KR20220144608A - Microparticle-based detecting kit for nucleic acid and Method for detecting nucleic acid amplification product - Google Patents

Abstract

The present invention relates to a microparticle-based nucleic acid detecting kit and a nucleic acid amplification product detecting method. In the microparticle-based nucleic acid amplification product detection method according to the present invention, after a target nucleic acid is amplified, aggregation of a nucleic acid amplification product captured in a microparticle can be visually detected only by adding the microparticle without a separate process or device, and the amplification product of the target nucleic acid can be detected quickly and easily with high sensitivity. Accordingly, the microparticle-based nucleic acid amplification product detection method according to the present invention is expected to be useful for rapidly diagnosing infectious diseases caused by hepatitis C virus, hepatitis B virus, influenza virus, human immunodeficiency virus (HIV), Mycobacterium tuberculosis, severe acute respiratory syndrome coronavirus (SARS-CoV), or SARS-CoV-2. The nucleic acid amplification product detecting method comprises the following steps of: synthesizing a cDNA from a sample; amplifying the cDNA; adding an microparticle; and detecting aggregation of the nucleic acid amplification product captured in the microparticle.

Description

Microparticle-based detecting kit for nucleic acid and Method for detecting nucleic acid amplification product}

The present invention relates to a microparticle-based nucleic acid detection kit, a nucleic acid amplification product detection method, and the like.

Recently, polymerase chain reaction (PCR) and real-time PCR techniques that can rapidly and simply amplify a DNA fragment of a specific nucleotide sequence have become common, making it easy to detect/diagnose infection. The PCR technique adds a nucleic acid fragment sequence (primer) that specifically binds to the nucleic acid sequence of a target pathogen, and repeats denaturation, recombination, and polymerization according to temperature to amplify a trace amount of pathogen nucleic acid. It is a technology that can detect the presence or absence of

In the practical aspect of clinical diagnosis, the PCR technique allows an objective large-scale test, unlike the cytological test of pap smear, and in terms of measurement principle, compared to cytology or liquid hybrid capture, test cost, experimental procedure, and detection sensitivity (sensitivity) ) and specificity, etc., are confirmed to have a relative advantage. However, since caution is required in designing the PCR technique and interpreting the results, an operator with high technical skills is required. The disadvantage is that it can be used only in limited places. In reality, when an infectious disease outbreak occurs, disease control must be carried out quickly, so disease diagnosis must be a fast, simple, inexpensive, and suitable method for a point-of-care (POCT) environment regardless of location.

In order to satisfy the above requirements, microfluidics, microelectronics, optical systems, and chip-based detection technologies have been developed, but most of them still require complex manufacturing processes and are limited to places that satisfy conditions such as analysis devices. It has the disadvantage that it can only be used in .

As an example, as a method for detecting human papillomavirus (HPV) DNA, a centrifugation-based detachable visual detection system (SPIN-DNA) has been developed, but the SPIN-DNA is used to separate DNA-bound beads from beads. There is a problem that a centrifugation device must be necessarily provided for this purpose.

Therefore, the need for the development of molecular point of care testing (molecular point of care testing, molecular POCT) has emerged. In order to develop a diagnostic technology applicable to molecular point of care testing (molecular POCT), first, in order to enable an immediate response to a disease, it is possible to immediately detect the DNA or RNA of a pathogen in the field, or RNA must be analyzed to obtain results quickly, so it must be detected visually or visually without additional measuring equipment or knowledge, and results can be obtained within a short time of less than 30 minutes, and additional process steps such as washing steps Existing DNA diagnostic method discovery strategies are limited as inexpensive, accurate, and excellent reproducibility are required without measuring devices or measuring instruments.

In this regard, in previous studies, a paper-based speedy separation of amplified DNA (PASS-DNA) was used, but this detection system detects DNA-bound microparticles from non-DNA-bound microparticles. Paper and particles were required to separate the particles.

Hybridization tests based on metal nanoparticles are attractive systems for diagnostic applications, as they are simple to implement and visually read through color changes that occur due to plasmon interactions between probes. Applying a small force such as capillary force for the assay) is a more feasible approach.

On the other hand, it has been reported that hepatitis C virus (HCV) infection is caused by blood transfusion and community-acquired, and about 70% of infections are caused by kidney dialysis. Once infected with the hepatitis C virus, about 20% will develop acute hepatitis with cirrhosis within 5 years and metastasize to liver cancer. This high chronic infection rate is unusual for RNA viruses, and it is evidence that the hepatitis C virus is a carrier that causes a high rate of liver cancer. In recent years, all blood is tested for hepatitis C virus well, so the number of infections caused by blood transfusions has significantly decreased. It is estimated that about 71 million people are affected by HCV.

