WO2002053768A2 - Procedes et appareil de detection immune rapide d'adn - Google Patents

Procedes et appareil de detection immune rapide d'adn Download PDF

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Publication number
WO2002053768A2
WO2002053768A2 PCT/CA2002/000012 CA0200012W WO02053768A2 WO 2002053768 A2 WO2002053768 A2 WO 2002053768A2 CA 0200012 W CA0200012 W CA 0200012W WO 02053768 A2 WO02053768 A2 WO 02053768A2
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nucleic acid
reagent
capture
zone
target nucleic
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PCT/CA2002/000012
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English (en)
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WO2002053768A3 (fr
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Manuel Campos
Nisha Vikas Padhye
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Diagnosticart Llc
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Publication of WO2002053768A2 publication Critical patent/WO2002053768A2/fr
Publication of WO2002053768A3 publication Critical patent/WO2002053768A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

Definitions

  • a nucleic acid detection system, apparatus and method for Point of Care Diagnostic Testing ("POCT”) procedures or applications is provided.
  • the POCT procedures for nucleic acid detection can be used in medical, veterinary, forensic, environmental and/or agricultural applications. Description of the Related Art
  • Target nucleic acid sequence a specific nucleic acid sequence of interest
  • the target nucleic acid sequence may be only a small portion of the total amount of nucleic acid present, so that it may be difficult to detect its presence using nonisotopically labeled probes.
  • the quantity of target nucleic acid sequences may need to be increased relative to the quantity of other nucleic acid sequences in the clinical or experimental sample.
  • Selective amplification techniques which increase the quantity of target nucleic acid sequences relative to the quantity of non-target nucleic acid sequences, therefore, have become more important in clinical and experimental settings.
  • PCR polymerase chain reaction
  • Each of the oligonucleotide primers specifically anneals or hybridizes to its complementary sequence which can be part of or near the target nucleic acid sequence. If the target nucleic acid is double stranded (i.e. DNA) it would be necessary, prior to primer annealing or hybridizing, to denature the double stranded target nucleic acid sequence under denaturation conditions. Under such denaturation conditions, double stranded nucleic acid sequences separate into complementary strands and do not rehybridize. Following the denaturation step, annealing or hybridizing of the oligonucleotide primers to each of the strands of the target nucleic acid sequence can proceed.
  • double stranded i.e. DNA
  • a polymerization agent is used to extend each of the oligonucleotide primers using the target nucleic acid sequence as a template by sequentially adding the appropriate complementary nucleotides, producing an extension product.
  • the extension product of each primer is complementary to the sequence of the specific target nucleic acid strand to which the primer binds.
  • Suitable polymerization agents include enzymes such as R coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, reverse transcriptases, and other enzymes, including heatstable enzymes, which will facilitate combination of the nucleotides in the proper manner to form the.
  • primer extension products which are complementary to the nucleic acid sequence of each target strand.
  • the extension products are DNA.
  • primer extension is then terminated.
  • the extension products synthesized from each of the oligonucleotide primers are then themselves separated from their complementary target strand under denaturing conditions (i.e. high temperature) so that the extension products themselves can serve as templates for the synthesis of additional extension products of oligonucleotide primers complementary thereto.
  • thermostable polymerase such as T4 DNA polymerase for example, allows the high temperature denaturation of the extension products from their templates into single stranded nucleic acid sequences, and subsequent hybridization of the primers to the single stranded extension products with minimal loss of enzymatic activity. As a result, the process can be automated resulting in significant increases in efficiency.
  • PCR allows target nucleic acid sequences, usually those target nucleic acid sequences of fewer than 3000 base pairs (bp), to be amplified approximately one million fold. As such, PCR can be used to enable detection and/or characterization of target nucleic acid sequences associated with infectious diseases, genetic or cellular disorders such as cancer.
  • PCR is also useful when the amount of target nucleic acid sequences available for analysis is very small or the analysis is to be done on a limited or small amount of sample using non-radioactive detection techniques which may be inherently insensitive, or where radioactive techniques are being employed but where rapid detection is desirable. Therefore, the identification and/or detection of the target nucleic acid sequences can more readily be determined, making PCR an effective technique for pathogen detection and/or the diagnosis of genetic disorders. Given the possibility of automating the process as well as the efficiencies associated with the automated process, PCR is now used as an effective research and diagnostic tool. The use of PCR as a research and diagnostic tool, however, suffers from shortcomings.
  • PCR One of the most significant shortcommings with PCR is a potential lack of specificity. This lack of specificity can arise due to non-specific hybridization of the oligonucleotide primers with those nucleic acid sequences which are not of interest from either a research or diagnostic perspective, namely non-target nucleic acid sequences. This lack of specificity is particularly problematic when PCR is used in a diagnostic setting. If PCR results in the amplification of non-target nucleic acid sequences, there is the possibility of the incorrect detection of a target nucleic acid sequence, so called “false positives". The amplification of non-target nucleic acid sequences can also result in decreased signal to noise ratio, so called "high background", which makes detection difficult and further increases the possibility of an inaccurate diagnosis.
