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WO2018128286A1 - Procédé de détection rapide d'acide nucléique, et procédé de diagnostic rapide d'une maladie l'utilisant - Google Patents

Procédé de détection rapide d'acide nucléique, et procédé de diagnostic rapide d'une maladie l'utilisant Download PDF

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Publication number
WO2018128286A1
WO2018128286A1 PCT/KR2017/014363 KR2017014363W WO2018128286A1 WO 2018128286 A1 WO2018128286 A1 WO 2018128286A1 KR 2017014363 W KR2017014363 W KR 2017014363W WO 2018128286 A1 WO2018128286 A1 WO 2018128286A1
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Prior art keywords
nucleic acid
disease
tag
pcr
capture component
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English (en)
Korean (ko)
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배진현
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Individual
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Priority claimed from KR1020170166156A external-priority patent/KR102135979B1/ko
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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

Definitions

  • the present invention relates to a rapid detection method of a nucleic acid and a rapid diagnosis method of a disease using the same, and more particularly, (a) a tagged primer having a sequence complementary to a region of a target nucleic acid sequence (tagged primer) Using, to perform a polymerase chain reaction (PCR) with a fluorescence probe that binds to the double stranded nucleic acid; (b) inducing a coupling reaction between the tag and the capture component by treating the PCR product generated in step ( a ) on a solid on which the capture component to which the tag is attached is accumulated at a specific position; And (c) detecting fluorescence from the solid phase of step (b), and a method for rapidly detecting a target nucleic acid and a method for rapidly diagnosing a disease using the method.
  • PCR polymerase chain reaction
  • pandemic caused by renal and mutant influenza viruses as in the case of the H1N1 influenza virus, current vaccine development techniques cannot develop preventive vaccines in early time.
  • Ant iviral treatments have a limited efficacy that requires them to be administered early in the infection (within at least 48 hours after infection).
  • pandemic influenza occurs, in order to maximize the efficacy of antiviral treatment until the development of preventive vaccine, it is possible to determine the infection in real time without going through a medical institution and receive appropriate treatment.
  • pandemic influenza in order to maximize the efficacy of antiviral treatment until the development of preventive vaccine, it is possible to determine the infection in real time without going through a medical institution and receive appropriate treatment.
  • RIDT rapid influenza test
  • Realt ime-RT-PCR real time polymerase chain reaction
  • RIDT has not been used to confirm the influenza infection of patients, indicating that it is not suitable for the diagnosis of swine flu. "As a result, the diagnosis of influenza viruses with RIDT will get faster results
  • the sensitivity is lower than that of Viral culture and Real RT-PCR, and if necessary, retesting by Viral culture and Real RT-PCR is necessary for patients with negative judgment by RIDT. (Research on rapid diagnosis for diagnosis of influenza, 2012, pp.1-5).
  • PCR is a molecular diagnostic method having a relatively high reliability, but there is a problem that must be relied on a specific expert in deriving, analyzing and interpreting the results by the PCR product.
  • an object of the present invention is to (a) polymerase chain reaction (PCR) with a fluorescent probe that binds to a double-stranded nucleic acid by using a tagged primer having a sequence complementary to a partial region of the target nucleic acid sequence.
  • PCR polymerase chain reaction
  • step (b) treating the PCR product generated in step (a) on a solid on which the capture component to which the tag is attached is integrated at a specific position to induce binding reaction between the tag and the capture component; And (c) detecting fluorescence from the solid phase of step (b). It is another object of the present invention to (a) bind to a double-stranded nucleic acid using a tagged primer having a sequence complementary to a region of a disease marker nucleic acid with respect to a nucleic acid sample obtained from a patient sample.
  • PCR polymerase chain reaction
  • step (b) treating the PCR product generated in step (a) on a solid on which the capture component to which the tag is attached is integrated at a specific position to induce binding reaction between the tag and the capture component; And (c) fluorescence from the solid phase of step (b). It provides a method for rapid diagnosis of a disease, including the step of detecting.