Molecular detection and quantification of the HCV genome by real-time polymerase chain reaction (PCR) represents the gold standard for HCV diagnosis and plays an important role in the management of treatment regimens. However, it is estimated that up to about 80% of infected patients are unaware of the infection, especially in low- and middle-income families. Accordingly, it is essential to increase diagnosis through a universal approach that can be connected to on-site diagnosis and treatment at low cost.

Therefore, the dominant model of centralized laboratory testing is not suitable for treating chronic hepatitis C patients in resource-constrained settings because they are often unavailable due to a lack of infrastructure and/or trained operators. As timely detection and diagnosis is important for infection control, antiviral therapy, and epidemiological studies for HCV, an alternative, inexpensive and point-of-care rapid diagnostic test is particularly useful.

Accordingly, the present inventors have developed not only for the diagnosis of infectious diseases such as hepatitis C virus, but also for all diseases that can be tested for nucleic acid amplification without other special devices such as paper, electrophoresis or centrifugation/column, signal detection such as fluorescence. An attempt was made to develop a simpler method that can rapidly detect amplified DNA with the naked eye.

Korean Patent No. 10-1507914

The present inventors have developed a detection method for target DNA that visually visualizes amplified DNA only by adding microparticles and does not require additional centrifugation, probe conjugation, long incubation time, signal generation or dilution steps. Thus, the present invention was completed.

Accordingly, it is an object of the present invention to provide a method for detecting a microparticle-based nucleic acid amplification product and a kit for detecting a microparticle-based nucleic acid.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned may be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. There will be.

In order to achieve the above object, the present invention provides a method for detecting a nucleic acid amplification product based on microparticles, comprising the following steps:

(a) synthesizing cDNA by isolating RNA from a sample collected from a subject;

(b) biotin, digoxigenin, an antigen, a target molecule, and a forward primer and a reverse primer each labeled with any one selected from the group consisting of oligonucleotides, and amplifying the cDNA using a primer set that specifically binds to a nucleic acid to be detected;

(c) an antibody capable of binding to the amplified cDNA through an antigen-antibody reaction to streptavidin, avidin, and an antigen bound to the primer, specifically to a target molecule bound to the primer adding a microparticle coated with any one selected from the group consisting of an aptamer binding to and an oligonucleotide complementary to an oligonucleotide binding to the primer; and

(d) detecting aggregation of the nucleic acid amplification product captured by the microparticle.

However, the centrifugation step between steps (c) and (d) is not included.

In addition, the present invention provides an information providing method for diagnosing an infectious disease, comprising the steps of:

(a) synthesizing cDNA by isolating RNA from a sample collected from a subject;

(b) biotin, digoxigenin, an antigen, a target molecule, and a forward primer and a reverse primer each labeled with any one selected from the group consisting of oligonucleotides, and amplifying the cDNA using a primer set that specifically binds to a nucleic acid to be detected;

(c) an antibody capable of binding to the amplified cDNA through an antigen-antibody reaction to streptavidin, avidin, and an antigen bound to the primer, specifically to a target molecule bound to the primer adding a microparticle coated with any one selected from the group consisting of an aptamer binding to and an oligonucleotide complementary to an oligonucleotide binding to the primer; and

(d) detecting aggregation of the nucleic acid amplification product captured by the microparticle.

However, the centrifugation step between steps (c) and (d) is not included.

In addition, the present invention consists of a forward primer and a reverse primer each labeled with any one selected from the group consisting of biotin, digoxigenin, antigen, target molecule and oligonucleotide. and a primer set that specifically binds to a nucleic acid to be detected; and

Streptavidin, avidin, an antibody capable of binding to an antigen bound to the primer through an antigen-antibody reaction, an aptamer that specifically binds to a target molecule bound to the primer, and A kit for detecting nucleic acids based on microparticles, comprising microparticles coated with any one selected from the group consisting of oligonucleotides complementary to the oligonucleotides bound to the primers,

The kit provides a kit, characterized in that it is used to detect aggregation of nucleic acids captured by microparticles without centrifugation.

In one embodiment of the present invention, the primer set may be composed of a forward primer and a reverse primer each labeled with any one selected from the group consisting of biotin and digoxigenin, but is not limited thereto.

