WO2018128286A1 - Method for rapidly detecting nucleic acid, and method for rapidly diagnosing disease using same - Google Patents

Abstract

The present invention relates to a method for rapidly detecting a target nucleic acid and a method for rapidly diagnosing a disease using said method, the method for rapidly detecting a target nucleic acid comprising the steps of: (a) by using a primer having a tag attached thereto (a tagged primer) and having a sequence complementary to a region of a target nucleic acid sequence, carrying out a polymerase chain reaction (PCR) with a fluorescent probe which binds to a double-stranded nucleic acid; (b) treating, with the PCR product produced in step (a), a solid phase having a capture element, to which the tag is to be attached, agglomerated at a predetermined position, and thereby inducing a binding reaction between the tag and the capture element; and (c) detecting fluorescence from the solid phase of step (b). The present invention provides a method for quickly and rapidly confirming a PCR result, and thus enables a rapid diagnosis in an emergency and on-site by analyzing the result in a short time even in the absence of a specialist and particular equipment. In particular, the present invention can create synergy in combination with the recent development of portable PCR devices.

Description

 [Specifications

 [Name of invention]

 Rapid Detection of Nucleic Acids and Rapid Diagnosis of Diseases Using the Same

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.

Background Art

This application claims priority to Korean Patent Application No. 10-2017-0002346, filed January 6, 2017 and Korean Patent Application No. 10—2017-0166156, filed December 5, 2017, and the entirety of the above specification. Is a reference of the present application. Respiratory viruses are the leading cause of acute respiratory infections and are the most common diseases regardless of age or gender. In particular, infants, the elderly, and people with reduced cardiopulmonary and immune function are known to cause sequelae and death. Diseases associated with respiratory viruses most often begin with cold symptoms, ranging from pharyngitis, exacerbation of chronic asthma, and pneumonia. Early diagnosis of these respiratory viruses can prevent the abuse of unnecessary antibiotics, and early detection of the type of virus can provide appropriate patient care. Especially in the case of 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). When 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. There is an urgent need for the development of a device that can quickly diagnose early. The development of such a device can be said to have a great ripple effect both nationally and socially, such as relieving anxiety and reducing medical expenses. Conventional methods of diagnosing such respiratory viruses include cell culture and rapid antigen testing. In the case of cell culture method, it takes more than 5 ~ 9 days to confirm the test result, which makes it difficult to diagnose and treat a respiratory virus infection early. In order to secure these disadvantages, the sensitivity is confirmed within 24 ~ 72 hours. Although R-mix virus culture method similar to traditional culture method is being implemented, it is also limited in early diagnosis and treatment. Rapid antigen test method can confirm the results in a short time, but the sensitivity is lower than the traditional culture method has a lot of false negatives. More specifically, a doctor who needs to decide on the rapid use of antiviral and anti-influenza drugs or a guardian who is waiting for the confirmation result of the swine flu infection can check the influenza right away on the spot. As a test method, the rapid influenza test (RIDT), an antibody-based immunochromatography (fast antigen test) that detects antigens of influenza, is most commonly used. However, RIDT is not as sensitive as seasonal influenza virus virus culture (Cold standard) or real time polymerase chain reaction (Realt ime-RT-PCR). (50-70% sensitivity compared to Viral culture) has been pointed out as a precaution before diagnosis of influenza. In fact, since the 2009 influenza outbreak, many cohorts using various RIDTs have evaluated sensitivity and specificity for the diagnosis of swine flu, and as a result, various ranges of sensitivity (11-70%) were shown. 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 However, due to the nature of the test method, 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). However, in the case of using the PCR, 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.

[Detailed Description of the Invention]

 [Technical problem]

Accordingly, the inventors of the present invention, while trying to devise a disease detection method that is easy to derive the results and short test time, like the existing rapid antigen diagnosis method while high accuracy and reliability in detecting the presence of the disease, the rapid nucleic acid of the present invention Using the detection method, it was confirmed that the disease can be detected with high sensitivity quickly and easily, and completed the present invention. Accordingly, 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. Performing); (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. Performing a polymerase chain reaction (PCR) together with a fluorescent probe; (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.

Technical Solution

In order to achieve the above object, 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). In order to achieve another object of the present invention, 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). Hereinafter, the present invention will be described in detail. The present invention,

 (a) Performing polymerase chain reaction (PCR) with a fluorescent probe that binds to a double stranded nucleic acid using a tagged primer having a sequence complementary to a region of the target nucleic acid sequence 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; And

 (c) detecting fluorescence from the solid phase of step (b)

It provides a, a rapid detection method of the target nucleic acid. In the present invention, the term "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. More specifically, 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. In the present invention, 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. When the target nucleic acid is 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. For example, 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. For example, the bacteria include Pseudomonas, Excherichia and Klebsilla. Klebsiel la, Enterobacter, Proteus, Serrat ia, Candida, Staphylococci, Streptococci, Chlamydia, Mycoplasma (Mycoplasma) genus, including but not limited to. 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.

