WO2008045819A2 - Détection et identification d'organismes par analyse de séquences d'acides nucléiques - Google Patents

Détection et identification d'organismes par analyse de séquences d'acides nucléiques Download PDF

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WO2008045819A2
WO2008045819A2 PCT/US2007/080685 US2007080685W WO2008045819A2 WO 2008045819 A2 WO2008045819 A2 WO 2008045819A2 US 2007080685 W US2007080685 W US 2007080685W WO 2008045819 A2 WO2008045819 A2 WO 2008045819A2
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primer
amplicon
nucleic acid
organisms
sample
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WO2008045819A3 (fr
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Mekbib Astatke
Charles Quentin Davis
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Bioveris Corp
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Bioveris Corp
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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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features

Definitions

  • the present invention in various embodiments, relates to methods, compositions, and kits for detecting and identifying organisms in a sample by identifying single or multiple nucleotide variations in a nucleotide sequence of interest.
  • the methods combine amplifying target nucleic acid sequences, digesting the nucleic acids with restriction enzymes and identifying the organism(s)/strain(s) in the sample based on the pattern of digestion predicted from the target nucleotide sequences.
  • the kits and compositions provide the reagents needed to perform these methods.
  • the present invention can be used on nucleic acids prepared from different types of organisms.
  • the clinician determines which bacteria are present in the sample. In this system, however, waiting for the results of growth on one selective medium before proceeding to the next medium based on that result can lead to long diagnosis times. Conversely, the clinician may try to minimize diagnosis time by using all available selective growth media for simultaneous testing. This strategy leads to a wasteful use of growth media.
  • the invention can provide a higher sensitivity and a faster diagnosis time than media- based methods.
  • Immunoassays use polyclonal or monoclonal antibodies that specifically bind to a particular organism. Though these assays decrease the time needed to detect an infection, they nonetheless suffer from a lack of specificity. Antibodies raised to one organism can cross-react with other related organisms. Moreover, immunoassays require the production of specialized reagents for each particular organism to be detected.
  • the present invention employs a minimal number of reagents to maximize the amount of information on organisms present in a sample. In the present invention, there is no need to prepare a reagent that is specifically designed to detect each organism.
  • samples suspected of harboring an organism may also be tested for the ability to cause disease in animals. Although this technique improves the sensitivity of detecting organisms, it is costly and requires enough time for symptoms to develop in the animals. In addition, while animal tests may identify the microorganism generally, these tests do not provide information on the strain of organism present in the sample.
  • the present invention solves this problem by using restriction enzymes to digest nucleic acid sequences amplified from an organism. Restriction enzymes digest nucleic acid on a sequence-specific basis and can detect a change of as little as one base pair at the enzyme's digestion site. This sequence sensitivity allows the invention to distinguish between very closely related organisms in a sample.
  • blotting techniques also require time-consuming steps, including resolving DNA or RNA from an organism by gel electrophoresis and then detecting species-specific sequences with a labeled probe. Although such methods may be used with the invention, the methods of the instant invention do not require analysis on a gel.
  • the instant invention facilitates the detection and identification of organisms in a sample by using a single set of reagents to identify a potentially large number of organisms.
  • the invention circumvents the need to identify a unique nucleic acid sequence marker for each organism.
  • the invention identifies an organism without the need for gel electrophoresis and allows for rapid results.
  • the invention provides a nucleic acid-based technique that identifies one or more organisms in a sample.
  • the organisms can be, for example, different strains of one organism, different species, or different genera.
  • the invention comprises an amplification step using a pair of primers, one of which can be linked to a solid support and the other of which can be labeled with a detectable label.
  • amplification occurs.
  • an aliquot of the amplicon(s) produced in the amplification reaction can be used in separate digestion reactions by exposing the amplicon(s) to a specific restriction enzyme in each reaction.
  • the restriction enzyme cuts the amplicon, thereby separating the detectable label from the rest of the amplicon, which can be attached to the solid support.
  • Each organism gives rise to an amplicon with a nucleotide sequence that yields a unique restriction digestion pattern based on the set of restriction enzymes used. These unique digestion patterns for each organism can be used to specifically identify organisms in a sample.
  • the invention provides a method of identifying at least one organism in a sample comprising the steps of:
  • step (e) generating an actual digestion pattern of the at least one amplicon based on the determination in step (d) for each enzyme
  • the presence of at least one amplicon in the sample can be detected before the at least one restriction enzyme is added. In certain embodiments, the presence of at least one amplicon in the sample can be determined at the same time the determination in step (d) is performed.
  • the target nucleic acid sequence can be amplified using polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), self-sustained sequence replication (SSSR), or Nucleic Acid Sequence-Based Amplification (NASBA ® ).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • SSSR self-sustained sequence replication
  • NASBA ® Nucleic Acid Sequence-Based Amplification
  • at least one target nucleic acid sequence can be amplified using a first primer and a second primer, wherein a portion of the at least one target nucleic acid sequence can be substantially identical to (i) a portion of the first primer or its complement and (ii) a portion of the second primer or its complement.
  • the terms “first” and “second” are used for identification purposes only and do not indicate any particular order of the primers.
  • the first primer and the second primer can be used to amplify several target nucleic acid sequences in the sample.
  • the first primer can comprise a nucleic acid sequence that can be linked to a solid support.
  • the first primer can comprise a nucleic acid sequence and a solid support.
  • the second primer can comprise a detectable label.
  • the second primer can bind to a detectable label.
  • kits comprising one or more primers of the invention and at least one restriction enzyme can be used to practice the methods of the invention.
  • Figure 1 depicts one embodiment of the invention.
  • Figure 2 shows a picture of amplification and digestion products derived from K. oxytoca (panel A) and E. coli (panel B) templates. Following amplification, aliquots of the amplicon were digested using BseMII, Bbvl, SSPI, Xmnl, Ase I and Gsul as indicated by the lane headers.
  • Figure 3 shows bar graphs of the raw electrochemiluminescent measurement of the digested and undigested amplicons derived from K. oxytoca (A) and E. coli (B) chromosomal DNA. Following the amplification, aliquots of the amplicon were digested using BseMII, Bbvl, SSPI, Xmnl, Ase I and Gsul as indicated in the figure.
  • the NC bar shows results without primer addition.
  • the PC bar shows results without restriction enzyme addition.
  • Figures 4A and 4B show bar graphs of measurements of the digested and undigested amplicons derived from 4 strains of N. meningitidis.
  • Figure 4A shows raw electrochemiluminescent signals.
  • Figure 4B normalizes these values as a percentage of the ratio of the difference of signal and the negative control to the difference of the positive and negative controls.
  • Figure 5 shows a picture of amplification and digestion products derived from N. meningitidis templates. Following amplification, aliquots of the amplicon were digested using BseMII, Bbvl, SSPI, Xmnl, Ase I and Gsul as indicated by the panel headers. DESCRIPTION OF THE INVENTION
  • target nucleic acid refers to any nucleic acid that can be amplified to form an amplicon.
  • Target nucleic acid can include, but is not limited to, chromosomal DNA, mitochondrial DNA, messenger RNA, ribosomal RNA, transfer RNA, and extrachromosomal DNA such as virulence plasmids.
  • primer refers to a relatively short oligonucleotide that is complementary to a portion of the sequence of interest (the sequence of interest can be a fragment of a larger nucleic acid sequence).
  • a primer can be the 5' terminus of one strand of the resulting extension product.
  • a primer that is complementary at its 3' terminus to the target nucleic acid sequence on the template strand can be extended using a polymerase to synthesize a sequence complementary to the template. Modifications to the 3' end can affect the ability of an oligonucleotide to function as primer.
  • LNA Locked Nucleic Acid
  • the length of the primer can be adjusted depending upon the particular application, but 15-30 nucleotides can be a common length. In some embodiments of the invention, the primer can be from about 6 to about 150 nucleotides in length. In some embodiments, the primer can be from about 10 to 40 nucleotides in length. In some embodiments, the primer can be from about 16 to 25 nucleotides in length.
  • Primers can be used in pairs to amplify the nucleic acid sequence that falls between the two primer binding sites on the sequence of interest.
  • a primer can comprise at least 18 contiguous nucleotides from the sequence of interest.
  • Primers can comprise nucleic acids, modified nucleic acids, and/or nucleic acid analogs.
  • nucleic acid refers to a nucleobase sequence containing an oligomer, polymer, or polymer segment, having a backbone formed solely from naturally occurring nucleotides or unmodified nucleotides.
  • Nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • Non- limiting examples of naturally occurring nucleobases include: adenine, cytosine, guanine, thymine, and uracil.
  • modified nucleic acid means an oligomer, polymer, or polymer segment comprising at least one modified nucleotide.
  • modified nucleotides include: 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5- methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopuhne, N9- (2-amino-6-chloropurine), N9-(2,6-diaminopuhne), hypoxanthine, N9-(7-deaza- guanine), N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).
  • suitable modified nucleobases include those nucleobases illustrated in Figures 2(A) and 2(B) of Buchardt et al. (US 6,357,163).
  • nucleic acid analog refers to synthetic molecules that can bind to a nucleic acid.
  • a nucleic acid analog primer can comprise peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or any derivatized form of a nucleic acid.
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • peptide nucleic acid or PNA means any oligomer or polymer comprising at least one or more PNA subunits (residues), including, but not limited to, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in United States Patent Nos.
  • PNA also applies to any oligomer or polymer segment comprising one or more subunits of those nucleic acid mimics described in the following publications: Lagriffoul et al., Bioorg. Med. Chem. Lett. A: 1081 -82 1994; Petersen et al., Bioorg. Med. Chem.