In another embodiment of the present invention, the microparticles may be coated with streptavidin or anti-digoxigenin antibody, but is not limited thereto.

In another embodiment of the present invention, the sample may be one or more selected from the group consisting of whole blood, serum, plasma, urine, feces, and saliva, but is not limited thereto.

In another embodiment of the present invention, the primer set in step (b) may be prepared so that the size of the nucleic acid amplification product is 100 to 200 bp, but is not limited thereto.

In another embodiment of the present invention, the amplification in step (b) is at least one method selected from the group consisting of polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), and isothermal amplification method. It may be made by, but is not limited thereto.

In another embodiment of the present invention, the isothermal amplification method comprises recombinant enzyme polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification. amplification, HDA) may be at least one selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, the diameter of the microparticles may be 0.25 to 10 μm, but is not limited thereto.

In another embodiment of the present invention, the microparticles may be magnetic beads or polystyrene particles, but is not limited thereto.

In another embodiment of the present invention, the detection limit concentration of the cDNA may be 1 to 15 IU/ml, but is not limited thereto.

In another embodiment of the present invention, the infectious disease is hepatitis C, hepatitis B, influenza, human immunodeficiency virus (HIV) induced AIDS, tuberculosis, severe acute respiratory syndrome coronavirus (SARS-CoV) infection , and may be one or more selected from the group consisting of Corona Virus Infectious Disease-19 (COVID-19), but is not limited thereto.

The microparticle-based nucleic acid amplification product detection method according to the present invention amplifies a target nucleic acid and then only adds microparticles to the amplification product of the nucleic acid captured by the microparticles without a separate process or device such as centrifugation, additional probe, culture, or dilution. Aggregation can be detected with the naked eye, and an amplification product of a target nucleic acid can be detected easily and quickly with high sensitivity. Accordingly, the microparticle-based nucleic acid amplification product detection method according to the present invention is a hepatitis C virus, hepatitis B virus, influenza virus, human immunodeficiency virus (HIV), Mycobacterium tuberculosis, severe acute respiratory syndrome coronavirus (SARS-CoV), or It is expected to be useful for rapidly diagnosing infectious diseases caused by SARS-CoV-2 and the like.

1 schematically shows the principle of a microparticle-based visual detection system for amplified cDNA as a method for detecting a nucleic acid amplification product according to an embodiment of the present invention.
2 is a view confirming the aggregation result by adding streptavidin-coated microparticles and anti-digoxigenin antibody-coated microparticles according to the presence or absence of amplified HCV cDNA according to an embodiment of the present invention.
3 is a view showing optimization conditions and results for nucleic acid detection through aggregation in a microparticle-based visual detection system of amplified cDNA according to an embodiment of the present invention.
4a and 4b show streptavidin-coated microparticles and anti-digoxigenin antibody coating in the presence ( FIG. 4a ) and in the absence ( FIG. 4b ) of amplified HCV cDNA according to an embodiment of the present invention. It is a diagram confirming the aggregation result by the addition of microparticles enlarged at 400x and 1000x magnification.
5 is a graph showing the concentration of HCV cDNA after adding streptavidin-coated microparticles and anti-digoxigenin antibody-coated microparticles using microparticle-based visual detection of amplified cDNA according to an embodiment of the present invention. It is a figure confirming the aggregation result.
6 shows the results of electrophoresis with actual clinical samples (HCV positive 4, 5, 6; negative, N4, N5, N6) (left diagram) and microparticle-based visual detection of amplified cDNA according to an embodiment of the present invention; It is a diagram comparing the results of confirming aggregation after adding the streptavidin-coated microparticles and the anti-digoxigenin antibody-coated microparticles using the (right figure).
7 is a view confirming the sensitivity of the microparticle-based visual detection system of amplified cDNA according to an embodiment of the present invention compared to the case of using the conventional nucleic acid amplification detection method.

The present invention provides a method for detecting a microparticle-based nucleic acid amplification product comprising the steps of:

(a) synthesizing cDNA by isolating RNA from a sample collected from a subject;

(b) biotin, digoxigenin, an antigen, a target molecule, and a forward primer and a reverse primer each labeled with any one selected from the group consisting of oligonucleotides, and amplifying the cDNA using a primer set that specifically binds to a nucleic acid to be detected;

(c) an antibody capable of binding to the amplified cDNA through an antigen-antibody reaction to streptavidin, avidin, and an antigen bound to the primer, specifically to a target molecule bound to the primer adding a microparticle coated with any one selected from the group consisting of an aptamer binding to and an oligonucleotide complementary to an oligonucleotide binding to the primer; and

(d) detecting aggregation of the nucleic acid amplification product captured by the microparticle.