  • the present invention (a) by using a tagged primer having a sequence complementary to some region of the target nucleic acid sequence, with a fluorescent probe that binds to a double stranded nucleic acid Performing polymerase chain reaction (PCR); (b) treating the PCR product generated in step (a) on a solid on which the capture component to which the tag is attached is integrated at a specific position to induce binding reaction between the tag and the capture component; And (c) detecting fluorescence from the solid phase of step (b).
  • PCR polymerase chain reaction
  • the present invention (a) using a tagged primer having a sequence complementary to a region of the disease marker nucleic acid for a nucleic acid sample obtained from a patient sample Performing a polymerase chain reaction (PCR ) with a fluorescence probe that binds to the double stranded nucleic acid; (b) treating the PCR product generated in step (a) on a solid on which the capture component to which the tag is attached is integrated at a specific position to induce a coupling reaction between the tag and the capture component; And (c) detecting fluorescence from the solid phase of step (b).
  • PCR polymerase chain reaction
  • step (b) inducing binding reaction between the tag and the capture component by treating the PCR product generated in step (a) on a solid on which the capture component to which the tag is attached is integrated at a specific position;
  • step (c) detecting fluorescence from the solid phase of step (b)
  • target nucleic acid in the present invention, as a substance to be detected whether or not present in a sample, refers to a plurality of nucleic acid polymers constituting a specific sequence (sequence).
  • the type of the nucleic acid material is not limited thereto, but may be DNA, RNA, peptide nucleotide nucleic acid (PNA), locked nucleic acid (LNA), or the like, and most preferably DNA or RNA.
  • the target nucleic acid is a bio-material, including a thing derived from or similar to an organism, or produced in vitro
  • the DNA includes cDNA, genomic DNA, oligonucleotide
  • RNA is genomic RNA, mRNA, oligonucleotide Nucleotides and the like.
  • the target nucleic acid functions as a marker, and the presence or absence of a specific disease (or condition) can be confirmed by detecting the presence or absence of the target nucleic acid in a sample.
  • the nucleic acid detection method of the present invention is available for the detection of various diseases (or conditions), and the types of the diseases are not particularly limited, but include, for example, infectious diseases; Or diseases involving gene mutations (including mutant diseases) such as cancer, inflammatory diseases, metabolic diseases, nervous system diseases, musculoskeletal diseases, digestive system diseases, allergic diseases, immune related diseases, endocrine diseases, cardiovascular diseases , It may be selected from the group consisting of urogenital diseases, respiratory diseases and skin diseases. Depending on the type of disease to be detected, one skilled in the art can easily configure the target nucleic acid as a disease marker.
  • the disease marker nucleic acid includes, but is not limited to, a foreign gene from an infectious agent or an internally mutated nucleic acid (gene) of an individual.
  • the infectious agent is not particularly limited as long as it is a known pathogenic microorganism, and includes, for example, viruses, bacteria, fungi or parasitic layers.
  • the virus may be influenza virus, respiratory syncyt ial virus (RSV), zika virus, metapneumovirus, adenovirus, parainfluenza virus, rhinovirus, adenovirus, hepatitis virus, rotavirus, HIV Viruses include but are not limited to norovirus, coxsackievirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, and the like.
  • the bacteria include Pseudomonas, Excherichia and Klebsilla.
  • the endogenous mutant nucleic acid refers to a mutation of an endogenus gene of an individual in a specific disease (or condition) state, and is naturally occurring in the individual, such as known cancer markers. Or may be generated by introduction of the foreign gene into an individual (in a cell).
  • the term "mutation" as used in the present invention refers to a mutation occurring at the genetic level compared to a control that does not cause mutation, and this mutation causes a difference in the characteristics of the phenotype (especially disease state).
  • the mutations found are, of course, encompassing all artificially introduced mutations. Mutations include all mutations occurring at random or at specific positions, and by addition, deletion, and / or substitution of nucleotides constituting the gene.