  • PCR is employed as the initial amplification step only, using specific oligonucleotide primers.
  • the presence of extension products which correspond to the target nucleic acid sequences are then identified and/or confirmed by a variety of detection systems.
  • the identification and/or confirmation of the presence of extension products which correspond to target nucleic acid sequences usually involves multiple steps, which are expensive and/or time-consuming.
  • One common detection procedure is electrophoretic separation of the extension products on an agarose gel so as to separate the extension products on the basis of size.
  • the target nucleic acid sequences can then be detected by labeled nucleic acid probes which are known sequences of nucleic acid that hybridize with the target nucleic acid sequences.
  • the hybridization of the labeled nucleic acid probe with the target nucleic acid sequence can be demonstrated by traditional electrophoresis and blotting techniques well known in the art, e.g. Southern blotting.
  • the extension products produced during PCR can also be digested or cut at sequence specific sites with particular restriction endonucleases such as EcoRL BamH, Hindlll, and the like. The resulting restriction fragments of the extension products can then be separated according to size using electrophoretic techniques.
  • the fragments derived from the digestion can then be compared with the fragments expected based on the nucleotide sequence ("restriction maps") to determine whether the target nucleic acid sequence was, in fact, present in the sample.
  • POCT procedures refer to diagnostic tests performed at or near the site of care.
  • POCT procedures can be used in medical, veterinary, forensic, environmental, and/or agricultural applications at the site at which the information is required.
  • diagnostic tests can include, but are not limited to, analytical testing activities provided within an institution, but performed outside the physical facilities of a clinical laboratory.
  • POCT procedures may include kits and instruments which are either hand carried or transported to die vicinity of care for immediate testing at that site. POCT procedures are particularly important, but not limited to, critical care, intensive care, neonatal intensive care and emergency departments, as well as operating rooms and field testing in agriculture and veterinary medicine.
  • nucleic acid detection techniques have, to a large extent, failed to reach POCT applications, as they are expensive and cumbersome to use, as well as slow to provide an answer. As such, the nucleic acid detection technologies discussed above, most notably PCR, have not been applied to POCT procedures.
  • DMG technology DNA Methyltransferases
  • DNA Methyltransferase Gene-typing technology DNA Methyltransferases Gene-typing technology
  • DMG technology has been described as an alternative to conventional PCR product-detection systems, as described in Lopez et al., International Patent Application No. PCT/US98/17859 published as WO 99/10540, the entire disclosure of which is incorporated herein by reference.
  • the DMG detection system described therein is based on the introduction of a methyl group into a specific 2 to 8 base pair nucleic acid sequence through the use of sequence or site specific DNA methyltransferases ("DNA MTase").
  • Each restriction endonuclease has a companion sequence or site specific DNA MTase which recognizes the same nucleic acid sequence or site as its companion restriction endonuclease.
  • Many DNA MTases can be obtained as a side fraction from commercial restriction endonuclease preparation. Several hundred DNA MTase specificities are known. There are also several DNA MTases that recognize 2 to 4 base pair nucleic acid sequences, such as the CG dinucleotide sequence, whereas there are no known restriction endonucleases that have such short recognition sequences.
  • DNA MTases are a constituent part of bacterial restriction-modification systems, which are able to catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) onto the substrate DNA.
  • SAM S-adenosylmethionine
  • Modification of symmetrically positioned nucleotides of the recognition sequence by the DNA MTase protects the DNA from cleavage by the complementary endonuclease of the DNA MTase.
  • Three types of methylated nucleotides have been found when analyzing the recognition sites modified by site-specific DNA MTase. Cytosine residues can be methylated at either the C5- position ( m 5C) or the N4-position ( m 4C), while adenine residues can be methylated at the N6- position ( m 6A).
  • DMG technology replaces the use of restriction endonucleases with DNA MTases, which transfer methyl groups to the nucleotides at specific recognition sites rather than digest the DNA at the specific recognition sequence. Instead of cutting the extension product into fragments with endonucleases for subsequent electrophoresis and hybridization analysis, the extension product is methylated at the specific recognition sequence of the particular DNA MTase.
  • the disclosure of WO 99/10540 provides that the methylated extension products can be detected using either radiolabelling techniques that are well known or immunochemical techniques using antibodies reactive against methylated nucleosides in a "dot blot" type of analysis.
  • DMG technology does not overcome the problems associated with nucleic acid detection techniques, which prevent these techniques from reaching POCT procedures. More specifically, the use of radiolabelling with DMG technology is not well suited for POCT procedures. Furthermore, the immunochemical use of antibodies reactive against methylated DNA in dot blots analysis requires cumbersome equipment and cannot provide a rapid test. Most immunodiagnostics methods have been developed to detect proteins, peptides or amino acid sequences using antigen-specific antibodies and are widely used to detect the presence of given pathogen antigen in biological samples. As such, immunodiagnostic methods have not been widely adapted to detect nucleic acids. As well, these immunological assays can be cumbersome, time consuming and require specialized equipment and training. The use of DMG technology itself, therefore, does not render nucleic acid detection techniques applicable to POCT procedures.