  • LNA locked nucleic acid
  • LNA subunit means a ribonucleotide containing a methylene bridge that connects the 2'-oxygen of the ribose with the 4'-carbon. See generally, Kurreck, Eur. J. Biochem. 270:1628-44 2003.
  • Bases in a primer can be joined by a linkage other than a phosphodiester bond, so long as it does not prevent hybridization.
  • primers can have constituent bases joined by peptide bonds rather than phosphodiester linkages.
  • a primer can bind to a target nucleic acid under certain conditions.
  • bind is synonymous with “hybridize” or “anneal”.
  • anneal When two molecules hybridize, they can form a combination of the two molecules through one or more types of Watson-Crick or non-Watson-Crick base pairing.
  • complementary refers to nucleobases that can hybridize to each other. For example, adenine is complementary to thymine and cytosine is complementary to guanine.
  • a primer can contain a mixture of nucleic acids, modified nucleic acids, or nucleic acid analogs as long as the primer specifically binds to its target sequence.
  • a primer nucleotide sequence can be substantially identical to a portion of a polynucleotide sequence of interest, or its complement, such that the primer specifically binds to the portion of the organism's target nucleic acid sequence to be amplified.
  • substantially identical means that two polynucleotides hybridize under high stringency conditions.
  • high stringency generally refers to hybridization at 5 0 C to 15 0 C less than the temperature of dissociation or melting temperature (T m ).
  • T m can be dependent upon, among other things, the polynucleotide's base pair composition, the length of the hybridized sequence, primer concentration, salt concentration, and on the solvent used.
  • high stringency conditions employ hybridization at 64 0 C - 72 0 C in a 10 mM Tris-HCI pH 8.3, 50 mM KCI, 2 mM MgCI 2 solution.
  • a primer need not be a perfect complement for successful hybridization and amplification to take place.
  • primers can contain some mismatched nucleotides in comparison to the organism's corresponding sequence and still specifically bind to the area of the organism's genome to be amplified.
  • the optimum amount of identity between the primer and the target for successful specific amplification depends on a variety of readily controlled factors including the annealing temperature, the salt concentration, primer length, and the location of mismatches, if any. If the primer is an imperfect complement to the target, an extension product can result that incorporates the primer sequence, and during a later cycle, the complement to the primer sequence can be incorporated into the template sequence.
  • a primer can incorporate any nucleic acid base, any modified nucleic acid, or any nucleic acid analog so that the primer extension product will incorporate these features to permit separation and detection of the primer extension product. While this amplification of imperfect complements is a detriment to prior art methods, in this invention specificity can be primarily achieved through the use of restriction enzymes. In some embodiments, imperfect complements can be expressly desired to be amplified, in order to reduce the number of unique primers.
  • restriction enzyme and “restriction endonuclease” are synonymous and refer to any of a group of enzymes that catalyze the sequence- specific cleavage of molecules that comprise nucleic acids, modified nucleic acids, and/or nucleic acid analogs.
  • amplicon refers to a nucleotide sequence that can be amplified.
  • an amplicon can be an amplified target nucleic acid sequence.
  • the amplicon can be from about 50 base pairs to about 3000 base pairs in length or any length in between.
  • the amplicon can be from about 50 base pairs to about 1000 base pairs in length or any length in between.
  • the amplicon can be from about 50 base pairs to about 800 base pairs in length or any length in between.
  • the amplicon can be from about 50 base pairs to about 600 base pairs in length or any length in between.
  • the amplicon can be from about 50 base pairs to about 400 base pairs in length or any length in between. In some embodiments, the amplicon can be from about 50 base pairs to about 200 base pairs in length or any length in between. In some embodiments, the amplicon can be from about 50 base pairs to about 100 base pairs in length or any length in between.
  • primer-based methods of amplification such as but not limited to PCR
  • an amplicon includes the sequences of the two primers and the sequence of the nucleic acid that lies between the two primer binding sites. Techniques for amplifying a DNA sequence are well known in the art. See, e.g., Saiki R.K.
  • An amplicon nucleotide sequence can contain additions, deletions, or substitutions of nucleotide bases in comparison to the analogous sequence present in an organism's nucleic acid so long as the additions, deletions, or substitutions do not eliminate the recognition site for a restriction enzyme used in the methods of the invention.
  • an amplicon nucleotide sequence can contain from 0 to about 2, 0 to about 10, 0 to about 20, 0 to about 30, or 0 to about 40 nucleotide additions, deletions, or substitutions of nucleotide bases in comparison to the detectable portion of a polynucleotide sequence of interest.
  • linking refers to an association between two moieties.
  • hybridization can be a form of linking in that it involves the association of complementary oligonucleotides and/or polynucleotides.
  • binding partners refers to a first entity that can bind (or become linked) to a second entity.
  • such complexes are characterized by a relatively high affinity and a relatively low to moderate capacity.
  • specific interactions between the two members of a pair or binding partners can occur when the affinity constant Ka is higher than about 10 6 M "1 , or is higher than about 10 8 M "1 .
  • Ka is higher than about 10 6 M "1
  • 10 8 M "1 indicates greater affinity, and thus greater specificity.
  • antibodies can bind antigens with an affinity constant in the range of 10 6 M “1 to 10 12 M "1 or higher. If desired, nonspecific binding can be reduced without substantially affecting specific binding by varying the binding conditions using routine techniques known in the art.
  • the conditions can be defined, for example, in terms of molecular concentration, ionic strength of the solution, temperature, time allowed for binding, or concentration of other molecules in a binding reaction.
  • a pair of binding partners can permit reversible linking between the primer and the solid support.
  • the interaction between the first member and the second member of a pair of binding partners can be (1 ) unstable at temperatures used for amplifying DNA, such that the first and second members will not stably bind to each other during the amplification reaction; and (2) stable at or below the temperature used for digesting DNA with one or more restriction enzymes.
  • a pair of binding partners can be nucleic acid sequences that are sufficiently complementary to each other but not complementary to a nucleic acid sequence in the amplicon. Sufficiently complementary nucleic acid sequences can hybridize as DNA/DNA hybrids, RNA/RNA hybrids, or DNA/RNA hybrids
  • adenosine nucleotides in one DNA sequence can hybridize with thymidine nucleotides in another DNA sequence or uridine nucleotides in an RNA sequence.
  • guanine nucleotides in one DNA sequence can hybridize with cytosine nucleotides in another DNA sequence.
  • the nucleic acid sequences used for the pair of binding partners can be a mixture of nucleotides to provide, for example, the desired thermal stability and unique binding for measuring one or more target sequences.
  • binding between a small number of adenine and thymidine residues can be temperature sensitive.
  • the temperature at which complementary nucleotide strands dissociate from each other is the melting temperature (T m ) of the strands.
  • T m melting temperature
  • an approximate primer T m can be calculated by adding 2 0 C for each A or T in the primer and 4 0 C for each G or C in the primer.
  • T m can be calculated using the following formulas.
  • T m (wA+xT) x 2 + (yG+zC) x 4.
  • T m 64.9 + 41 x (yG + zC - 16.4)/(wA + xT + yG + zC).
  • w, x, y, and z are the number of As, Ts, Gs, and Cs in the nucleotide sequence for which the T m can be calculated.
  • the T m of a pair of binding partners can be lower than the lowest temperature used in an amplification reaction.
  • the pair of binding partners can bind, linking the amplicon to the solid support.
  • the solid support can be modified with a poly T tail and the primer can have a poly A tail, wherein the shorter of the two tails can be about 10, about 20, about 40, or about 50 residues long or any intermediate length.
  • the poly A tail can be linked to the solid support and the oligonucleotide primer can have a poly T tail at its 5'-end.
  • a pair of binding partners can be comprised of biotin and avidin or streptavidin.
  • the solid support can be dehvatized with avidin and the primer can be biotinylated.
  • the solid support can be dehvatized with streptavidin and the primer can be biotinylated.
  • the biotinylated primer can be linked to the solid support before the amplification reaction begins.
  • the primer can be covalently linked to the solid support via a crosslinker before the amplification reaction begins.
  • Solid supports can be, but are not limited to, beads, membranes, synthetic organic polymers, microfuge tubes and inorganic oxides.
  • Beads can be, but are not limited to, polystyrene beads.
  • Beads can be magnetizable beads.
  • the term "magnetizable” as used herein refers to a property of matter wherein the permeability of the matter differs from that of free space.
  • Magnetizable beads include, but are not limited to, paramagnetic and superparamagnetic beads. Beads can also be metallic beads, including gold beads.
  • beads can have a diameter in the range of about 0.01 ⁇ m - about 100 ⁇ m, about 0.1 ⁇ m - about 50 ⁇ m, about 1 ⁇ m - about 20 ⁇ m, about 0.5 ⁇ m - about 10 ⁇ m, about 0.05 ⁇ m - about 5 ⁇ m, or about 1 ⁇ m - about 3 ⁇ m.
  • centhfugation techniques and/or filtration techniques can be used to isolate the amplicon from the amplification reaction.
  • Membranes that can be used as solid supports in the invention comprise, for example, nitrocellulose, nylon, polyvinylidene fluoride (PVDF) or carboxylated polyvinylidene (U.S. Patent No.: 6,037,124).