  • the disease marker nucleic acid may be a foreign gene from an infectious agent or an endogenous mutant nucleic acid by introduction of said foreign gene.
  • the specific sequence of the target nucleic acid or disease marker nucleic acid can be determined by those skilled in the art according to the type of the specific disease for which information is to be obtained in diagnosis or the like.
  • the art discloses that the influenza virus and the antigenic protein hemagglutinin (Nemagulutin) or neuraminidase encoding nucleic acid can be used as a nucleic acid for diagnosis of influenza infection. .
  • sample refers to an analysis object containing a target nucleic acid to be detected, and may be, for example, obtained from a sample of an individual (or patient) suspected of having a specific disease. .
  • the sample may be a specimen itself, or may be a workpiece in which the specimen is subjected to any treatment such as grinding, extraction, or purification.
  • the sample means a material required for the test, and includes, but is not limited to, tissue, cells, whole blood (blood), serum, plasma, saliva, sputum, cerebrospinal fluid or urine. Preferably blood or saliva.
  • step (a) polymerase chain reaction (PCR) is performed with a fluorescent probe that binds to the double-stranded nucleic acid using a tagged primer having a sequence complementary to a partial region of the target nucleic acid sequence.
  • PCR polymerase chain reaction
  • the term "primer” refers to a short nucleic acid sequence having a free 3 'terminal hydroxyl group, capable of forming base pairs with complementary templates and serving as a starting point for template strand copying. Short nucleic acid sequence. Primers can initiate DNA synthesis in the presence of four nucleoside triphosphates that are different from reagents for polymerization reaction (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.
  • the primer is a pair of sense and antisense nucleic acids (i.e. forward primers and reverse primers) consisting of 5 to 50, preferably 10 to 30 nucleotides, the basic properties of the primer acting as a starting point for DNA synthesis. Additional features can be incorporated as long as they do not change.
  • the term “complementary” means having complementarity enough to selectively hybridize to the target nucleic acid sequence described above under certain specific hybridizations or annealing conditions. It may have one or more mismatch sequences as long as it can be selectively localized / preferably the primer may be perfect ly complementary to the desired nucleic acid sequence.
  • the primer is characterized in that a tag (tag) is attached to the 5 'end, the tag refers to a substance that attaches to or binds to the capture element (capture element) in step (b) described later
  • the tag may be attached to or included in the primer by any suitable method, and the tag may itself be bound to the primer or through a linker molecule.
  • the bond may be covalent or non-covalent, and its attachment method according to the specific component of the tag material can be easily determined by those skilled in the art.
  • the tag allows the tagged primer to be directly or indirectly bound to the capture component (or receptor) attached to the solid support by starting the tagged primers by PCR.
  • the capture component typically interacts with the tag It is either very specific or selected (or designed) to maintain binding during the next processing step.
  • the tag may be positioned at a spatially defined location on a solid support, with the nucleic acid sequence extended to the starting point of the tagged primer. Thus, different tags can place different nucleic acid sequences at different spatially defined locations on the solid support.
  • the tag may be determined by a person skilled in the art according to the capture ingredient material used together, and the tag or capture ingredient is not limited thereto, and specifically, the polynucleotide, polypeptide, peptide nucleic acid, locked nucleic acid, Oligosaccharides, polysaccharides, antibodies ⁇ affibodies, antibody mimetics, cell receptors, ligands, lipids, any fragment or derivative of these structures.
  • a tag is configured to not interact internally with itself or with a primer molecule attached to the tag.
  • the combination of the tag and the capture component is biotin-avidin bond, biotin-streptavidin bond, biotin- liquid stravidine bond, biotin-neutravidin bond, benzyl Guanine-SNAP bond, benzylcytosine CLIP bond, enzyme-substrate bond, antigen-antibody bond, metal-histidine bond, metal compound-protein bond, protein-protein bond, or complementary bond between nucleic acids, preferably Preferably it may be by biotin-avidin bond, biotin-streptavidin bond, biotin-extravidin bond or biotin-neutravidin bond.