  • nucleic acid detection methods in human health, forensic medicine, animal health, and environmental and agricultural testing, particularly POCT procedures, would be a great benefit.
  • the use of nucleic acid detection in POCT procedures could be greatly improved by developing diagnostic apparatuses, systems, kits and methods that are user friendly, inexpensive, versatile and fast. , Better diagnostic apparatuses, systems, kits and methods would allow practitioners in these arts to make rational, knowledge-based decisions for the care of human and veterinary patients as well as intervention in forensic, environmental and/or agricultural areas.
  • DMG and RIM immunochromatography provides significant improvements over the previously known methods of detecting and/or analyzing target nucleic acid sequences.
  • the invention described in this application relates to the integration of DMG and RIM immunochromatography technologies to develop inexpensive, versatile and rapid nucleic acid detection techniques that can be used in laboratory settings and/or POCT applications.
  • the invention allows reduction of the cost and complexity of nucleic acid testing and makes nucleic acid testing a suitable alternative for the development of POCT applications.
  • the integration of DMG and RIM inimunoclrromatography technologies was conceived to meet the needs not met by currently available nucleic acid detection technologies. These include many instances where nucleic acid detection needs to be performed without die use of sophisticated equipment and/or elaborate training.
  • An object of the present invention is to provide apparatuses, systems and methods that combine DMG and RIM immunochromatography diagnostic assays for nucleic acid detection.
  • Another object of the present invention is to apply these apparatuses, systems and methods that combine DMG and RIM immunochromatography diagnostic assays to the development of POCT applications.
  • step (b) exposing the product of step (a) to conditions in which the DNA extension product is methylated by a DNA methyltransferase specific for a recognition site within the
  • step (c) determining if a methylated DNA extension product is obtained in step (b) using immunochromatography.
  • step (b) Another embodiment of the present is the use of rapid immune migration immunochromatography in determining if a methylated DNA extension product is obtained in step (b), the rapid immune migration immunocl romatogrpahy comprising: (d) exposing the DNA extension product of step (b) to a capture reagent which is specific for methylated DNA so that the capture reagent binds to the methylated DNA extension product to form a complex;
  • step (e) exposing the product of step (d) to conditions in which the complex migrates into a test zone, the test zone containing a test reagent which is specific for the tag and specifically binds to the tag;
  • step (e) exposing the product of step (e) to conditions in which the capture reagent migrates into a control zone, the control zone containing a control reagent which is specific for the capture reagent and specifically binds to the capture reagent; and
  • the imunocassette comprises a strip of material, preferably a porous material, capable of transporting fluids, usually by capillary action, and one or more diagnostic reagents immobilized on the material of the strip.
  • One of the reagents a capture reagent, is capable of specifically binding to the methylated extension products, which correspond to the target nucleic acid.
  • another reagent a control reagent, may be used which specifically binds to the capture reagent used to detect the methylated extension products.
  • a further reagent, a test reagent may be used which specifically binds to the tagged primer.
  • the method of using the immunocassette involves adding an aliquot of the sample, which may contain the target nucleic acid, to a sample or capture zone in the immunocassette containing a target nucleic acid capture reagent under conditions that promotes transport by way of capillary action of the complex formed by the target nucleic acid and the capture reagent and further interaction with the other reagents in the device.
  • kits for use in determining the presence of a target nucleic acid in a sample comprising:
  • a strip of material capable of transporting fluids having a proximal and a distal end;
  • a capture zone provided near the proximal end of the strip adapted to permit application of a liquid sample containing methylated nucleic acid sequences which correspond to the target nucleic acid sequences, to the strip to permit transportation of the methylated nucleic acid sequences complexed with the capture agent along the material from near the proximal end to near the distal end;
  • control zone distinct from the capture zone provided near the distal of the strip having a control reagent immobilized thereon, wherein the control reagent binds specifically to the capture reagent
  • test zone provided between the capture zone and the control zone but distinct from the capture zone and control zone, having a test reagent immobilized thereon, wherein the test reagent binds specifically to the tagged primer
  • kits for use in determining the presence of a target nucleic acid in a sample comprising:
  • a strip of material capable of transporting fluids having a proximal and a distal end
  • a capture zone provided near the proximal end of the strip and having a capture reagent which is specific for methylated DNA, adapted to permit application of a liquid sample containing methylated nucleic acid sequences which correspond to the target nucleic acid sequences, to the strip to permit transportation of the methylated nucleic acid sequences complexed with the capture agent along the material from near the proximal end to near the distal end
  • a control zone distinct from the capture zone provided near the distal along the strip having a control reagent immobilized thereon, wherein the control reagent binds specifically to a portion of the capture reagent
  • a test zone provided between the capture zone and the control zone but distinct from the capture zone and control zone, having a test reagent immobilized thereon, wherein the test reagent binds specifically to the tag.
  • the systems, apparatuses and methods disclosed in t is invention are also useful to develop many other immunological based assays to detect target nucleic acid sequences and can be adapted to detect target nucleic acids from any source using antibody-based immune detection systems.