  • Membranes can be coated with various materials, including polyvinyl benzyl dimethyl hydroxyethyl ammonium chloride, polyvinyl benzyl benzoyl aminoethyl dimethyl ammonium chloride, polyvinyl benzyl tributyl ammonium chloride, copolymers of polyvinyl benzyl trihexyl ammonium chloride and polyvinyl benzyl tributyl ammonium chloride, copolymers of polyvinyl benzyl benzoyl dimethyl ammonium chloride and polyvinyl aminoethyl dimethyl ammonium chloride, and copolymers of polyvinyl benzyl phenyl ureidoethyl dimethyl ammonium chloride or polyvinyl benzyl benzoyl dimethyl ammonium chloride (U.S. Patent No.: 5,336,596).
  • antibody refers to an immunoglobulin or a part thereof, and encompasses any polypeptide (with or without further modification by sugar moieties (mono and polysaccharides)) comprising an antigen-binding site regardless of the source or method of production.
  • the term includes, for example, polyclonal, monoclonal, monospecific, polyspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and CDR-grafted antibodies as well as fusion proteins.
  • a part of an antibody can include any fragment which can bind antigen, including but not limited to Fab, Fab', F(ab') 2 , Facb, Fv, ScFv, Fd, V H> and V L .
  • specimen refers to any material taken from a biological or environmental source that may contain one or more target nucleic acid sequences from at least one organism of interest. Specimens can be drawn from any source upon which analysis is desired. For example, the specimen can arise from a host or biological fluids from a host, such as blood, plasma, serum, milk, semen, amniotic fluid, cerebral spinal fluid, sputum, bronchoalveolar lavage, nasopharyngeal wash, tears, urine, saliva, nasal swabs, throat swabs, or stool.
  • a host or biological fluids from a host such as blood, plasma, serum, milk, semen, amniotic fluid, cerebral spinal fluid, sputum, bronchoalveolar lavage, nasopharyngeal wash, tears, urine, saliva, nasal swabs, throat swabs, or stool.
  • the specimen can be a water sample obtained from a body of water, such as lake or river, or it may be from a source of drinking water, such as a tap, aquifer, reservoir, or water purification system.
  • Specimens can be taken by a variety of means, including swabs of a surface.
  • a surface can be swabbed, and then a sample prepared by washing the swab with a liquid, thereby transferring an analyte from the surface into the liquid.
  • Specimens can also be taken from the air.
  • sample refers to the material actually tested.
  • a sample comprises a specimen. Samples in most cases are in liquid form.
  • liquid comprises — in addition to the more traditional definition of liquid — colloids, suspensions, slurries, and dispersions of particles (including beads) in a liquid wherein the particles have a sedimentation rate due to earth's gravity of less than or equal to about 1 mm/s.
  • the sample can also be prepared by dissolving or suspending a specimen in a liquid, such as water or an aqueous buffer.
  • a liquid such as water or an aqueous buffer.
  • the air can be filtered, and the filter washed by a liquid, thereby transferring an analyte from the air into the liquid.
  • Exemplary hosts include, but are not limited to, humans, animals, and plants.
  • organism refers to living matter and viruses comprising nucleic acid that can be detected and identified by the methods of the invention.
  • Organisms include, but are not limited to, bacteria, archaea, prokaryotes, eukaryotes, viruses, protozoa, mycoplasma, and fungi. Different organisms can be different strains, different varieties, different species, different genera, different families, different orders, different classes, different phyla, and/or different kingdoms.
  • organisms include bacterial pathogens such as: Aeromonas hydrophila and other species (spp.); Bacillus anthracis; Bacillus cereus; Botulinum neurotoxin producing species of Clostridium; Brucella abortus; Brucella melitensis; Brucella suis; Burkholderia mallei (formally Pseudomonas mallei); Burkholderia pseudomallei (formerly Pseudomonas pseudomallei); Campylobacter jejuni; Chlamydia psittaci; Clostridium botulinum; Clostridium botulinum; Clostridium perfringens; Coccidioides immitis; Coccidioides posadasii; Cowdria ruminantium (Heartwater); Coxiella burnetii; Enterovirulent Escherichia co// group (EEC Group) such as Escherichia coli - enterotoxigenic (
  • viruses such as: African horse sickness virus; African swine fever virus; Akabane virus; Avian influenza virus (highly pathogenic); Bhanja virus; Blue tongue virus (Exotic); Camel pox virus; Cercopithecine herpesvirus 1 ; Chikungunya virus; Classical swine fever virus; Coronavirus (SARS); Crimean-Congo hemorrhagic fever virus; Dengue viruses; Dugbe virus; Ebola viruses; Encephalitic viruses such as Eastern equine encephalitis virus, Japanese encephalitis virus, Murray Valley encephalitis, and Venezuelan equine encephalitis virus; Equine morbillivirus; Flexal virus; Foot and mouth disease virus; Germiston virus; Goat pox virus; Hantaan or other Hanta viruses; Hendra virus; Issyk-kul virus; Koutango virus; Lassa fever virus; Louping ill virus; Lumpy skin disease virus; Lymphocytic chor
  • organisms include parasitic protozoa and worms, such as: Acanthamoeba and other free-living amoebae; Anisakis sp. and other related worms Ascaris lumb ⁇ coides and T ⁇ chu ⁇ s t ⁇ chiura; Cryptosporidium parvum; Cyclospora cayetanensis; Diphyllobothrium spp.; Entamoeba histolytica; Eustrongylides sp.; Giardia lamblia; Nanophyetus spp.; Shistosoma spp.; Toxoplasma gondii; and Trichinella.
  • parasitic protozoa and worms such as: Acanthamoeba and other free-living amoebae; Anisakis sp. and other related worms Ascaris lumb ⁇ coides and T ⁇ chu ⁇ s t ⁇ chiura; Cryptosporidium parvum; Cyclospora cayetan
  • analytes include allergens such as plant pollen and wheat gluten.
  • organisms include fungi such as: Aspergillus spp.; Blastomyces dermatitidis; Candida; Coccidioides immitis; Coccidioides posadasii; Cryptococcus neoformans; Histoplasma capsulatum; Maize rust; Rice blast; Rice brown spot disease; Rye blast; Sporothrix schenckii; and wheat fungus.
  • detectable label refers to an atom, moiety, functional group, molecule, or collection of molecules that can be attached to or incorporated in an oligomer or polymer to thereby render the oligomer or polymer detectable by an instrument or method.
  • a detectable label can be, for example, a fluorophore, a chromophore, a spin label, a radioisotope, Quantum Dot, beads, aminohexyl, pyrene, biotin, an antigenic determinant detectable by an antibody, a chemiluminescence compound, or a electrochemiluminescent (ECL) moiety.
  • a detectable label can be directly attached to a primer.
  • a detectable label can be indirectly attached to a primer.
  • a detectable label can be attached to a primer using a linker.
  • a hapten can be attached to a primer and can be recognized by a detectably labeled antibody.
  • a primer attached to a hapten can be used in a PCR reaction to produce an amplicon. After the PCR reaction, a labeled antibody that recognizes the hapten binds to the amplicon, thereby labeling the amplicon.
  • Haptens can be, but are not limited to, dinitrophenyl (DNP), fluorescein isothiocyanate (FITC), 5(6)-carboxyfluorescein, 2,4-dinitrophenyl, rhodamine, bromodeoxy uridine, acetylaminofluorene, mercury trinitrophenol, estradiol, and biotin.
  • DNP dinitrophenyl
  • FITC fluorescein isothiocyanate
  • 5(6)-carboxyfluorescein 2,4-dinitrophenyl
  • rhodamine bromodeoxy uridine
  • acetylaminofluorene mercury trinitrophenol
  • estradiol and biotin.
  • Detectable labels can also be fluorophores.
  • Fluorophores that can be used in the method of the present invention include, but are not limited to, infrared (IR) dyes, Dyomics dyes, phycoerythrine, cascade blue, Oregon green 488, pacific blue, rhodamine derivatives such as rhodamine green, 5(6)-carboxyfluorescein, cyanine dyes (i.e., Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy 7) (diethyl-amino)coumarin, fluorescein (i.e., FITC), tetramethylrhodamine, lissamine, Texas Red, AMCA, TRITC, bodipy dyes, and Alexa dyes.
  • IR infrared
  • Dyomics dyes e.g., DIR
  • phycoerythrine phycoerythrine
  • cascade blue Oregon green 488
  • pacific blue pacific blue
  • rhodamine derivatives such as rhodamine
  • Fluorophores with large Stoke's shifts can be used in the present invention, for example, by utilizing at least two fluorophores in a fluorescent resonant energy transfer (FRET) arrangement.
  • the fluorophores can be located in the same bead (see, e.g., U.S. Patent No. 5,326,692), or they can be covalently coupled (e.g., Tandem dyes, U.S. Patent Nos. 5,783,673; 5,272,257; and 5,171 ,843 such as Alexa Fluor ® APC-Alexa Fluor 750 (Molecular Probes; Carlsbad, CA, USA)).
  • Detectable labels can also be beads or other containers of detectable labels.
  • U.S. Patent Application Publication No. 2006/0078912 discloses containers of ECL moieties comprising more than 10 9 moieties that can be linked to a binding partner
  • U.S. Patent No. 5,326,692 discloses fluorescently labeled microparticles that incorporate multiple labels to increase both the signal generated and its Stoke's shift. See, for example, TransFluoSpheres ® (Molecular Probes; Eugene, OR, USA).
  • the detectable label can be a molecular beacon (i.e., a conformation-sensitive label attached to a hairpin loop- containing oligonucleotide) as described, for example, in Kostrikis, L. et al., Science 279:1228-29 1998 and in Tyagi, S. et al., Nat. Biotechnol. 16:49-52 1998.
  • This type of label can be used, for example, as a probe rather than a primer.