  • the capture component may be a biotin binding protein such as avidin, streptavidin, nutravidin, axtravidin.
  • the capture component may be biotin and the tag may be a biotin binding protein.
  • the capture component is chelated with metal ions such as nickel, cobalt, iron or nitri lotriacet ic acid (NTA) Nitrilotriacetic acid chelated with any other metal ion capable of forming a poly-histidine and a coordination compound.
  • NTA nitri lotriacet ic acid
  • the tag and capture component may be reversed.
  • protein may be used interchangeably with “polypeptide” or “peptide”, for example, in natural proteins. Refers to a polymer of amino acid residues as is found in the contrary.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleotides or ribonucleotides in the form of single- or double-stranded nucleic acids, unless otherwise specified, in a manner similar to naturally occurring nucleotides. Also known are analogues of natural nucleotides that are common to the term 'polymerase chain react ion (PCR)' or 'nucleic acid amplification reaction' as used herein in the context of amplifying a particular target nucleic acid molecule using a thermostable DNA polymerase.
  • PCR 'polymerase chain react ion
  • 'nucleic acid amplification reaction as used herein in the context of amplifying a particular target nucleic acid molecule using a thermostable DNA polymerase.
  • PCR includes PCR (eg, primers, forward primers, reverse primers), deoxynucleotide mixtures (dNTP mixture), and Mg2 + that can specifically hybridize to target nucleic acids.
  • a reaction buffer containing a divalent ion is used, etc.
  • a nucleic acid molecule (DNA produced or extended by the PCR reaction) is used.
  • RNA is referred to herein as "amplification product” or "PCR product.”
  • the type of PCR is not particularly limited as long as it is known as a PCR method for amplifying a specific genetic material (target nucleic acid) to be detected.
  • a reverse transcriptase polymerase chain reaction that synthesizes complementary DNA using reverse transcriptase from a single PCR, nested PCR, multiple PCR, micro PCR, and RNA and performs a polymerase chain reaction using the template as a template transcriptase polymerase chain react ion (RT-PCR).
  • PCR reaction conditions adopted in the detection method of the present invention may be carried out by adopting or partially modifying conventional PCR reaction conditions according to the type of PCR such as single PCR, nested PCR, multiple PCR, micro PCR, and the like. Those skilled in the art fall into a category that can be easily configured.
  • PCR in step (a) of the present invention is characterized in that it is performed with a fluorescence probe that binds to the double-stranded nucleic acid.
  • the fluorescent probe that binds to the double-stranded nucleic acid is not particularly limited as long as it is a fluorescent dye known in the art to specifically bind to a nucleic acid double strand when the nucleic acid sequence is stretched.
  • the fluorescent probe that binds to the double-stranded nucleic acid is provided in a PCR reaction mixture with a tagged primer, DNA polymerase, deoxynucleotide mixture (dNTP mixture), etc., and the double-stranded nucleic acid synthesized by PCR reaction
  • the fluorescence signal is generated by binding to the sequence (DNA or RA sequence). The presence of the target nucleic acid (target gene) can be seen from the fluorescence signal.
  • the amplification reaction of the target nucleic acid sequence occurs through step (a), a fluorescent probe is attached to the double-stranded nucleic acid sequence, and the PCR product tagged with the 5 'end is generated.
  • Step (b) is a step of inducing binding reaction between the tag and the capture component by treating the PCR product generated in step (a) on a solid on which the capture component to which the tag is attached is accumulated at a specific position.
  • Types of the capture components and tags, combinations thereof, and binding relationships are as described above.
  • the term 'integration' refers to a state in which a large amount of capture components are fixed and gathered in a specific shape at a specific position of a solid phase (sol id phage). It can be integrated into various shapes.
  • the solid phase is characterized by constituting part or all of the "test strip".
  • the term test strip may be used interchangeably with terms such as an assay strip herein.
  • the solid phase (sol id phage) is generally used in immunochromatography analysis, if the solid support means, such as pads, membranes known in the art is not particularly limited in kind.