However, the centrifugation step between steps (c) and (d) is not included.

In the present invention, "primer" is a nucleic acid sequence having a short free 3 hydroxyl group, which can form a complementary template and base pair, and serves as a starting point for template strand copying. It refers to a short nucleic acid sequence that functions. The primers are capable of initiating DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerate or reverse transcriptase) in an appropriate buffer and temperature. The primer can be variously modified according to methods known in the art within the range that does not interfere with hybridization with the polynucleotide to be detected. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and fluorescence or enzymatic binding of a labeling material.

In the present invention, the primer set may be composed of a forward primer and a reverse primer each labeled with any one selected from the group consisting of biotin and digoxigenin, and according to an embodiment of the present invention, the primer set is biotin It may consist of a forward primer labeled with and a reverse primer labeled with digoxigenin, and the size of the nucleic acid amplification product is 100 bp to 200 bp, 100 bp to 180 bp, 100 bp to 160 bp, 120 bp to 200 bp, It may be designed to be 120 bp to 180 bp, 120 bp to 160 bp, 140 bp to 200 bp, 140 bp to 180 bp, or 140 bp to 160 bp, but is not limited thereto.

In the present invention, “microparticle” means a particle having a diameter of 0.1 μm to 1000 μm, and according to an embodiment of the present invention, the microparticle has a diameter greater than 0.1 μm. When it is small, it is difficult to observe with the naked eye, and preferably 0.1 μm to 10 μm, 0.1 μm to 7 μm, 0.1 μm to 5 μm, 0.1 μm to 3 μm, 0.1 μm to 1 μm, 0.2 μm to 10 μm, 0.2 μm to 7 μm, 0.2 μm to 5 μm, 0.2 μm to 3 μm, 0.2 μm to 1 μm, 0.25 μm to 1 μm, 0.25 μm to 0.3 μm, 1 μm to 10 μm, or 1 μm. .

In the present invention, the microparticles may be magnetic beads or polystyrene particles coated with streptavidin or anti-digoxigenin antibody, wherein the polystyrene particles may be blue polystyrene particles, It is not limited thereto. In addition, the microparticles are 1 mg/ml to 20 mg/ml, 1 mg/ml to 17 mg/ml, 1 mg/ml to 15 mg/ml, 1 mg/ml to 12 mg/ml, 1 mg/ml to 10 mg/ml, 3 mg/ml to 20 mg/ml, 3 mg/ml to 15 mg/ml, 3 mg/ml to 12 mg/ml, 3 mg/ml to 10 mg/ml, 5 mg/ml to 20 mg/ml, 5 mg/ml to 15 mg/ml, 5 mg/ml to 12 mg/ml, 5 mg/ml to 10 mg/ml, 3 mg/ml to 7 mg/ml, 7 mg/ml It may be added at a concentration of 12 mg/ml, 5 mg/ml, or 10 mg/ml, but is not limited thereto.

In the present invention, "antibody" refers to a polypeptide comprising a framework region derived from an immunoglobulin gene or fragment thereof that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include numerous immunoglobulin variable region genes, including kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes. Light chains were classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, consequently defining the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody becomes most critical in binding specificity and affinity. In some embodiments, antibodies or fragments of antibodies may be from different organisms, including humans, mice, rats, hamsters, camels, and the like. Antibodies of the invention are modified or mutated at one or more amino acid positions to improve or modulate a desired function of the antibody (eg, glycosylation, expression, antigen recognition, effector function, antigen binding, specificity, etc.). may include

Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids mainly responsible for antigen recognition. The terms variable light (VL) and variable heavy (VH) chains refer to these light and heavy chains, respectively. An Fc (ie, fragment determinable region) is the “base” or “tail” of an immunoglobulin and typically consists of two heavy chains contributing to two or three constant domains, depending on the class of antibody. By binding to a specific protein, the Fc region ensures that each antibody generates an appropriate immune response to a given antigen. The Fc region also binds various cellular receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

In the present invention, "sample" means any amount of material derived from an organism, for example, the sample may be one or more selected from the group consisting of whole blood, serum, plasma, urine, feces, and saliva, but is limited thereto. doesn't happen In the present invention, the sample may be isolated from a subject, and the subject is, for example, a human or non-human primate, a mouse, a rat, a rabbit, a dog, a cat, a horse, and a mammal, including cattle, and the like. may be, but is not limited thereto.