  • the probe spans across a restriction enzyme site so the molecular beacon's conformational shape is dependent on the presence of the restriction enzyme site and the proper restriction enzyme.
  • Radioisotopes that can be used in the present invention include, but are not limited to 32 P and 35 S.
  • Chemiluminescent compounds that can be used in the present invention include, but are not limited to adamantyl 1 ,2-dioxetane arylphosphate, other 1 ,2-dioxetanes, achdinium esters, and achdinium sulphonamides.
  • ECL moieties that can be used as detectable labels in the present invention can comprise a metal.
  • the metal can be selected from ruthenium, rhenium, osmium, lanthanum, and europium. Representative ECL moieties are described in U.S. Patent Nos. 5,221 ,605; 5,591 ,581 ; 5,858,676; and 6,808,939.
  • the ECL moiety can be ruthenium(ll) ths-bipyridyl ([Ru(bpy) 3 ] 2+ ), also known as BV-TAGTM.
  • the ECL moiety can be [Ru(sulfo-bpy) 2 bpy] 2+ (also known as BV-TAG Plus) whose structure is provided by:
  • W is a functional group attached to the ECL moiety capable of reacting with a biological material, binding reagent, enzyme substrate or other assay reagent, thereby forming a covalent linkage.
  • the covalent linkage may be chosen from a NHS ester, an activated carboxyl, an amino group, a hydroxyl group, a carboxyl group, a hydrazide, a maleimide, and a phosphoramidite.
  • ECL labels can be used with coreactants.
  • coreactant refers to a chemical compound that either by itself or via its electrochemical reduction oxidation product(s), participates in the ECL reaction sequence.
  • coreactants can be chemical compounds that, upon electrochemical oxidation / reduction, can yield, either directly or upon further reaction, strong oxidizing or reducing species in solution.
  • a coreactant can be peroxodisulfate (i.e., S 2 O 8 2" , persulfate), which can be irreversibly electro- reduced to form oxidizing SO 4 '" ions.
  • the coreactant can also be oxalate (i.e., C 2 O 4 2" ), which can be irreversibly electro-oxidized to form reducing CO 2 '" ions.
  • a class of coreactants that can act as reducing agents is amines or compounds containing amine groups, including, for example, tri-n-propylamine (i.e., N(CH 2 CH 2 CH 2 ) 3 , TPA).
  • amines or compounds containing amine groups including, for example, tri-n-propylamine (i.e., N(CH 2 CH 2 CH 2 ) 3 , TPA).
  • tertiary amines can be better coreactants than secondary amines.
  • secondary amines can be better coreactants than primary amines.
  • Coreactants include, but are not limited to, lincomycin; clindamycin-2- phosphate; erythromycin; 1 -methylpyrrolidone; diphenidol; atropine; trazodone; hydroflumethiazide; hydrochlorothiazide; clindamycin; tetracycline; streptomycin; gentamicin; reserpine; trimethylamine; tri-n-butylphosphine; piperidine; N 1 N- dimethylaniline; pheniramine; bromopheniramine; chloropheniramine; diphenylhydramine; 2-dimethylaminopyridine; pyrilamine; 2-benzylaminopyridine; leucine; valine; glutamic acid; phenylalanine; alanine; arginine; histidine; cysteine; tryptophan; tyrosine; hydroxyproline; asparagine; methion
  • ECL coreactants include, but are not limited to, 1 -ethylpipehdine; 2,2- Bis(hydroxymethyl)-2,2',2"-nitrilothethanol (BIS-TRIS); 1 ,3- bis[tris(hydroxymethyl)methylamino]propane (bis-Tris propane) (BIS-TRIS propane); 2- Morpholinoethanesulfonic acid (MES); 3-(N-Morpholino)propanesulfonic acid (MOPS); 3-Morpholino-2-hydroxypropanesulfonic acid (MOPSO); 4-(2-Hydroxyethyl)piperazine- 1 -(2-hydroxypropanesulfonic acid) (HEPPSO); 4-(2-Hydroxyethyl)piperazine-1 - propanesulfonic acid (EPPS); 4-(N-Morpholino)butanesulfonic acid (MOBS);N,N-Bis(2- hydroxyethyl
  • the invention provides a method for detecting and identifying one or more organisms in a sample by combining a nucleic acid amplification reaction, a detectable label, and the use of restriction enzymes. In certain embodiments, the invention provides a method of identifying at least one organism in a sample comprising the steps of:
  • step (e) generating an actual digestion pattern of the at least one amplicon based on the determination in step (d) for each enzyme
  • the invention provides the method of paragraph [065] wherein the target nucleic acid sequence can be amplified by a method selected from PCR, LCR, SDA, and SSSR. In certain embodiments, a preliminary step of amplifying a target nucleic acid sequence with NASBA can be performed.
  • the invention provides the method of paragraph [065], wherein amplification of the target nucleic acid sequence can be achieved using (1 ) a first primer comprising a nucleic acid sequence that can be linked to a solid support and (2) a labeled second primer, wherein (i) a first portion of the at least one target nucleic acid sequence can be substantially identical to at least 10 consecutive nucleotides, preferably at one end, of the first primer or its complement, and (ii) a second portion of the at least one target nucleic acid sequence can be substantially identical to at least 10 consecutive nucleotides, preferably at one end, of the second primer or its complement.
  • the invention provides the method of paragraph [065], wherein the actual restriction enzyme digestion pattern can be determined by measuring a decrease in the label signal.
  • the methods of the invention can be used to distinguish organisms at different levels of resolution.
  • the invention can distinguish between organisms in a sample that are in different genera.
  • the invention can distinguish between organisms in a single genus that are of different species.
  • the invention can distinguish between organisms that are different strains of a single genus and species.
  • the invention can distinguish between strains of an organism that are resistant to various forms of treatment such as drug treatment.
  • the invention can distinguish between homozygotes and heterozygotes.
  • this invention provides advantages over gel-based methods because this invention detects enzyme digestion more directly.
  • Gel-based methods measure band positions that can be related to the length of the amplicon or digestion products.
  • organisms that have different length amplicons or digestion products appear differently.
  • this invention is more robust to length changes so long as those changes do not affect the primer sites, restriction enzyme sites, or labeling sites. This can even be an advantage for strain-level determinations, if naturally (or otherwise) occurring deletions and mutations significantly affect amplicon or digestion product length.
  • the methods of the invention entail amplification and labeling of a target nucleic acid sequence from one or more organisms in a sample.
  • a single pair of primers can amplify target nucleic acid sequences from more than one organism. Each possible labeled amplicon can be different from the others by one or more base pairs.
  • the method of the invention can identify one organism in a sample, by using a pair of primers to amplify one target nucleic acid sequence in the sample.
  • the method of the invention can identify two organisms in a sample, by using a pair of primers to amplify two target nucleic acid sequences in the sample.
  • the method of the invention can identify three organisms in a sample, by using a pair of primers to amplify three target nucleic acid sequences in the sample. In various embodiments, the method of the invention can identify several organisms in a sample, by using a pair of primers to amplify several target nucleic acid sequences in the sample.
  • the invention uses restriction enzymes, which can detect differences between nucleic acid sequences of as little as one base pair. The set of restriction enzymes used can be chosen so that the predicted target nucleic acid sequence of each possible organism shows a unique restriction pattern.
  • a target nucleic acid sequence can be predicted based on the known nucleic acid sequences present in the organisms that may be in the sample. An organism can be identified based on which restriction enzymes do or do not cut the amplified target nucleic acid sequence.
  • a genomic nucleic acid sequence for amplification, a sequence of interest can be identified by aligning all or a portion of the genomic nucleic acid sequences derived from an organism with sequences available in a nucleic acid sequence database such as Genbank or EMBL. The genomic sequence can be compared with sequences of other organisms in the database in order to find homologous sequences.
  • the set of aligned sequences can be used to design a common primer set that can be used to amplify a sequence of interest derived from each of the organisms, with a minimal level of primer-dimer or nonspecific by-product formation.
  • the sequence of each amplicon that can be produced using the selected primer set can be analyzed to develop a restriction map for each amplicon.
  • the unique combination of restriction enzyme sites in each amplicon that are used gives rise to a "predicted restriction enzyme digestion pattern.”
  • the target nucleic acid sequence of interest can be chosen to produce a unique set of restriction enzyme recognition sites to allow identification of each amplicon sequence from other sequences amplified by the same pair of primers.
  • the target amplicon sequence can be highly conserved in all species of one genus while the corresponding sequence in the other genus/genera consistently differs from that highly conserved sequence.
  • the target sequence can be highly conserved in all members of one species but differ in members of other species in the same genus.
  • the target nucleic acid sequence for amplifying can be conserved within a particular strain but differ among other strains in the same genus and species so as to produce a different digestion pattern, depending on which restriction enzymes are used.
  • the number of primer / restriction enzyme combinations depends in part on the desired results. For example, to choose 1 organism out of O candidate organisms in a sample, there are O possible outcomes (plus a "no organism present" outcome and optionally plus a "data are inconsistent with any one of the candidate organisms" outcome). For example, to choose 1 or 2 organisms out of O candidate organisms in a sample, there are (O 2 + O)/2 possible outcomes (plus a "no organism present" outcome and optionally plus a "data are inconsistent with any one or two of the candidate organisms" outcome).
  • the primer / restriction enzyme combinations can be selected to uniquely identify all the possible outcomes.
  • the primer / restriction enzyme combinations can be selected to maximize the smallest difference between the predicted restriction enzyme digestion patterns for any two of the possible outcomes.