  • FIGURE 1 represents a photograph of an apparatus of the present invention demonstrating the RIM immunocl romatograpliic detection of bovine viral diarrhea virus (BVDV) amplicon after DMG.
  • Lane 1 represents a positive control PCR reaction using a BVDV amplicon.
  • Lane 2 represents a negative control PCR reaction having no BVDV amplicon.
  • target nucleic acid sequence is any nucleic acid sequence, the presence of which is to be detected or determined in a sample.
  • a target nucleic acid sequence may be a naturally occurring nucleic acid, may be the product of nucleic acid amplification methods such as PCR or a transcription-based amplification method (i.e., an "amplicon"), or may be produced synthetically.
  • the "target nucleic acid sequence” may comprise the entire nucleic acid sequence to be determined or may be a portion thereof.
  • non-target nucleic acid sequence is defined as any nucleic acid sequence which is not a target nucleic acid sequence.
  • DNA mefhyltransferase or “DNA MTase” refers to an enzyme which transfers a methyl group to a specific nucleotide or nucleosides, within a specific 2 to 8 nucleic acid base pair recognition sequence.
  • oligonucleotide as used herein is defined as a polymer of two or more nucleotides. Oligonucleotides are preferably polymers of 2 to 25 nucleotides.
  • the nucleotides can comprise deoxyribonucleotides or ribonucleotides. The number of nucleotides will depend on many factors, which i turn depend on the ultimate function or use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation for primer extension when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and a polymerization agent such as T4 polymerase and at a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent.
  • the exact lengths of the primers will depend on many factors, including temperature and source of primer.
  • the oligonucleotide primer typically contains 15 to 25 or more nucleotides, although it may contain greater or fewer nucleotides.
  • a primer is selected to be sufficiently complementary to a strand of a target nucleic acid sequence to be amplified so as to hybridize therewith under conditions that permit selective amplification. Therefore, the primer sequence need not be exactly complementary to the sequence of the template. For example, a non-complementary oligonucleotide fragment may be attached to the 5' end of a primer, with the remainder of the primer being sufficiently complementary to the strand to hybridize therewith under amplification conditions.
  • non-complementary sequences of one or more nucleotides can be interspersed within the primer, provided that the entire sequence of the primer has sufficient complementarity with the sequence of the strand to be amplified so that the primer can hybridize therewith and thereby form a template for synthesis of the extension product of the other primer.
  • restriction endonucleases and “restriction enzymes” refer to enzymes of which digest or cut nucleic aid sequences at or near a specific nucleotide sequence.
  • the terms "5' end” and "3' end” are defined as the 5' phosphate or tire 3' oxygen of a mononucleotide pentose ring, respectively.
  • An end of an oligonucleotide, target or non-target nucleic acid sequence is referred to as the "5' end” if its 5'phosphate or hydroxyl group is not linked to the 3' oxygen of a mononucleotide pentose ring or as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
  • This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along a nucleic acid strand.
  • the terms “complementary” or “complementarity” are used in reference to oligonucleotides, target nucleic acid sequences or non-target nucleic acid sequences related by the Watson-Crick base-pairing rales.
  • sequence 5'-A-G-T-3' is complementary to the "sequence 3'-T-C-A-5'.
  • Complementarity may be "partial,” in which only some of the nucleic acid's bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • substantially complementary refers to any primer that can hybridize to either or both strands of the target nucleic acid sequence under conditions which permit primer hybridization with its target and primer extension.
  • the term “correspond” refers to nucleic acid sequences which are either identical to each other or complementary.
  • an extension product or primer corresponds to a target nucleic acid sequence when it is either identical thereto or complementary thereto.
  • hybridization is used in reference to the pairing of complementary nucleic acid sequences. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between die nucleic acids, stringency of the conditions involved, the T m (thermal melting point) of the formed hybrid, and the G:C ratio within the nucleic acids.
  • Hybridization stringency conditions can be generally varied by manipulation of temperature, salt concentration, and formamide concentration.
  • temperature high temperatures increase strmgency, while low temperature decrease stringency.
  • salt concentrations high salt concentrations decrease stringency, while low salt concentrations increase stringency.
  • Formamide decreases the T m of DNA, thus lowering the temperature at wliich DNA sequences will hydridize. Thus, raising amounts of formamide increases strmgency.
  • tl e terms "amplification” or “selective amplification” refer to any method through which the quantity of extension products wliich correspond to the target nucleic acid sequences can be selectively increased in relation to the amount of extension products which correspond to the non-target nucleic acid sequences present in a sample.
  • a preferred example is Polymerase Chain Reaction (PCR).
  • PCR Polymerase Chain Reaction
  • ligand refers to a first molecule that specifically binds to a second molecule.
  • ligands include antigens, haptens, lectins, carbohydrates, glycoproteins, substrates and other molecules well known in die art.
  • molecules that bind to ligands include oligonucleotides, antibodies, receptors, lectins, chelators, enzymes and other molecules well l ⁇ iown in the art.
  • sample as used herein is used in its broadest sense.