  • the test strip specifically binds to the tag used in step (a). If it is configured using a capture component to be not limited to the specific manufacturing form and manufacturing method, and includes a sample pad (conjugate pad), a conjugate pad (conjugate pad) and an absorbent pad (absorbent pad).
  • the test strip may be referred to a method for constructing an immunochromatography test strip known in the art.
  • immunochromatography analysis includes an assay strip including any reflective material capable of reacting with an analyte to be detected and displaying a detection signal, or an analytical device in the form of a device equipped with the assay strip in a plastic case. It is commonly used.
  • Conventional assay strips are conjugated samples (samples) that contain liquid samples (samples), conjugates with ligands such as antigens, antibodies, and the like that generate signals that can be detected using the naked eye or sensors.
  • Conjugate pad containing, an analyte in a sample and / or a porous membrane pad (conjugate pad) on which a binding agent (antibody or antigen) is specifically bound to the conjugate, and a hygroscopic pad which finally receives a liquid sample.
  • a binding agent antibody or antigen
  • a hygroscopic pad which finally receives a liquid sample.
  • the test strip used in step (b) does not require a separate conjugate pad as compared to test strips commonly used in immunochromatography.
  • step a) is due to the non-re-process the probe binding to double-stranded nucleic acid in performing PCR.
  • Heunhap liquid sample and the conjugate to which is fixed in a dry form to the polymer pad is moved by capillary action
  • the binding with the conjugate may not be uniform, which may cause variation in individual assay strips, and may result in denaturation of the antibody (generally protein) used as a conjugate, which may reduce the accuracy and reproducibility of the sample analysis. Possible problems are reduced.
  • the test strip of the present invention may be preferably made by sequentially connecting a sample pad, a conjugate pad, and an absorbent pad.
  • the conjugate pad is characterized in that the capture component forming a complex with the tag particles is fixed.
  • the conjugate pad may be preferably immobilized with a capture component on a Nitrocel lurose membrane or a PVDF membrane, but is not limited thereto.
  • the treatment of the PCR product includes all forms in which the capture component is directly or indirectly processed in a solid phase, ie, a conjugate gate pad, integrated at a specific position.
  • the indirect treatment may include dropping a PCR product into a sample pad on a test strip (analysis strip), and passing the PCR product through the conjugate pad by capillary action on the pad.
  • the tagged PCR product is specifically present only at the corresponding position according to the shape of the capture component present on the test strip, and is applied by the fluorescent probe attached to the PCR product. Only at these locations will the fluorescence signal of a particular intensity be emitted.
  • step (c) fluorescence is detected from the solid phase of step (b).
  • the detection of fluorescence can be easily determined by those skilled in the art according to the type and nature of the fluorescence probe used in step (a) described above.
  • a mecury lamp, a metal hydride lamp, a Xenon lamp, a UV lamp, an LED lamp, a halogen lamp, or a laser beam may be used, but is not limited thereto.
  • the light source may be used to irradiate light of a specific wavelength suitable for emitting or developing the fluorescent probe.
  • the rapid detection method of the target nucleic acid of the present invention is based on the PCR method, while the specific and target detection accuracy of the target nucleic acid is high, and the amplification result of the PCR product is located through a specific color reaction (fluorescence) reaction on the test strip.
  • the advantage is that it can be analyzed very quickly visually.
  • test strips are small in size and are easy to carry and use. This is in contrast to the need for a significant additional analysis time and professional staff in the case of using a specific analysis device such as electrophoresis or ELISA in the conventional PCR product analysis. In case of using the electrophoresis method, a gel, etc.
  • the rapid detection method of the target nucleic acid of the present invention comprising the steps (a), (b) and (c) may be performed further comprising the following step (d);
  • step (d) determining that the target nucleic acid is present if the fluorescence detection result of step (c) matches the location where the capture component is accumulated in step (b).