Preferably 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. For example, 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. . As used herein, the term "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. In 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. Perform. As used herein, 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. In the present invention, 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 ᅳ affibodies, antibody mimetics, cell receptors, ligands, lipids, any fragment or derivative of these structures. In general, a tag is configured to not interact internally with itself or with a primer molecule attached to the tag. Combination of the capture component and the tag substance specifically binding thereto, preferably, 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. When the tag is biotin, the capture component may be a biotin binding protein such as avidin, streptavidin, nutravidin, axtravidin. In contrast, the capture component may be biotin and the tag may be a biotin binding protein.

When the tag is polyhist idine, 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. Likewise, the tag and capture component may be reversed. As used herein, the term "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. As used herein, “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. In addition to 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. For example, 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).

The 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. For example, SYBR green, Pi cogreen, Evagreen, Hoechst 33258, Hoechst 33342, ethidium bromide, acridine orange, propidium iodide, mithramycin, TO-PRO-3, T0T0, YOYO, YOPRO-1 and the like. 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. In the present invention, 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.

In general, 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. These functional pads are connected and attached in some superimposed form in the order listed above and arranged continuously. When the analysis strip is mounted inside the plastic case and used, a sample inlet for loading a sample (sample) into the sample (sample) pad at the top of the case, and a test at a position where the binder of the porous membrane pad is fixed A result confirmation window is formed to confirm the result. As described above, in an immunochromatography assay using an assay strip, when a liquid sample is dropped on a sample pad, the liquid sample moves through the conjugate pad and the porous membrane pad (conjugate pad) by capillary action. Finally it is housed in a moisture absorption pad. At this time, the conjugate contained in the conjugate pad also moves with the liquid sample, and if the substance to be analyzed exists in the sample, the conjugate binds to the binder fixed to the porous membrane pad through the analyte (usually, " Sandwich reactions are known as 'competitive ion reactions' because the conjugates and analytes are competitively bound to the binder (commonly referred to as “compet it ion reactions”). In the present invention, the test strip used in step (b) does not require a separate conjugate pad as compared to test strips commonly used in immunochromatography. in step _a) is due to the non-re-process the probe binding to double-stranded nucleic acid in performing PCR. Thus in the conventional analytical strips, Heunhap liquid sample and the conjugate to which is fixed in a dry form to the polymer pad is moved by capillary action As a result, 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. Therefore, 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. Preferably, 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. Through the coupling reaction between the tag and the capture component, 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.

In 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. Depending on the type of fluorescent probe, for example, 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. Typically, 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. must be manufactured, and a special device for transferring electricity is required, as well as a relatively considerable time is required for the separation of nucleic acids according to molecular weight. The ELISA method also requires a separate ELISA kit and reader device. The devices used in these methods are usually stored in specialized laboratories, and are immediately used when on-site diagnosis or other urgent diagnosis is required when entering overseas. There is a problem that is difficult to apply to. On the contrary, the present invention is advantageous in the field application because it uses a test strip, and especially when used in the grand field with a portable PCR device (eg Arambio Palm PCR), etc., has a synergistic effect in terms of rapid and accurate detection of the disease. . 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);

(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

 (a) Polymerizing a nucleic acid sample obtained from a patient's sample with a fluorescent probe that binds to a double stranded nucleic acid using a tagged primer having a sequence complementary to a region of the disease marker nucleic acid. Performing enzyme chain reaction (PCR);

 (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; And

 (c) detecting fluorescence from the solid phase of step (b)

 It includes, and provides a method for providing information for the rapid diagnosis of the disease. The method also

 (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;

 It may further include. The disease marker and steps (a) to (d) are as described above. Advantageous Effects

 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. In addition, 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. By simplifying the process of analyzing results, results can be easily analyzed in the field and diagnosed in a grand situation. In addition, it is possible to use the advantages of PCR regardless of time and space limitations by enabling reliable results analysis without the need for an expert. This can be especially synergistic with the recent development of portable PCR devices.

According to 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 nazica virus and a severely damaging virus to suspected patients when entering a local hospital or an overseas traveler. Help with the patient's prognosis Can give In addition, when the diagnosis of a sexually transmitted disease is sent to a specialist to confirm the diagnosis usually takes a week's time, even in this case, by early diagnosis and confirmation through the present invention can prevent disease spread. In addition, it is possible to minimize the damage of livestock farmers due to the spread of disease by quickly conducting on-site inspections for livestock diseases such as AI, and expecting to have various ripple effects by addressing the demands required for further diagnosis and on-site diagnosis. do.

[Brief Description of Drawings]

 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.

[Form for implementation of invention]

 Hereinafter, the present invention will be described in detail.

 However, the following examples are merely to illustrate the present invention, the contents of the present invention is not limited to the following examples.

<Example 1>

PC and immunochromatography based. Rapid nucleic acid detection method <1-1> Avidin-based Test Strip Manufacturing

 After attaching the nitrocelose membrane (180 sec Nitrocel lulose, Mi ll ipore, Germany) and the absorption pad (Mi 11 ipore, Germany) used as the detection layer to the plastic pad (Mi ll ipore, Germany), 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. Sample pads (C068, Mi 11 ipore, Germany) were soaked in 0.5% Tween 20, 5% sucrose, 5% dextran, 0.05% sodium azide solution and dried It was produced by cutting to about mm. Finally, the 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).