  • a sample either containing or suspected of containing a nucleic acid may comprise, but is not restricted to, a eukaryotic or prokaryotic cell or cell suspension, a cell line, a sample of a tissue from a human, plant or animal, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA, RNA, cDNA and the like. It may also include soil, water, and forensic samples.
  • the terms "detection” and “determination” as used herein refer to quantitatively or qualitatively identifying tlie presence of a target nucleic acid sequence within a sample.
  • anti-methylated DNA antibodies refers to antibodies, which specifically recognize or bind to methylated DNA, including methylated nucleotides and/or modified nucleosides. These anti-methylated DNA antibodies can be produced by conventional technology (i.e. monoclonal or polyclonal technology) or by recombinant antibody teclmology including eukaryotic, prokaryotic and plants expression systems.
  • immunochromatography refers to antibody-based assays in which one or more analytes of a sample are analyzed by having the sample flow on or through a membrane, usually by capillary action to (a) mix one or more analytes with the one or more diagnostic reagents of the assay and (b) separate tlie diagnostic reagents and/or tlie analytes.
  • RIM Rapid Immune Migration
  • Inmiunochromatographic assays typically involve dispensing an aliquot of the sample, which may or may not containing an analyte of interest, onto a sample or capture zone. Either before dispensing tlie aliquot of the sample or while in the capture zone, any analyte present in the sample can be mixed widi a capture reagent to form an analyte-capture reagent complex.
  • the analyte-capture reagent complex can then flow out of the capture zone by way of capillary action.
  • the analyte then flows across a membrane, where it can bind with the test and/or control reagents in the test and control zones, respectively.
  • tlie analyte If tlie analyte is present in the sample, it binds to tl e test reagent, forming a test line and a positive result is indicated.
  • the intensity of tlie test line can be proportional to tlie concentration of analyte in the sample.
  • the detection of a target nucleic acid sequence, particularly a target DNA sequence, in a sample using immunochromatography format in a single step procedure can be conducted in approximately 20 minutes.
  • Iimiiunochromatograpliic assays have tlie benefits of a user-friendly format, short reaction time, long-term stability over a wide range of climates and low manufacturing costs.
  • Different formats can be designed to combine DMG with RIM immunochromatography in order to develop a fast one-step diagnostic kit.
  • the invention is amenable to tlie development of diagnostic products where detection of a target nucleic acid sequence is required.
  • the process of target nucleic acid sequence detection is typically initiated by collecting a sample.
  • Samples can originate from a number of sources including, but not limited to, humans, plants or animals, human, animal or plant products, soils, water, and the environment at large.
  • the sample is then process to release the total amount of nucleic acid present in the sample.
  • tlie sample may need to be pre-processed in order to enrich the amount of nucleic acid present in the sample. Enrichment procedures may involve partial purification of special culture conditions but other known methods of enrichment are also included.
  • the nucleic acid suitable for detection is then extracted and processed for PCR and/or other suitable amplification procedures.
  • Methods and modifications of the techniques to amplify nucleic acids, particularly DNA, are very well known in the art.
  • Several strategies for the amplification of nucleic acids have been described, including PCR amplification, amplification of a nucleic acid probe (e.g., ligase chain reaction, Q ⁇ replicase), signal amplification (e.g., branched-probe DNA assay) and the like.
  • PCR is preferred.
  • Primers needed to initiate the reaction, enzymes, buffers and general amplification conditions for either PCR or the other amplification methods indicated can vary for different target nucleic acid sequences and many of them are known in the art and or can be easily develop following standard procedures familiar to those skill in tl e art.
  • the 5' and 3' primers are selected to be complementary to sequences within the target sequence or which flank the target nucleic acid sequence.
  • one or both of the 5' or 3' primers are to be "tagged" with a ligand that binds to a specific binding partner.
  • a ligand can consist of either a non-nucleic acid moiety, preferably biotin, or can consist of a known nucleic acid sequence.
  • a nucleic acid tag could be synthesized to be complementary to or specific for a general nucleic acid sequence attached to an inert support so that the nucleic acid can be used in a variety of assays.
  • the nucleic acid tag does not need, however, to be synthesized specially for a particular target nucleic acid sequence. Instead, a general nucleic acid tag can be used.
  • the 5' end primer can be tagged with either the same or different tag as the 3' end primer.
  • Many such tags and methods for tagging ligands to nucleotides are known in the art, as disclosed in Y. M. D. Lo et al, Nucleic Acids Research, 16, 8719 (1988), European Patent Application Serial No. EP 0 192 168 and U.S. Patent No. 5,629,158, the contents of each of which are hereby incorporated by reference. It should be clear that these examples do not limit the invention since additional methods to accomplish "tagging" could be design and develop by those familiar in the art.
  • the tagged primers can then be used in the amplification procedure, preferably PCR.
  • each of the extension products corresponding to the target nucleic acid sequences will be tagged.
  • Ho ⁇ vever, differently tagged 5' and 3' end primer could also be used to expand the versatility of the assay.
  • the 5' tag could consist of biotin
  • the 3' tag could consist of Protein A. 'It would be obvious to a person skilled in the art that many suitable tags could be used in combination.