  • the system can be made robust to noise by making the Hamming distance between the predicted restriction enzyme digestion patterns for any two of the possible outcomes be larger than the Hamming distance arising from errors for those two outcomes.
  • 4 restriction enzymes R1 , R2, R3, and R4 can be used to determine two species, species A or B.
  • Species A could have a digestion pattern of + + + - for R1 -R4 respectively, with a + indicating enzyme digestion and a - indicating no enzyme digestion.
  • Species B could have a digestion pattern of + - + - or — +.
  • the second digestion pattern for species B ( — +) has a greater (and the greatest) Hamming distance, while the first digestion pattern for species B (+ - + -) has the smallest nonzero Hamming distance.
  • the amplicon can be digested with one or more restriction enzymes that can cut DNA in a sequence-specific manner.
  • the restriction enzymes can be selected with at least one of the following criteria in mind: 1 ) an enzyme that digests each of the predicted amplicons, i.e., that can act, for example, as a positive control for digestion; 2) an enzyme that can be specific to the nucleic acid sequence of each amplicon produced; 3) commercial availability of the restriction enzyme; 4) the specificity and enzymatic activity of the enzyme; and 5) the optimal temperature of the digestion reaction.
  • a restriction enzyme can be chosen based upon a restriction enzyme recognition site that is common to all the strains of a given species sequence (to, for example, avoid false results due to mutational variation with a given species) and not present in at least one of the other genera to be distinguished.
  • restriction enzyme recognition site refers to the DNA nucleotide sequence position/region at which the restriction enzyme binds to and cleaves the DNA molecule.
  • Restriction enzymes that can be used with the invention include, but are not limited to, Acyl, Bbvl, Narl, Tfil, Tsel, BseMII, BsrDI, Haell, Kpnl, Taul, Hindll, Hin4l, Bfil, and Bsrl. Additional restriction enzymes are well known in the art and can be found in, for example, the New England Biolabs catalog and its REBASE website. See also, Roberts R. J. et al., Nucleic Acid Research, VoI 33, p. D230 - D232, 2005. The instant invention need not be limited to the use of restriction enzymes. Any reagent that cleaves a nucleic acid in a sequence-specific manner can be used with the invention, for example, integrase, recombinase, transposase, topoisomerase, etc.
  • High-temperature tolerant restriction enzymes can also be used, for example, AcII, ApaLI, ApeKI, Avrll, BamHI, BcII, BgIII, BIpI, BsaXI, BsoBI, BsrFI BstBi, BstEII, BstNI, BstUI, BstZ17l, BtsCI, CspCI, Hpal, Kpnl, Mwol, Nb.
  • restriction enzymes can be useful, for example, because they can survive the high temperatures associated with PCR or other amplification techniques.
  • restriction enzymes are either in the amplification mixture during amplification or located sufficiently close to the amplification mixture that they are exposed to high temperatures (e.g., > 60 0 C, > 70 0 C, > 80 0 C, > 90 0 C) during amplification.
  • one restriction enzyme can be used. In some embodiments, more than one restriction enzyme can be used.
  • An amplicon can be detected by a variety of means well known to persons skilled in the art.
  • a detectable label can be incorporated into the amplified DNA during synthesis by using labeled nucleotides.
  • a detectable label can be associated with one of the oligonucleotide primers. Exemplary techniques for analyzing, staining, and labeling nucleic acids are well known in the art and can be found, for example, in Biren, B., Green, E.
  • At least one ECL label can be incorporated into one of the PCR primers.
  • a primer can be labeled with an amino group introduced during synthesis using tag NHS and tag phosphoramidite, respectively, where the "tag” can be an ECL moiety.
  • At least one fluorescent or chemiluminescent label can be incorporated into one of the PCR primers.
  • a primer can be labeled with an amino group introduced during synthesis using tag NHS and tag phosphoramidite, respectively, where the "tag" can be a fluorophore or chemiluminescent moiety.
  • a labeled amplicon can be rendered detectable by incorporating a binding moiety into a primer that is not labeled or, if labeled nucleotides are used, into either primer. Binding moieties that can be used in the present invention include, but are not limited to, biotin and digoxigenin.
  • a digoxigenin label can be detected through a variety of reactions including, but not limited to, fluorescent reactions, chemiluminescent reactions, and ECL reactions.
  • a biotin label can be detected through at least one of a labeled form of streptavidin (i.e., alkaline phosphatase streptavidin or FITC-streptavidin) or a labeled anti-biotin antibody.
  • an amplicon can be detected by incorporating a primer comprising a detectable label and then measuring the labeled amplification product.
  • a labeled amplicon can be separated from unincorporated primer comprising the detectable label by physical means such as centrifugation, dialysis, column purification, or exposure to magnetic or electric fields.
  • amplicons can be attached to a solid support.
  • Magnetizable beads can be the solid support and labeled amplicons can be separated from unincorporated labeled primers by exposure to a magnet.
  • Amplicons can comprise one or more labels. In some embodiments using amplicons linked to a solid support, at least one candidate restriction enzyme site can be located between at least one label and the solid support. Thus, enzymatic cutting of the amplicon by the restriction enzyme changes the amount of label linked to the solid support thereby making the presence of the restriction enzyme site measurable.
  • Amplicons can also be labeled with probes that span a candidate restriction enzyme site. Upon heating and cooling the amplicon, the probe can preferentially bind to uncut amplicons. The probe-amplicon complex can be measured through a solid support system as described above or with a homogeneous method such as a molecular beacon. In some embodiments, amplicons are not labeled with probes that span a restriction enzyme site.
  • a target nucleic acid sequence can be amplified by several methods.
  • Methods of nucleotide sequence amplification include, but are not limited to, the polymerase chain reaction (PCR), nucleic acid sequence based amplification (NASBA; U.S. Pat No 5,409,818), ligase chain reaction (LCR; Wu, D.Y. et al., Genomics 4:560- 569 1989), strand displacement amplification (SDA; Walker et al, Nucleic Acids Res. 20:1691 -96 1992), and self-sustained sequence replication (SSSR; Guatelli, J.C. et al., Proc. Natl. Acad. Sci. USA 87:1874-78 1990).
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence based amplification
  • LCR ligase chain reaction
  • SDA Walker et al, Nucleic Acids Res. 20:1691 -96 1992
  • SSSR self-sus
  • one target nucleic acid sequence can be amplified from the sample.
  • two target nucleic acid sequences can be amplified from the sample.
  • three target nucleic acid sequences can be amplified from the sample.
  • several target nucleic acid sequences can be amplified from the sample (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 75, 100, 200, 300, 400, 500, 600, 1000, 10000, 20000, 50000, 100000, 1000000 or more target sequences or any number in between).
  • only one set of primers is used.
  • multiple sets of primers are used (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, 75, 100, 200, 500, 1000 or more primer sets or any number in between).
  • they can target the same or different organisms.
  • the method of the invention comprises extracting at least one target nucleic acid from a sample, wherein the nucleic acid can be DNA or RNA.
  • the two primers can be mixed with DNA extracted from a sample and with a DNA polymerase, deoxyhbonucleotide triphosphates, buffer, and salts and placed in a thermal cycler instrument in a typical PCR reaction.
  • RNA can be extracted from a sample and converted to DNA using reverse transcriptase before mixing the resulting DNA with a DNA polymerase, deoxyhbonucleotide triphosphates, buffer, and salts for amplification.
  • the test substance contains an organism of interest, thousands to millions of copies of the amplicon can be synthesized.
  • amplification is not performed; rather, only an extension is performed.
  • Extension-only methods require the detection of fewer labeled amplicons, but have the advantage of not having to amply the nucleic acid.
  • the resulting products are extension products. These extension products can be used in an analogous manner to labeled amplicons for determining the presence of an organism.
  • Extension-only methods require one or more primers. After the primer and a target nucleic acid hybridize, a polymerase is used to build a double-stranded nucleic acid that, depending on the target nucleic acid sequence, may contain one or more restriction enzyme sites that can be used to identify the organism.
  • an amplicon can be linked to a solid support. This linking serves at least two purposes: (1 ) to facilitate separation of the labeled amplicon from labels that are not incorporated into amplicons; and (2) to allow removal of labels separated from amplicons via restriction enzyme digestion.
  • covalent bonds can link the amplicon to a solid support.
  • an amplicon can be linked to a solid support through a pair of binding
  • the pair of binding partners can permit reversible linking between the solid support and the amplicon, for example, the pair of binding
  • nucleic acids can comprise nucleic acids, modified nucleic acids, and/or nucleic acid analogs in such a configuration that have a melting temperature above room
  • amplification and/or extension temperatures e.g., ⁇ 50 0 C, ⁇ 60 0 C, ⁇ 70 0 C, 80 0 C, ⁇ 90 0 C, and/or ⁇ 98 0 C.
  • a primer can be linked to a solid support. This linking serves at least two purposes: (1 ) to facilitate separation of the labeled amplicon from labels that are not incorporated into amplicons; and (2) to allow removal of labels separated from amplicons via restriction enzyme digestion.
  • covalent bonds can link the primer to a solid support.
  • the primer can be linked to a solid support through a pair of binding partners.
  • the pair of binding partners can permit reversible linking between the solid support and the primer
  • the pair of binding partners can comprise nucleic acids, modified nucleic acids, and/or nucleic acid analogs in such a configuration that have a melting temperature above room temperature (e.g., > 30 0 C, > 40 0 C, > 45 0 C, > 50 0 C, and/or > 60 0 C) and less than amplification and/or extension temperatures (e.g., ⁇ 50 0 C, ⁇ 60 0 C, ⁇ 70 0 C, 80 0 C, ⁇ 90 0 C, and/or ⁇ 98 0 C).