<1-2> Production of biotinylated nucleic acid via polymerase chain reaction (PCR)

From samples in which nucleic acids of various genes were mixed, it was intended to detect whether a gene of SITKNCBI sequence ID: XM_005660221, SEQ ID NO: 1) exists. As shown in Table 1 below, 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.

Table 1

Table 2

 Total 20 ul

<l-3> Specific detection of the target nucleic acid on the test strip

 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) . As a result, as shown in Figure 4, it was confirmed that the instant fluorescence signal in the avidin (avidin) integration part. In addition, as shown in Figure 5, 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.

 Thus, the above example suggests that the PCR result can be confirmed quickly and quickly according to the method of the present invention.

Industrial Applicability

As described above, 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. Performing a 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), 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. According to the present invention, 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. In addition, when the diagnosis of a sexually transmitted disease is sent to a specialist to confirm the diagnosis, it usually takes a week, but even in this case, by early diagnosis and confirmation through the present invention can prevent disease spread. In addition, it is possible to minimize the damage of livestock farmers due to the spread of disease by quickly conducting on-site inspections for livestock diseases such as AI, and it is expected to have various ripple effects by addressing the demands requiring further diagnosis and on-site diagnosis. Industrial use is highly likely.

Claims

[Range of request]  [Claim 1]  (a) performing polymerase chain reaction (PCR) with a fluorescent probe that binds to a double-stranded nucleic acid using a tagged primer having a sequence complementary to a region of the desired nucleic acid sequence 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 ᅳ  (c) detecting fluorescence from the solid phase of step (b)  A rapid detection method of the target nucleic acid comprising a. [Claim 2]  According to claim U, the method  (d) determining that the target nucleic acid exists if the fluorescence detection result of step (c) matches the location where the capture component is accumulated in step (b);  The method for the rapid detection of the target nucleic acid, characterized in that it further comprises. [Claim 3]  The method of claim 1, wherein the binding of the tag and the capture component is biotin-avidin bond, biotin-streptavidin bond, biotin-axtravidin bond, biotin-neutravidin bond, benzyl guanine-SNAP bond benzylcytosine-CLIP bond ， Enzyme-substrate binding, antigen-antibody binding, metal-histidine binding, metal compound-protein binding, protein-protein binding, or complementary binding between nucleic acids. [Claim 4] The method of claim 1, wherein the tag is a polynucleotide, a polypeptide (polypeptide), peptide nucleic acid (peptide nucleic acid), locked nucleic acid (locked nucleic acid), oligosaccharide (polysaccharide), polysaccharide (polysaccharide), antibody ( antibody, affibody, antibody mimic, cell receptor, ligand, lipid, biotin, avidin, streptavidin ), One or more selected from the group consisting of extravidin, neutravidin, metal, and histidine. How to [Claim 5]  The method of claim 1, wherein the capture component is polynucleotide, polypeptide, peptide nucleic acid, locked nucleic acid, jl¾- (oliggosaccharide), polysaccharide (polysaccharide), Antibodies, Afbodies, Antibody Mimics, Cell Receptors, Ligands, Lipids, Biotin, Avidins, Streps At least one member selected from the group consisting of: stavidavidin, actavividin, neutravidin, metal, and histidine. [Claim 6]  The method of claim 1, wherein the fluorescence probe binding to the double-stranded nucleic acid is SYBR green, Picogreen, Evagreen, Hoechst 33258, Hoechst 33342, ethidium bromide, acridine orange, propidium iodide, mithramycin, TO-PRO-3, T0T0, YOYO and Y0PR0-1, characterized in that selected from the group consisting of. [Claim 7]  (a) To a nucleic acid sample obtained from a patient's sample, by using a tagged primer having a sequence complementary to a region of a disease marker nucleic acid, and adding a fluorescent probe that binds to a double-stranded nucleic acid. Performing enzyme chain reaction (PCR);  (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 integrated at a specific position; And  (c) detecting fluorescence from the solid phase of step (b)  Including, a rapid diagnosis method of the disease. [Claim 8]  The method of claim 7, wherein the method (d) The fluorescence detection result of step (c) is integrated into the capture component in step (b). Determining that a disease marker nucleic acid is present if it matches the identified position; It characterized in that it further comprises, Rapid diagnosis method of the disease. 【Claim 9】.  The method of claim 17, wherein the disease is an infectious disease, cancer, inflammatory disease, metabolic disease, nervous system disease, musculoskeletal disease, digestive system disease, allergic disease, immune related disease, endocrine system disease, cardiovascular disease, genitourinary system disease, respiration Method selected from the group consisting of mechanical diseases and skin diseases. [Claim 10]  8. The method of claim 7, wherein said disease marker nucleic acid is a foreign gene from an infectious agent or an endogenous mutant nucleic acid of an individual.