  • the tagged extension products can then be identified using the specific binding partner of the tag being used.
  • the specific binding partner of the tag being used For example, if biotin is used as a tag, Streptavidin can be used as the binding partner to bind the tagged extension products.
  • extension products that correspond to both target and non-target nucleic acid sequences will contain tlie tag.
  • tlie tags alone cannot detect the presence of any extension products that correspond to tl e target nucleic acid sequence.
  • the confirmation or detection step is provided by sequence specific methylation and the analysis which follows the amplification step, as provided below.
  • the sequence specific methylation step involves the following.
  • Hie- sequence specific DNA MTases can be added to the sample containing tlie extension products which correspond to both target and non-target nucleic acid sequences in Hie presence of S-Adenosyl- Methionine (SAM).
  • SAM S-Adenosyl- Methionine
  • the specific DNA MTase to be employed in accordance with the invention is selected on tl e basis that it will selectively methylate a specific site or sites within the target nucleic acid sequence preferentially.
  • the remaining reaction conditions such as reaction time and appropriate temperature for the methylation reaction can all be established by those skilled on the art.
  • extension products tiiat correspond to the target nucleic acid sequence can be methylated on the basis of the known, proposed or suspected nucleotide sequence of the target nucleic acid sequence.
  • extension product Following methylation of the extension product, the methylated extension product which corresponds to the target nucleic acid sequence can be identified. This identification occurs by way of immunochromatography assays. Extension products have differential reactivity to reagents specific for methylated DNA due to their methylation state. More specifically, extension products that correspond to the target nucleic acid sequence will have cytosiiies and/or adenines of tlie DNA MTase restriction site selectively or preferentially methylated over extension products that correspond to the non-target nucleic acid sequences.
  • the reagents specific for me ⁇ iylated DNA preferably an anti-methylated DNA antibody
  • the reagents are first coupled or conjugated with a signaling agent, h a preferred embodiment, the signaling agent is coupled or conjugated with the anti-methylated DNA antibodies so as not to perturb the antibodies binding characteristics but enable the presence of the antibodies to be identified either visually or by an appropriate detection technology as outline below.
  • the choice of signaling agents is influenced by factors such as ease and sensitivity of detection, as well as the ability to readily incorporate such signaling agent into tlie anti-methylated DNA antibodies.
  • the signaling agent can be any material having a detectable physical or chemical property. Such signaling agents have been well developed in the field of immunochromatography and, in general, any label useful in such methods can be applied to the present invention.
  • Useful signaling agents can include magnetic beads, chemiluminescent or fluorescent compounds (e.g., luciferin, fluorescein isothiocyanate, texas red, rhodamme, acridinium esters, and lanthanide chelates), radioactive isotopes (e.g.
  • enzymes e.g., horse radish peroxidase, and alkaline phosphatase
  • colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • colloid gold is used as the signaling agent.
  • a gold colloid is prepared using gold chloride and sodium citrate.
  • anti- methylated DNA antibodies such as polyclonal rabbit anti- m 6A, are passively conjugated with the gold colloid suspension. It will be understood by those skilled in the art that other anti-methylated DNA antibodies including anti- m 5C and anti- m 4C antibodies can be used.
  • conjugated antibodies are then recovered by centrifugation.
  • the conjugate antibodies can be mixed directly with the methylated sample under conditions that allow the conjugate antibodies to form complexes with those extension products that have been previously methylated
  • the conjugated antibodies can be provided in tl e capture zone of the immunocassette, as provided below. In that case, once an aliquot of the methylated sample is added to the capture zone, the methylated extension products and the anti-methylated DNA antibodies form complexes within the capture zone. In either case, the formed complexes can migrate along the membrane. From the discussion provided earlier in tlie disclosure, Hie extension products that correspond to the target nucleic acid sequences will have been selectively methylated.
  • extension products that correspond to the target nucleic acid sequence will be complexed with conjugate antibodies. Extension products that are complexed with conjugate antibodies can then be visualized within the test zone as disclosed below and in Figure 1. As shown in Figure 1, two immunocassettes 10 and 12 are each provided with strips 14 and
  • the immunocassette 10 is used with a positive sample, while the immunocassette 12 is used with a negative sample.
  • the positive sample used in immunocassette 10 is a sample that contains the target nucleic acid sequence.
  • tlie negative sample does not contain the target nucleic acid sequence.
  • extension products will be produced which correspond to tlie target nucleic acid sequence.
  • the extension products from the positive sample will be methylated and, under appropriate conditions, will form complexes with the conjugated antibodies.
  • no extension products should be produced which correspond to the target nucleic acid sequence. As such, the extension products from the negative sample will not be methylated and will not fonn complexes with the conjugated antibodies.
  • strips 14 and 14' which are made of a membranes 18 and 18', absorbent wicks and stiff backing material (not shown) to provide support for the wicks and the membranes 18 and 18'.
  • the membranes 18 and 18' can be made of any solid or semisolid material, preferably porous to which proteins, peptides, amino acid sequences or nucleic acids can be immobilized and which allows liquid migration, preferably by capillary action.