  • a melting temperature above room temperature e.g., > 30 0 C, > 40 0 C, > 45 0 C, > 50 0 C, and/or > 60 0 C
  • amplification and/or extension temperatures e.g., ⁇ 50 0 C, ⁇ 60 0 C, ⁇ 70 0 C, 80 0 C, ⁇ 90
  • Amplicons can be attached to a solid support via a primer used to make the amplicon in a variety of ways.
  • a primer can be covalently linked to a solid support.
  • a primer can be covalently linked to the solid support, for example, by synthesizing a primer with a primary amine at the 5' end and by reacting the primer with a carboxylated solid support.
  • a primer can be covalently linked to the surface of the solid support by a variety of well-known chemistries, such as carbodiimide coupling (see, e.g., Katz et. al., 1994, J. Electroanal. Chem.
  • the primer can be covalently linked to a solid support before beginning the amplification reaction.
  • the primer, contained in an amplicon can be covalently linked to a solid support after the amplification reaction using, for example, amino linkers such as aminohexyl.
  • the primer and the solid support can be chemically modified with moieties that allow the primer to be cross-linked to the solid support.
  • Such moieties can be, for example, photoactivatable crosslinkers such as phenyl azide, nitrophenyl azide, psoralen, azido-methylcoumarin, and any other crosslinkers known to the art.
  • the cross-linker can be [1 -ethyl- 3-(dimethylaminopropyl)carbodiimide] hydrochloride.
  • the primer can be cross-linked to the solid support after the amplification reaction.
  • the primer can be linked to a magnetizable bead.
  • An amplicon comprising the primer linked to the magnetizable bead can be isolated by subjecting the beads to a magnetic field.
  • Figure 1 illustrates embodiments of the invention in which a sample tube containing one or more organisms' target nucleic acid can be amplified using one primer that can be labeled and another primer that can be linked to a magnetizable bead. If a target nucleic acid sequence of interest is present in the sample, amplification occurs to form amplicons. Aliquots of the amplicon preparation are added to tubes containing different restriction enzymes and one or more tubes lacking restriction enzymes. After permitting the restriction enzymes to cut amplicons having their respective recognition sequences, a magnetic field is used to separate labels that are linked to the beads through an intact amplicon from other labels. The linked labels are measured to determine (a) if an amplicon was formed (from the tubes lacking restriction enzymes) and (b) which restriction enzymes cut the amplicons. Thus, the process described in this paragraph generates an actual restriction enzyme digestion pattern.
  • restriction enzyme digestion of an amplicon can be detected by measuring a loss of signal from the support-bound amplicon.
  • digestion of the amplicon by a restriction enzyme can result in cleavage of the amplicon, separating the label from the rest of the amplicon, which is still attached to a solid support.
  • the labeled portion of the amplicon can be removed from the sample containing the support-bound amplicon and the sample can then be analyzed for a loss of signal from the label.
  • the detached segments can be removed and the amount of detectable label remaining can be measured and compared with the label signal of an amplicon that is not digested with a restriction enzyme.
  • restriction enzyme digestion of an amplicon can be detected by measuring a partial decrease in the signal from the detectable label as opposed to a total loss of signal if the label were completely removed by restriction enzyme digestion.
  • labeled nucleotides can be used during amplification, and the resulting labeled amplicon digested with one or more restriction enzymes. If an amplicon contains a restriction enzyme recognition site, the signal from the detectable label decreases in proportion to the portion of amplicon removed from the solid support due to enzymatic cleavage. The cleaved portion of the amplicon can be removed from the sample and the sample then analyzed for a reduction in the signal from the label. This reduction of signal can be detected by comparing the signal of the digested sample to the signal of an undigested amplicon preparation.
  • the sample is split before amplification.
  • the sample can be split into at least RP containers (e.g., P+RP or more than P+RP containers if replicates are desired).
  • RP containers can be used if there is a separate method to determine if amplicons were formed (e.g., incorporation of radioactive nucleotides into acid-precipitable material); otherwise, P+RP is a more practical lower limit.
  • a potential advantage of splitting the sample before amplification is a reduction in the number of times additions have to be made to the reaction mixtures: the restriction enzymes may be added simultaneously with the amplification reagents.
  • the sample is split after amplification.
  • the amplified sample can be split into least RP containers (e.g., P+RP or more than P+RP containers if replicates are desired).
  • RP containers can be used if there is a separate method to determine if amplicons were formed (e.g., incorporation of radioactive nucleotides into acid- precipitable material); otherwise, P+RP is a more practical lower limit
  • Potential advantages of splitting the sample after amplification can be (a) a reduction in counting (Poisson) variability if the copy number of the target nucleic acid is small, (b) a reduction in variability in amplicon concentration due to having fewer (e.g., one) mixture undergoing amplification.
  • the sample is split after partial amplification.
  • RT-PCR real-time PCR
  • the sample can be split after e.g., 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16 cycles. These first cycles can reduce the counting (Poisson) variability, while maintaining the ability of RT-PCR to estimate concentrations in part by determining the cycle numbers needed to reach a certain signal level.
  • Aliquots of amplicons can be split into multiple containers by pipetting into and out of individual containers (e.g., tubes).
  • a single cartridge can have multiple containers fluidically connected so that amplicons are not exposed to the exterior of the cartridge when the amplicons are split into separate containers having restriction enzymes.
  • digested or undigested amplicons can be quantified, and the quantitative results can be used to help determine the presence of multiple organisms in the sample.
  • different target nucleic acid sequences present in the organisms of a sample can be amplified via PCR with one primer labeled with a detectable label and another primer linked to a bead.
  • the amplicon can be isolated via centrifugation, filtration, or column purification.
  • aliquots of the amplicon preparation can be added to several tubes, each tube containing a different restriction enzyme, chosen by the criteria described above.
  • the restriction digests can then be incubated under conditions that permit the restriction enzymes to digest the amplicon at the enzyme recognition sites.
  • the nucleic acids that remain linked to the solid support after enzyme digestion can be separated from the labeled nucleic acids via centhfugation, filtration, or column purification.
  • the samples can then be analyzed to determine whether the detectable label remains associated with the solid support via the amplicon (i.e., a recognition site for the restriction enzyme was not present in the amplicon) or whether the detectable label has been removed due to digestion by the restriction enzyme.
  • the organisms that the target nucleic acid was derived from in the sample can be identified by comparing the actual pattern of enzymatic digestion obtained by analyzing the samples to a predicted pattern of enzymatic digestion based on the nucleotide sequence of the amplicon from each possible target organism.
  • the label can be an ECL label and the samples can be analyzed on an M1 M analyzer.
  • different target nucleic acid sequence(s) of organism(s) in a sample can be amplified with one primer comprising a detectable label and the other primer coupled to one member of a pair of binding partners, wherein the other member of the pair of binding partners can be linked to a magnetizable bead.
  • a separate amplification reaction can be prepared, one amplification reaction for each restriction enzyme to be used in the second reaction.
  • the amplicon(s) can be isolated from each amplification reaction by exposing the amplification reaction to a magnet.
  • the captured amplicon(s) can then be digested with a set of restriction enzymes, chosen by the criteria described above.
  • a different enzyme can be added to each of the isolated amplification reactions.
  • the nucleic acids that remain linked to the solid support after enzyme digestion can be cleaved from the labeled nucleic acids by exposure to a magnet.
  • the samples can then be analyzed to determine whether the detectable label remains associated with the solid support via the amplicon or whether the detectable label has been removed due to digestion by the restriction enzyme. If an amplicon contains a recognition site that can be cleaved by a specific restriction enzyme, the detectable label is removed from the bead, causing the amplicon to lose its detectable signal.
  • the organisms in the sample can be identified by comparing the actual pattern of enzymatic digestion obtained by analyzing the samples to the pattern of enzymatic digestion predicted from the nucleotide sequences of the target amplicons.
  • target nucleic acid sequence(s) of organism(s) in a sample can be amplified with one chemically modified primer capable of crosslinking to a bead, and a labeled second primer.
  • the amplicons can be captured onto chemically modified beads to crosslink the amplicon with the bead via the modified primer.
  • the beads are magnetizable beads in which case the amplicons can be isolated by exposure to a magnet.
  • aliquots of the amplicon preparation can be added to several tubes, each tube containing a different restriction enzyme, chosen by the criteria described above.
  • the restriction digests can then be incubated under conditions that permit the restriction enzymes to digest the amplicon(s) at the enzyme recognition sites.
  • the nucleic acids that remain linked to the solid support after enzyme digestion can be cleaved from the labeled nucleic acids by exposure to a magnet.
  • the samples can then be analyzed to determine whether the detectable label remains associated with the magnetizable beads or whether the detectable label has been removed due to digestion of the amplicon by the restriction enzyme. For example, whether an amplicon has been digested can be determined by comparing the signal from an undigested sample to the signal from a sample incubated with a restriction enzyme.
  • the organisms in the sample can be identified by comparing the actual pattern of enzymatic digestion obtained by analyzing the samples to the pattern of enzymatic digestion predicted from the nucleotide sequences of the target amplicons.
  • the label can be an ECL label.
  • the samples can be analyzed on an M1 M analyzer.
  • different target nucleic acid sequences of organisms in a sample can be amplified with one chemically modified primer capable of crosslinking to a bead, labeled deoxyribonucleotides, and a second primer.
  • the amplicons can be captured onto chemically modified beads to crosslink the amplicon with the bead via the modified primer.