  • the step (d) is for distinguishing the fluorescence signal from the specific amplification of the target nucleic acid and the non-specific fluorescence signal, and when a fluorescence signal is detected at a position other than a predetermined position, that is, by the hybridization between nucleic acids other than the target nucleic acid. This step excludes any positive reactions that may occur.
  • the tagged PCR product is specifically present only at the corresponding position according to the shape of the capture component present on the test strip, and thus emits a fluorescence signal having a specific intensity only at that position.
  • the above-described method for rapidly detecting a target nucleic acid of the present invention has an advantage of providing information for quickly diagnosing a disease when the target nucleic acid to be detected is a marker indicating a specific disease (or condition). Therefore, the present invention
  • step (b) treating the PCR product generated in step (a) on the solid phase in which the capture component to which the tag is attached is integrated at a specific position to induce binding reaction between the tag and the capture component;
  • step (c) detecting fluorescence from the solid phase of step (b)
  • the method includes, and provides a method for providing information for the rapid diagnosis of the disease.
  • the method also includes, and provides a method for providing information for the rapid diagnosis of the disease. The method also includes, and provides a method for providing information for the rapid diagnosis of the disease. The method also includes, and provides a method for providing information for the rapid diagnosis of the disease. The method also includes, and provides a method for providing information for the rapid diagnosis of the disease. The method also includes, and provides a method for providing information for the rapid diagnosis of the disease. The method also
  • step (d) if the fluorescence detection result of step (c) is consistent with the location where the capture component is accumulated in step (b). Determining that a disease marker nucleic acid is present;
  • the present invention provides a method for quickly and rapidly checking PCR results using a tag-labeled primer, a fluorescence probe that binds to a double-stranded nucleic acid, and a test strip in which a capture component specific to the tag is fixed.
  • it is possible to analyze the results in a short time without the need for a specific facility, so that the diagnosis can be made in a grand and on-site.
  • results can be easily analyzed in the field and diagnosed in a grand situation.
  • the present invention it is possible to prevent the spread of the virus by making a short-term diagnosis of a highly contagious virus such as MERS michica virus and a severely damaging virus to suspected patients when entering a local hospital or an overseas traveler.
  • a highly contagious virus such as MERS michica virus
  • a severely damaging virus to suspected patients when entering a local hospital or an overseas traveler.
  • Help with the patient's prognosis Can give
  • FIG. 1 shows a binding relationship between a target nucleic acid (template nucleic acid) and a primer in PCR using a biot in a primer attached (labeled) as a tag.
  • Figure 2 shows a schematic diagram of the PCR product attached to the test line in which avidin (the capture component) is fixed.
  • Figure 3 is a schematic diagram showing the principle of the detection of fluorescence on the test strip in the present invention.
  • Figure 4 shows the results of testing the specific detection of SIT1 gene nucleic acid in accordance with the present invention, in a sample mixed with various nucleic acids.
  • Figure 5 shows the results of specific detection of the SIT1 gene nucleic acid on the test strip according to the present invention.
  • the dispenser Dispenser system (MDS) was used to draw 1 mg / mL solution of Avidin (A9275-lMG, Sigma-Aldrich, USA) in distilled water at 6 cm / sec. ) was formed. After drying the membrane, the membrane was cut into 3 mm intervals into a cutter.
  • MDS Dispenser system
  • test strip of the present invention consisted of attaching a sample pad at one end and an absorbent pad at the other end of the nitrocell drawn with a detection line with Avidin (see Fig. 3).
  • PCR was performed using a biotinylated forward primer and a reverse primer set having a sequence complementary to a portion of the dsDNA sequence of the SIT1 gene (see FIG. 1). As the PCR equipment, verit i of Appl ied Biosystems was used. PCR semi-aqueous solution was prepared using TOY0B0 SYBR Green Realt PCR Mastet Mix (QPK-201) with the composition shown in Table 2 below.
  • PCR reaction conditions were denatured at 95 ° C for 2 minutes, followed by a total of 35 cycles of denaturation (20 seconds at 95 ° C), binding (40 seconds at 55 ° C), and extension (1 minute at 72 ° C). It was done in a manner.