  • a cliromatograpliic material such as a nitrocellulose or a nylon membrane. It will be understood by those skilled in the art that immunocassettes could be employed using the present invention which would include a solid support containing materials such as biochips or microfluidic chips, which allow migration, preferably by capillary action.
  • the conjugate antibodies are provided within the capture zone.
  • the treated positive and negative samples * have been added to capture zones 16 or 16'; any methylated extension products which are complexed with conjugated antibody.
  • complexes will begin to migrate along the membranes 18 and 18', by way of capillary action, towards their respective test zones 20 and 20'.
  • the test zones 20 and 20' have a binding agent that can recognize the tag attached to either the 5' or 3' primer used during PCR.
  • Streptavidin is preferably immobilized to the membrane 18 and 18' in the test line 22 and 22'.
  • the Streptavidin immobilized in test line 22 and 22' of zones 20 and 20' will bind to all the tagged extension products of both tlie positive and negative samples.
  • tlie 5' or 3' primers are tagged with biotin.
  • biotinylated extension products will bind to Streptavidin immobilized in tlie test zone 20 and 20' of the positive and negative samples, thus stopping the migration of the extension products in the test zone 20 and 20'.
  • any conjugate antibodies which are complexed with the extension products and the Streptavidin of test line 22 and/or 22' will form the test line 22 and/or 22' within test zone 20 and/or 20 'resulting in a visible band or line seen due to the presence of die signaling agent.
  • the signaling agent is gold.
  • only the positive sample contained tlie target nucleic acid sequence. Consequently, the presence of the extension products which correspond to the target nucleic acid sequence can be easily determined by a visible signal 23 in test line 22 of test zone 20.
  • the negative control has no visible line at test line 22' indicating that the negative sample failed to contain any of tlie target nucleic acid sequence.
  • Each membrane 18 and 18' of the control zone 24 and 24' has a binding agent immobilized at control line 26 and 26'in control zone 24 and 24'.
  • the binding agent is selected on the basis of its ability to bind to the capture reagent, preferably a portion of anti-methylated DNA antibodies, more preferably the Fc portion of the antibody.
  • a preferred binding agent to be used in the control zone 24 and 24' is Protein A.
  • other binding agents could be used in the control zone, provided they are capable of binding to the capture agent.
  • the above assay fonnat is only offered as an example and should not limit the invention since several components of the assay could be easily substituted with out changing the principle of the invention.
  • the anti-methyl DNA antibodies could be coated to latex beads or any other particular matter that could allow visualization of the reaction.
  • the Strepavidin could be substituted by anti-biotin antibodies or if a different primer tag was to be selected, the test zone could be coated with an appropriate ligand to capture tlie methyl-DNA-particulate complex.
  • the protein A in the control zone could also be substituted with various other ligands capable of capturing tlie antibody complexes.
  • Other modifications will be readily apparent to those skilled in this art.
  • the combination of DMG and RIM immunochromatography can be used for any application requiring nucleic acid analysis.
  • the present invention could be used for the development of POCT diagnostic kits to detect mutations responsible for hereditary diseases in human beings and animals, to diagnose and/or fingerprint DNA from infectious agents or forensic materials, and to detect genes responsible for productions traits in crops and animals. Since the troublesome and expensive multiple step procedure of analyzing nucleic acid in samples is replaced by an inexpensive and fast one step procedure, other uses will be readily apparent to those skilled in this art.
  • Bovine viral diarrhea vims is a small, enveloped RNA virus classified in die family Flaviviridae, member of the genus Pestivirus. Significant genotypic and phenotypic differences have been shown to exist between different BVDV isolates that has warranted tl e classification of BVDV in type I and II groups.
  • RNA extraction Viral RNA of the BVDV isolates used was extracted from serum or tissue culture supernatant using tile QIAamp Viral RNA kit (QUIAGEN Inc.). Briefly, 140 ⁇ l of serum or cell culture supernatants were treated following the manufacturer directions and the total RNA was solubilized in 50 ⁇ l of water.
  • RNA 8 ⁇ l of total RNA was reverse transcribed to produce cDNA to the target mRNA using Superscript RT (GIBCO, Life Tech) in ANB.
  • the specific primer used was BVDC3: 5' GTA TAA AAG TTC ATT T 3'. This primer anneals with nucleotides 371 to 386 and 400 to 414 of the 5' terminal of the BVD RNA types I and II respectively.
  • the BVDC3 primer has a 100 % of identity with all the known isolates of BVDV genotype I and genotype II. The reaction was performed for 45 mill, and the enzyme inactivated for 15 min at 70°C. PCR
  • the cDNA obtained by reverse transcription was used as template for PCR in ANB buffer IX.
  • the primers used for amplification were BVD3for (5' TCC CTC TCA GCG AAG GCC G 3;), tagged at the 5' end with biotin (5' biotinylated) and BVD3rev (5' GCC CTC GTC CAC GTG GCA T 3'), untagged.
  • the BVD3for and BVD3rev primers have a T m 61.34°C and 60.33°C, respectively, at a salt concentration of 50 niM of NaCl.