In the present invention, “nucleic acid” refers to a molecule comprising one or more nucleotides, ie, ribonucleotides, deoxyribonucleotides, or both. The term includes polymers and monomers of ribonucleotides and deoxyribonucleotides, wherein the ribonucleotides and/or deoxyribonucleotides are linked together via 5' to 3' bonds in the case of polymers. Ribonucleotide and deoxyribonucleotide polymers may be single or double stranded. The nucleic acid includes all of DNA, mRNA, cDNA reverse transcribed from mRNA, DNA amplified from the cDNA, RNA transcribed from the amplified DNA, and the like.

In the microparticle-based nucleic acid amplification product detection method of the present invention, the type of the nucleic acid is not limited, for example, hepatitis C virus (HCV), hepatitis B virus (HBV), influenza ( influenza) virus, human immunodeficiency virus (HIV), mycobacterium tuberculosis, severe acute respiratory syndrome coronavirus (SARS-CoV), or severe acute respiratory syndrome coronavirus 2 (Severe) acute respiratory syndrome coronavirus 2, SARS-CoV-2) nucleic acid.

In the present invention, "amplification" means constructing a nucleic acid molecule into multiple copies or constructing multiple copies complementary to the nucleic acid molecule using one or more nucleic acid molecules as a template.

In the present invention, the amplification in step (b) can be made by one or more methods selected from the group consisting of polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), and isothermal amplification method, , the isothermal amplification method is from the group consisting of recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification (HDA). It may be one or more selected, but is not limited thereto.

In the present invention, "detection" is meant to include both measuring and confirming the presence of a nucleic acid amplification product, or measuring and confirming a change in the level (number of nucleic acids) of the nucleic acid amplification product. In the same context, in the present invention, measuring the presence level of the nucleic acid amplification product means measuring the presence (ie, measuring the presence or absence), or measuring the level of qualitative and quantitative change of the nucleic acid amplification product. it means. The measurement may be performed without limitation, including both a qualitative method (analysis) and a quantitative method. Types of qualitative and quantitative methods for measuring the presence or absence of nucleic acid amplification products are well known in the art, and the experimental methods described herein are included therein. Methods for comparing the level of specific nucleic acid amplification products for each method are well known in the art. According to an embodiment of the present invention, the detection of the nucleic acid amplification product may be measured by aggregation of the nucleic acid amplification product captured by microparticles.

In the present invention, the term “aggregation” means that two or more dispersed nucleic acids are gathered by being captured by microparticles to form an aggregated state.

In the present invention, the term “amplicon” refers to a target sequence amplified in a single or double-stranded form from a target polynucleotide in a nucleic acid amplification reaction.

In the present invention, the detection limit concentration of the cDNA is 1 IU / ml to 15 IU / ml, 1 IU / ml to 13 IU / ml, 1 IU / ml to 10 IU / ml, 3 IU / ml to 15 IU / ml, 3 IU/ml to 13 IU/ml, 3 IU/ml to 10 IU/ml, 5 IU/ml to 15 IU/ml, 5 IU/ml to 13 IU/ml, 5 IU/ml to 10 IU/ml ml, 7 IU/ml to 15 IU/ml, 7 IU/ml to 13 IU/ml, or 7 IU/ml to 10 IU/ml, but is not limited thereto, and detection of nucleic acid amplification products according to the present invention Through this method, the amplification product of cDNA can be detected even when the concentration is as low as 15 IU/ml or less.

In the present invention, the microparticle-based nucleic acid amplification product detection method may have a sensitivity of 90% or more, for example, 90% to 100%, 90% to 98%, 90% to 96%, 90% to 94%, 90% to 92%, 92% to 95%, 92% to 97%, 92% to 100%, 95% to 97%, 95% to 100%, 97% to 100%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, but is not limited thereto.

A microparticle-based nucleic acid amplification product detection method according to the present invention, wherein cDNA is synthesized through reverse transcription after RNA is extracted, amplified, and the amplified cDNA is treated with microparticles to visually detect amplified cDNA through aggregation. The particle-based visual detection system may be referred to as “STAT-DNA”.