  • the PCR product prepared in ⁇ 1-2> was treated with the test strip prepared in Example ⁇ 1-1> to induce binding reaction between biotin contained in the PCR product and avidin immobilized on the strip. Thereafter (see FIG. 2), the fluorescent pattern by SYBR green dye was detected and analyzed.
  • To the sample pad 150 ul of D.W (distilled water) was added to 20 ul of the PCR product, and after 1 minute, 470 nm LED light was irradiated to the pad to confirm the emi ss ion signal of SYBr green (FIG. 3). Reference) .
  • FIG. 3 a result, as shown in Figure 4, it was confirmed that the instant fluorescence signal in the avidin (avidin) integration part.
  • the immediate fluorescence signal appeared in the avidin (avidin) integrated portion, it was confirmed that the target nucleic acid is specifically detected at a specific detection position on the test strip of the present invention.
  • the present invention provides a polymerase with a fluorescent probe that binds to a double-stranded nucleic acid by using a tagged primer having a sequence complementary to a partial region of the target nucleic acid sequence.
  • a chain reaction PCR
  • step (b) treating the PCR product generated in step (a) on a solid on which the capture component to which the tag is attached is integrated at a specific position to induce binding reaction between the tag and the capture component; And (c) detecting fluorescence from the solid phase of step (b), and a method for rapidly detecting a target nucleic acid and a method for rapidly diagnosing a disease using the method.
  • the present invention provides a method for checking PCR results quickly and quickly, thereby providing a reliable analysis result in a short time without the need for specialists and special equipment Rapid diagnosis at the grand site is possible. This may be particularly synergistic with the development of portable PCR devices in recent years.
  • it is possible to prevent the spread of the virus by making a short-term diagnosis of susceptible virus such as MERS or Zika virus, and the severely damaging virus, for suspected patients when entering a local hospital or an overseas traveler. It can help the patient's prognosis.
  • susceptible virus such as MERS or Zika virus

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Abstract

La présente invention concerne un procédé de détection rapide d'un acide nucléique cible et un procédé de diagnostic rapide d'une maladie en utilisant ledit procédé, le procédé de détection rapide d'un acide nucléique cible comprenant les étapes de : (a) en utilisant une amorce ayant une étiquette fixée dessus (une amorce étiquetée) et ayant une séquence complémentaire d'une région d'une séquence d'acides nucléiques cible, la conduite d'une réaction en chaîne de la polymérase (PCR) avec une sonde fluorescente qui se lie à un acide nucléique double brin ; (b) le traitement, avec le produit de PCR produit dans l'étape (a), d'une phase solide ayant un élément de capture, auquel l'étiquette doit être fixée, agglomérée à une position prédéterminée, et l'induction ainsi d'une réaction de liaison entre l'étiquette et l'élément de capture ; et (c) la détection de fluorescence à partir de la phase solide de l'étape (b). La présente invention fournit un procédé de confirmation prompt et rapide d'un résultat de PCR, et permet ainsi un diagnostic rapide en urgence et sur un site d'analyse du résultat sur une courte durée même en l'absence d'un spécialiste et d'un équipement particulier. En particulier, la présente invention peut créer une synergie en combinaison avec le développement récent des dispositifs portables de PCR.
PCT/KR2017/014363 2017-01-06 2017-12-08 Procédé de détection rapide d'acide nucléique, et procédé de diagnostic rapide d'une maladie l'utilisant Ceased WO2018128286A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20170002346 2017-01-06
KR10-2017-0002346 2017-01-06
KR1020170166156A KR102135979B1 (ko) 2017-01-06 2017-12-05 핵산의 신속 검출법 및 이를 이용한 질병의 신속 진단 방법
KR10-2017-0166156 2017-12-05

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WO2018128286A1 true WO2018128286A1 (fr) 2018-07-12

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CN114686611A (zh) * 2022-04-24 2022-07-01 常州先趋医疗科技有限公司 用于检测单增李斯特菌的引物组及其应用

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