  • the BVD3for and BVD3rev primers are universal for both BVDV biotypes and produce a fragment of 176 bp and 179 bp for the type I and type II BVD, respectively.
  • Ten PCR cycles at 94 °C: 2 seg., 60 °C: 1 seg. and 72 °C: 3 seg were performed.
  • This procedure produced an amplicon of 176 bp for type I and 179 bp for type II BVDV.
  • the 176 bp and 179 bp amplicons contained a Rsal (GTAC) recognition site at position 153 and two Taql (TCGA) recognition sites at positions 174 and 199 of BVDV type I. Seventeen BVDV isolates from USA and Argentina show tlie presence of a Taql and a Rsal recognition sites in this area.
  • DNA restriction endonuclease and DNA methyltransferase reactions were used to produce a fragment of 176 bp and 179 b
  • Rabbit polyclonal antibodies ' were conjugated to gold micro particles using the standard procedures. Briefly, gold colloid was prepared using Gold Chloride and sodium citrate to which polyclonal rabbit anti-6 methyl Adenine was passively conjugated. The conjugated particles were then incubated with BSA to block and the conjugate was recovered by centrifugation. Conjugated particles were washed by centrifugation in a PBS based buffer containing BSA and detergent and stored at 2-8°C until use. To prepare the strips the nitrocellulose and an absorbent wick were placed on a stiff backer using automation. The nitrocellulose was striped at the test line position with 3 mg/nil of Streptavidin (Sigma cat. no S4762).
  • Protein A was striped at the control line position and tlie colloidal gold conjugate was striped at the capture zone.
  • the nitrocellulose was incubated at elevated temperature to immoblize the proteins. Storage was under desiccating conditions. Assemble of strips in devices was accomplished by overlaying the striped nitrocellulose/wick/backer card assembly and applying sample pad material. Strips were cut on an automated cutting instrument and assembled into plastic cassette housings. Detection of BVDV amplicon by RIO immunochromatography After PCR amplification and methylation of the amplicon with M.

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Abstract

Cette invention se rapporte à un système de détection d'ADN rapide, peu coûteux et facile à utiliser, destiné à des applications de tests génétiques chez l'être humain, les animaux, les plantes et dans l'environnement. Cette invention concerne des procédés, des systèmes et des kits qui permettent le développement d'applications de techniques au point de soins qui utilisent le diagnostic à base d'acide nucléique.
PCT/CA2002/000012 2001-01-05 2002-01-04 Procedes et appareil de detection immune rapide d'adn WO2002053768A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008080031A1 (fr) 2006-12-21 2008-07-03 Silver Lake Research Corporation Bandes pour test chromatographique d'un ou plusieurs analytes
CN111197110A (zh) * 2020-01-16 2020-05-26 浙江农林大学 用于检测猫泛白细胞减少症病毒的rpa-fld方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2722799A1 (fr) * 1994-07-21 1996-01-26 Parteurop Procede d'amplification d'acide nucleique a l'aide d'un nucleoside modifie, et detection du produit d'amplification a l'aide d'anticorps
WO1999010540A1 (fr) * 1997-08-29 1999-03-04 Lopez Osvaldo J Etablissement de genotype au moyen de la methyltransferase d'adn
EP1018649A2 (fr) * 1992-03-31 2000-07-12 Abbott Laboratories Dispositif immunochromatographique et procédé pour une détection multiplexe d'analytes multiples
EP1020726A1 (fr) * 1998-07-01 2000-07-19 Nitto Denko Corporation Test immunologique et kit de test immunologique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1018649A2 (fr) * 1992-03-31 2000-07-12 Abbott Laboratories Dispositif immunochromatographique et procédé pour une détection multiplexe d'analytes multiples
FR2722799A1 (fr) * 1994-07-21 1996-01-26 Parteurop Procede d'amplification d'acide nucleique a l'aide d'un nucleoside modifie, et detection du produit d'amplification a l'aide d'anticorps
WO1999010540A1 (fr) * 1997-08-29 1999-03-04 Lopez Osvaldo J Etablissement de genotype au moyen de la methyltransferase d'adn
EP1020726A1 (fr) * 1998-07-01 2000-07-19 Nitto Denko Corporation Test immunologique et kit de test immunologique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOU C ET AL: "DELETION DETECTION IN THE DYSTROPHIN GENE BY MULTIPLEX GAP LIGASE CHAIN REACTION AND IMMUNOCHROMATOGRAPHIC STRIP TECHNOLOGY" HUMAN MUTATION, WILEY-LISS, NEW YORK, NY, US, vol. 5, no. 1, 1995, pages 86-93, XP002046895 ISSN: 1059-7794 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008080031A1 (fr) 2006-12-21 2008-07-03 Silver Lake Research Corporation Bandes pour test chromatographique d'un ou plusieurs analytes
US7919331B2 (en) 2006-12-21 2011-04-05 Silver Lake Research Corporation Chromatographic test strips for one or more analytes
CN111197110A (zh) * 2020-01-16 2020-05-26 浙江农林大学 用于检测猫泛白细胞减少症病毒的rpa-fld方法

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