  • the beads are magnetizable beads, in which case the amplicons can be isolated by exposure to a magnet.
  • aliquots of the amplicon preparation can be added to several tubes, each tube containing a different restriction enzyme, chosen by the criteria described above.
  • the restriction digests can then be incubated under conditions that permit the restriction enzymes to digest the amplicon at the enzyme recognition sites.
  • the nucleic acids that remain linked to the solid support after enzyme digestion can be cleaved from the nucleic acids that have been cleaved by exposure to a magnet.
  • the samples can then be analyzed to determine whether a decrease in the signal from the detectable label associated with the magnetizable beads has occurred. For example, a decrease in signal can be determined by comparing the signal from an undigested sample to the signal from a sample incubated with a restriction enzyme.
  • the organisms in the sample can be identified by comparing the actual pattern of enzymatic digestion obtained by analyzing the samples to the pattern of enzymatic digestion predicted from the nucleotide sequences of the target amplicons.
  • the label can be an ECL label.
  • the samples can be analyzed on an M1 M analyzer.
  • kits comprising one or more primers of the invention and at least one restriction enzyme can be used to practice the methods of the invention.
  • the kit comprises at least one pair of primers, each primer pair comprising at least 12 contiguous nucleotides of a target nucleic acid sequence of interest or at least 12 contiguous nucleotides complementary to a target nucleic acid sequence of interest, wherein the primers specifically amplify the target nucleic acid sequence from at least one organism in the sample.
  • the kit further comprises reagents for carrying out one or more amplification reactions and one or more restriction enzyme digestions.
  • the kit further comprises a means for nucleic acid amplification and is portable.
  • a timed and temperature controlled flow design having multiple incubation chambers can be used. Following incubation in one chamber (extension reaction), the sample then flows into several chambers containing specific restriction enzymes. Following incubation with the restriction enzyme(s), the sample then flows into a detection chamber. In embodiments where a thermostable restriction enzyme is used, a single chamber can be used for the extension/digestion reactions. The sample can then flow into a detection chamber.
  • Each organism of interest and each primer / restriction enzyme pair can be analyzed to determine if (a) an amplicon will be created, and (b) if the restriction enzyme will or will not cut the amplicon.
  • This analysis can use sequence information from the organism available, for example, from databases such as Genbank or EMBL.
  • the collection of analyses for all the primer / restriction enzymes pairs for each organism gives rise to a "predicted enzyme digestion pattern.”
  • the predicted enzyme digestion pattern can be refined by determining the actual enzyme digestion pattern with a known nucleic acid sequence derived from a specific organism/strain. This refinement may be useful, for example, if one restriction enzyme used is not as efficient as the other restriction enzymes. In this case, not all of the amplicon that have restriction sites for that restriction enzyme may be cleaved; consequently, the signal change for the presence and absence of a particular restriction site may differ among the restriction enzyme digests.
  • each datum in the predicted enzyme digestion pattern comprises binary values for measurements that result from restriction enzymes, one value indicates that the restriction enzyme made a cut, and one value indicates that the restriction enzyme did not make a cut. For measurements that did not have restriction enzymes and had a primer, one value indicates that an amplicon was created and one value indicates that an amplicon was not created.
  • each datum in the predicted enzyme digestion pattern can be more than 2 values (e.g., greater than or equal to 3, 4, 10, 100, 2 7 - 1 , 2 8 - 1 2 11 - 1 , 2 12 - 1 , 2 14 - 1 , 2 15 - 1 , 2 16 - 1 , 10 5 , 10 6 , 2 23 - 1 , or 2 24 - 1 values).
  • each datum is the expected numerical value for that primer / restriction enzyme / organism combination.
  • each datum is the expected numerical value for that primer/organism combination.
  • each datum can be the result of simple or complex analysis steps.
  • each datum can be simply the average value of a photodetector from a measurement technique that uses a luminescent label.
  • multiple measurements can be combined for each datum.
  • a background measurement can be taken (e.g., in the absence of sample, or in the absence of primer), and each datum is taken to be the ratio or difference between the measurement and the background.
  • a measurement using primers and no restriction enzyme can be used with primer/restriction enzyme pair data (and optionally a background measurement) to produce a datum that is (a) the ratio or difference of the positive to the primer/restriction enzyme pair data or (b) the ratio of the difference between the positive and the background to the difference between the primer/restriction enzyme pair data and the background.
  • the goal is to determine which one organism, among a possibly large number of candidates, is found in a sample.
  • the analysis can also result in the conclusion that none of the candidate organisms are in the sample.
  • the analysis can also result in the conclusion that an unidentified organism or multiple types of organisms are present.
  • the analysis uses the actual enzyme digestion pattern and the collection of all of the predicted enzyme digestion patterns from all the candidate organisms. As described above, to choose 1 organism out of O candidate organisms in a sample, there are O possible outcomes (plus a "no organism present" outcome and optionally plus a "data are inconsistent with any one of the candidate organisms" outcome).
  • the analysis for a single organism compares the actual enzyme digestion pattern to each of the collection of all of the predicted enzyme digestion patterns for all the possible outcomes and chooses the best match.
  • the results can be analyzed by first choosing the predicted enzyme digestion pattern that minimizes the sum of the squared differences (SSD) between a particular predicted and the actual enzyme digestion patterns. Next, this minimum SSD can be compared to a threshold value. If the SSD is smaller than or equal to the threshold, report the organism that produced the minimum SSD; if the SSD is greater than the threshold, report that the data are inconsistent with any one of the candidate organisms.
  • SSD squared differences
  • the data can first be normalized to reduce the variations in SSD based on signal level changes.
  • the data can first be weighted in such a way that the expected standard deviation is constant for each of the values in the predicted enzyme digestion pattern (a coefficient of variation of 1 % yields a much bigger standard deviation for means of 100,000 than for means of 100).
  • the SSD method can be used.
  • the SSD method is equivalent to the sum of the exclusive or's (XOR) of the elements of the predicted and actual enzyme digestion pattern.
  • norms other than the square can be used on the differences: the sum of the absolute differences, the sum of the cube of the absolute differences, and the maximum of the absolute differences are a few more exemplary embodiments.
  • ratios rather than differences can be used.
  • the goal is to determine which combination of any of p or fewer organisms, among a possibly large number of candidates, is found in a sample.
  • the single organism determination can be considered a special case of this more general analysis.
  • the analysis can also result in the conclusion that none of the candidate organisms are in the sample. In some embodiments, the analysis can also result in the conclusion that an unidentified organism or more than p types of organisms are present.
  • the analysis uses the actual enzyme digestion pattern and the collection of all of the predicted enzyme digestion patterns from all the candidate organisms.
  • One potential issue with multi-organism detection can be a variation in the concentration of the organisms in the original sample.
  • the range of required scaling factors can be minimized or eliminated by the assay methods.
  • the number of PCR amplicons can be ultimately limited by the number of primers available.
  • the amplification efficiency can first decrease for nucleic acids that were originally high in concentration.
  • the range of amplicon concentration can be less that the range of the nucleic acid concentration.
  • a scaling factor is negative or zero, that outcome can be dismissed: the negative case is non-physical and the zero case would be duplicative of another outcome with fewer organisms.
  • outcomes that have a small positive scaling factor can also be dismissed.
  • the outcomes that produce a ratio of the largest to smallest scaling factors greater than the maximum possible corresponding amplicon concentration e.g., 10x, 100x, 1000x, 1000Ox
  • the maximum possible corresponding amplicon concentration e.g., 10x, 100x, 1000x, 1000Ox
  • the residual error can also be weighted by the number of organisms with non-zero scaling factors to compensate for the general numerical rule that greater degrees of freedom results in better fits, even if those fits are non-physical. If the (possibly weighted) residual error of the possible outcome with the smallest residual error is sufficiently small, that outcome can be reported as the organism or organisms present in the sample. Otherwise, the analysis can result in reporting a "data are inconsistent with any combination of p or fewer of the candidate organisms" outcome.
  • n represent the number of measurements in each enzyme digestion pattern. If each measurement can take on v statistically different values, then n has to be greater than or equal to log 2
  • the predicted enzyme digestion pattern for an organism can be considered as an n- dimensional vector.
  • the possible outcome currently under consideration has P organisms (1 ⁇ P ⁇ p).
  • a predicted matrix M can be constructed of the P n-dimensional vectors of the relevant predicted enzyme digestion patterns.
  • ⁇ represent the n-dimensional vector of the actual enzyme digestion pattern.
  • the residual mean-square error is ⁇ ⁇ ⁇ - ⁇ ⁇ Mf.
  • a partial gene sequence for a topoisomerase derived from Klebsiella oxytoca was used to find homologous DNA sequences in the NCBI databank using the BLAST algorithm (www.ncbi.nih.gov/BLAST/), for divergent sequences/discontiguous megablast.
  • BLAST algorithm www.ncbi.nih.gov/BLAST/
  • Several genes from different strains of K oxytoca and sequences derived from other bacteria were found to have significant homology to the sequence, as shown in Table 1. As shown in Table 1 , many organisms and strains share significant homology to the topoisomerase gene from Klebsiella oxytoca (Accession # AJ133197).
