 75 and 76 were selected to detect a target sequence corresponding to the 16S ribosomal RNA gene of various mycobacteria.
• LCR was performed as described in Example 1 except that cycling was performed in a Sutter dunker at 85 C for 55 sec. followed by 62 C for 85 sec. for a total of 40 cycles. Reaction products were analyzed as described in Example 1. Probes were labelled with carbazole and adamantane as described previously. Human placental DNA was present at 330 ng/reaction.
• Group 1 Probe A (SEQ ID NO. 35), and A' (SEQ ID NO. 36) from Set 8 0 (GSM1A);
• Probe A SEQ ID NO. 73
• A' SEQ ID NO. 74
• Group 2 Probe B (SEQ ID NO. 75), and B ' (SEQ ID NO. 76) from Set 16 5 (GSM1B).
• Probe A SEQ ID NO. 35
• probe A' SEQ ID NO. 36
• probe B SEQ ID NO.
• probe B' SEQ ID NO. 38 from Set 8 GSM1A.
• Table 16 shows the combination of probes used, and their concentrations.
• Results derived using each of these combinations are shown in Table 17, and illustrate that the combined probe sets of group 1 and 2 detected both M. tuberculosis and M. chelonae.
• Example 17 Detection of Bacteria of the Genus Mycobacterium Using A Mixture of Probe Set 8 (SEQ ID NOS. 35, 36, 37, and 38) and Probe Set 16 (SEQ ID NOS. 73, 74, 75 and 76) Oligonucleotide probe set 8 (SEQ ID NOS. 35, 36, 37, and 38) and oligonucleotide probe set 16 (SEQ ID NOS. 73, 74, 75 and 76) were used together to detect a target DNA corresponding to the 16S ribosomal RNA gene of various mycobacteria.
• LCR was performed as described in Example 5 except that all 8 probes were present at 5 x l ⁇ l 1 molecules/reaction, and cycling was performed at in a COY thermocycler at the following settings: 55 ° C, 30 sec, 85 C, 20 sec. for a total of 45 cycles. Reaction products were analyzed as described in Example 1. Probes were labelled with biotin and fluorescein. Results are shown in Table 18. Table 18
• Target DNA (5 pg) LMx® Rate (c/s/s)
• probe sets 8 SEQ ID NOS. 35, 36, 37, and 38
• 16 SEQ ID NOS. 73, 74, 75 and 76
• Probes From Probe Set 8 (SEQ ID NOS. 35, 36, 37 and 38) and 16 (SEQ ID NOS. 73, 74, 75 and 76)
• LCR was performed as in Example 1 except that the amount of oligonucleotide probes were as follows: GSMIA A/A' was 2.67 x l ⁇ l2/rxn, GSMIB A/A' was 1.33 x l ⁇ l2/rxn, GSMIB B/B' was 4 x l ⁇ l2/rxn, and human placental DNA was present at 330 ng/rxn. Cycling was performed in a Sutter dunker at 85 C for 85 sec,
• probe set 8 SEQ ID NOS. 35, 36, 37, and 38
• probe set 16 SEQ ID NOS. 73, 74, 75 and 76
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• ORGANISM Mycobacterium tuberculosis
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• ORGANISM Mycobacterium tuberculosis
• xi SEQUENCE DESCRIPTION: SEQ ID NO:16:
• MOLECULE TYPE synthetic DNA
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• ORGANISM Mycobacterium tuberculosis
• xi SEQUENCE DESCRIPTION: SEQ ID NO:21:
• MOLECULE TYPE Synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• ORGANISM Mycobacterium tuberculosis
• xi SEQUENCE DESCRIPTION: SEQ ID NO:39:
• MOLECULE TYPE Synthetic DNA
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• N stands for inosine
• N stands for inosine
• N stands for inosine
• N stands for inosine

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