  • the PCR primers used to amplify both the K. oxytoca and E. coli amplicon were: Forward Primer: ⁇ '-TTCTCCTACCGCTATCCGCTGGT-S' (SEQ ID NO. 1 ) Reverse Primer: ⁇ '-GAAGTTTGGTACCCAGTCAACGGT-S' (SEQ ID NO. 2) [0126]
  • the predicted nucleic acid sequence of the amplicon for detecting and identifying K. oxytoca was as follows, with the position of each primer identified for the
  • the predicted nucleic acid sequence of the amplicon for detecting and identifying E. coli was as follows, with the position of each primer identified for the forward primer (bold) and the reverse primer (underline):
  • the unique and common restriction enzymes for each amplicon were selected based on one or more of the following criteria: 1 ) common to each organism; 2) specific to each amplicon; 3) common to all the strains of a given species sequence; 4) commercial availability of the restriction enzyme; 5) the specificity and enzymatic activity of the enzyme; and 6) the optimal temperature of the digestion reaction.
  • Tables 2 and 3 have 8 restriction enzymes (Acyl, Haell, Hin4l, Hindll, Taul, Tfil, Tsel, and Xmnl) that recognize more than 1 sequence: sequence code N refers to A or C or G or T; R means A or G; S means C or G; V means A or C or G; W means A or T; and Y means C or T. Based on this analysis, the following restriction enzymes were chosen to differentially identify K. oxytoca and E. coli: BseM II, Bbv I, Ssp I, Xmn I, Ase I, and Gsu I.
  • the enzymes Bbvl and Gsul are common to both organisms, and therefore establish some level of certainty that an actual digestion pattern is the result of one of these two organisms.
  • the enzymes BseMII, SSPI, Xmnl, and Asel provide specificity to distinguish between the two organisms. In this example, only 1 strain of K. oxytoca and E. coli were desired to be distinguished, thus, the criteria of selecting restriction enzymes common to all the strains of a given species sequence was unnecessary. All the restriction enzymes were commercially available. All the restriction enzymes had notorious specificity and had comparable enzymatic activities on the unit enzyme scale. For this proof of concept, differences in required digestion temperatures among the restriction enzymes was not a criterion.
  • primer set A Two primer sets (SEQ ID NOs. 1 and 2), purchased from Biosource, were used in this example.
  • primer set A the forward and reverse primers were unlabeled.
  • primer set B the forward primer was labeled with an electrochemiluminescent label BV-TAG Plus (BioVeris Corp., Gaithersburg, MD) at the 5'-prime position and the reverse primer was labeled with biotin at the 5'-phme position.
  • PCR was used to incorporate the labeled primers into amplicons to form labeled amplicons.
  • PCR was used to create the labeled amplicons.
  • ECL signal is to be generated by coupling a magnetizable bead to the labeled amplicon and using a magnetic field to separate ECL label linked to the magnetizable bead from ECL label not linked (e.g., free in solution). If a labeled amplicon has a restriction enzyme site for a restriction enzyme present in solution, the ECL label is separated from the magnetizable bead, resulting in a reduction in ECL signal.
  • the predicted restriction enzyme digestion pattern also includes a negative control where no primers (or primer set A) are used that can generate labeled amplicons.
  • the predicted restriction enzyme digestion pattern also includes a positive control for the amplification reaction where no restriction enzymes are used.
  • Chromosomal DNA was prepared from 50 ⁇ l_ each of a K. oxytoca and an E. coli culture that was incubated at 37 0 C overnight.
  • 20 ⁇ l_ of bacterial culture was added to 180 ⁇ l_ TE buffer (1 mM Tris, 0.01 mM EDTA, pH 8.0). The mixture was incubated at 95 0 C for 10 min, and centrifuged in a microcentrifuge at 13,000 rpm for 5 minutes. The supernatant was transferred to a microfuge tube.
  • Nucleic acid was precipitated using DNA co-precipitating reagents from Bioline Inc., (Randolph, MA) per the manufacturer's instructions. Each chromosomal DNA preparation was reconstituted in TE buffer and stored at -20 0 C.
  • PCR reactions were performed using both primer set A and primer set B. All PCR reagents were from Roche Molecular (Catalog # 12032937001 ; Pleasanton, CA), except for the dNTPs which were from Bioline (Catalog # B10- 39045, Boston, MA). Five microliters of each DNA preparation was added to a reaction mix containing 2.5 mM MgCI 2 , each primer at a concentration of 200 nM, 200 ⁇ M dATP, 200 ⁇ M dGTP, 200 ⁇ M dCTP, and 200 ⁇ M dTTP in 1X PCR buffer.
  • Taq DNA polymerase was then added to a concentration of 2.5 unit/50 ⁇ l_ PCR reaction immediately before placing the reaction tubes in PTC-200 Peltier Thermal Cycler (MJ Research, Waltham, MA). The final volume of each PCR reaction was 50 ⁇ l_. The reactions were incubated for 35 cycles of 15 seconds at 95 0 C followed by 30 seconds at 55 0 C and then 30 seconds at 72 0 C. Prior to initiating the first cycle, the reaction mix was incubated at 95 0 C for 1 min to denature the double stranded template. Following PCR, the amplicons were purified using a PCR preparation kit from Qiagen (Cat. No. 28104), and each DNA preparation was reconstituted with 30 ⁇ l_ TE
  • Restriction enzymes were used as directed by the manufacturer's (New England Biolabs) instructions. Digestions were carried out using 1 unit of enzyme at 37 0 C for 1 hour. Either 3.0 or 1.5 ⁇ l_ of each amplicon preparation was used for each digestion reaction. Following the digestion reaction, the amplification and digestion samples from the amplification reaction performed with unlabeled primer were detected using ethidium bromide staining to detect and identify fragments derived from the amplification of K. oxytoca (Fig. 2A) and E. coli (Fig. 2B) DNA.
  • Each digestion reaction was loaded on a 2% agarose pre-cast gel (Invitrogen Corp.) and the bands were visualized on a UV transilluminator (Ultraviolet Products).
  • the low molecular weight ladder was purchased from Invitrogen. .
  • Amplification and digestion products from the amplification reaction performed with the ECL-labeled primer were detected using electrochemiluminescence as shown in Figure 3.
  • amplification products were digested using BseMII, Bbvl, SSPI, Xmnl, Ase I and Gsul as indicated in the figure.
  • Each restriction enzyme digest was mixed with 80 ⁇ l_ of 0.5 mg/mL streptavid in-coated M-280 magnetic beads (Dynal BioTech, part number 110029). If digestion with a particular enzyme occurred, the BV-TAGTM Plus and biotin labels were separated, removing the ECL label from the amplicon bound to the bead.
  • the negative control sample defined as NC
  • the positive control sample defined as PC
  • PC was a PCR reaction that was not treated with any restriction enzyme.
  • N. meningitides aspartate b-semialdehyde dehydrogenase gene (partial coding sequence) from GenBank with the accession number of AF205574.1 was used.
  • Two primer sequences were designed based on the published gene sequence and were used to amplify genomic DNA preparations derived from the four N. meningitides strains that are commercially available from ATCC with the following accession numbers: 53415D, 53416D, 53417D, and 53418D.
  • the gene sequence (AF205574.1 ) for the N. meningitides gene is given below. Also given is a primer pair that can be used to amplify the sequence.
  • each of the four genomic DNAs was used in PCR in order to verify that amplification of a similar length amplicon could be produced from each
  • primer set C the forward and reverse primers were unlabeled (primer set C).
  • primer set D the forward and reverse primers were labeled with a BV-TAGTM Plus and biotin at the 5'-postions, respectively.
  • the predicted PCR product for each strain of N. meningitides is shown in the sequence data above.
  • PC in the figures is a positive signal where no restriction enzyme was added to the PCR reaction.
  • the vertical axis in Figure 4A is ECL signal.
  • the vertical axis in Figure 4B is the ECL signal, after it has be normalized to a percentage of the positive control signal.
  • ECL signal was generated by coupling a magnetizable bead to the labeled amplicon and using a magnetic field to separate ECL label linked to the magnetizable bead from ECL label not linked (e.g., free in solution). If a labeled amplicon has a restriction enzyme site for a restriction enzyme
  • the predicted restriction enzyme digestion pattern also included a positive control for the amplification reaction where no restriction enzymes are used.
  • the predicted restriction enzyme digestion patterns for the 4 strains differ, which is the end-goal of the primer / restriction enzyme selection process.
  • This binary-value predicted restriction enzyme digestion pattern can be improved with experimental data.
  • the experimental data in the next section can be used to make these patterns more quantitative by using more than
  • Figure 4 shows an exemplary alternate analysis method. If binary valued data are desired, a threshold of 50% of the positive control signal can differentiate between "high” and “low” signals to compare with Table 7. Thus, if either of these samples were unknown, the predicted restriction enzyme patterns could be used to identify the organism.

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Abstract

La présente invention concerne des procédés, des compositions et des trousses permettant d'identifier un ou plusieurs organismes dans un échantillon. L'invention porte sur une combinaison d'amplification d'acide nucléique, de détection de marqueurs, et de digestion d'enzyme de restriction de l'acide nucléique amplifié.
PCT/US2007/080685 2006-10-11 2007-10-08 Détection et identification d'organismes par analyse de séquences d'acides nucléiques Ceased WO2008045819A2 (fr)

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US7314711B2 (en) * 1997-05-23 2008-01-01 Bioveris Corporation Assays employing electrochemiluminescent labels and electrochemiluminescence quenchers
US20020086289A1 (en) * 1999-06-15 2002-07-04 Don Straus Genomic profiling: a rapid method for testing a complex biological sample for the presence of many types of organisms
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CN112725534B (zh) * 2021-01-25 2022-05-06 中国疾病预防控制中心病毒病预防控制所 用于检测喀山病毒、哈扎拉病毒和伊塞克库尔病毒的引物探针、靶标组合、试剂盒和方法

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