In addition, the present invention consists of a forward primer and a reverse primer each labeled with any one selected from the group consisting of biotin, digoxigenin, antigen, target molecule and oligonucleotide. and a primer set that specifically binds to a nucleic acid to be detected; and

Streptavidin, avidin, an antibody capable of binding to an antigen bound to the primer through an antigen-antibody reaction, an aptamer that specifically binds to a target molecule bound to the primer, and A kit for detecting nucleic acids based on microparticles, comprising microparticles coated with any one selected from the group consisting of oligonucleotides complementary to the oligonucleotides bound to the primers,

The kit provides a kit, characterized in that it is used to detect aggregation of nucleic acids captured by microparticles without centrifugation.

In the present invention, the kit includes a container, instructions; and the primer set and microparticles according to the present invention. The container may serve to package the primer set and microparticles according to the present invention, and may serve to store and fix them. The material of the container may be, for example, plastic or a glass bottle, but is not limited thereto. The instructions may describe, for example, the characteristics, composition, or method of use of the kit.

In the present invention, the kit may further include materials commonly used for PCR or isothermal nucleic acid amplification known in the art in addition to the primer set and microparticles, for example, a test tube or other suitable container, a reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, or sterile water, and the like.

In addition, the present invention provides an information providing method for diagnosing or predicting the onset of a disease, comprising the steps of:

(a) synthesizing cDNA by isolating RNA from a sample collected from a subject;

(b) biotin, digoxigenin, an antigen, a target molecule, and a forward primer and a reverse primer each labeled with any one selected from the group consisting of oligonucleotides, and amplifying the cDNA using a primer set that specifically binds to a nucleic acid to be detected;

(c) an antibody capable of binding to the amplified cDNA through an antigen-antibody reaction to streptavidin, avidin, and an antigen bound to the primer, specifically to a target molecule bound to the primer adding a microparticle coated with any one selected from the group consisting of an aptamer binding to and an oligonucleotide complementary to an oligonucleotide binding to the primer; and

(d) detecting aggregation of the nucleic acid amplification product captured by the microparticle.

However, the centrifugation step between steps (c) and (d) is not included.

In the present invention, the disease is not limited in its type as long as it is a disease capable of nucleic acid amplification test, and may include an infectious disease or cancer. According to an embodiment of the present invention, the disease may be an infectious disease.

In the present invention, "infectious disease" means a disease caused by the growth of pathogenic microorganisms by settling on the tissues, body fluids, and surfaces of humans, animals, and plants, and can be divided into several types depending on the route of infection and whether it is contagious. . Such infections include viral infections, fungal infections, bacterial infections, protozoal infections and parasitic infections. In the present invention, the infectious disease is hepatitis C, hepatitis B, influenza, human immunodeficiency virus (HIV) induced AIDS (AIDS), tuberculosis, severe acute respiratory syndrome coronavirus (SARS-CoV) infection, and coronavirus Infectious disease-19 (severe acute respiratory syndrome coronavirus 2 infection) may be one or more selected from the group consisting of (SARS-CoV-2; COVID-19), but is not limited thereto.

In an experimental example of the present invention, after amplifying cDNA of HCV using the nucleic acid amplification product detection method according to the present invention, cDNA amplification when anti-digoxigenin antibody-coated microparticles and streptavidin-coated microparticles are added It was visually confirmed that aggregation occurred by the microparticles in the presence of the product. Here, the concentration of the microparticles was optimized to produce a clear visualization of amplicon aggregation, and the optimal concentration of microparticles was 2 μl of 10 mg/ml of magnetic streptavidin beads and 5 mg/ml of anti-digoxigenin alpha particles. It was determined to be 1 μl (see Experimental Example 1).

In another experimental example of the present invention, the reproducibility and limit of detection (LOD) of the nucleic acid amplification product detection method according to the present invention were confirmed using HCV cDNA at concentrations of 10 ¹ , 10 ² , 10 ³ and 10 ⁴ IU/ml, It was observed that aggregation was clearly seen even at about 10 ¹ IU/ml, so an analysis with a detection limit of less than 15 IU/ml was recommended (see Experimental Example 2).

In another experimental example of the present invention, it was confirmed that the nucleic acid amplification product can be detected with high sensitivity when the method for detecting the amplified nucleic acid product according to the present invention is used (see Experimental Example 3).

Hereinafter, preferred examples and experimental examples are presented to help the understanding of the present invention. However, the following Examples and Experimental Examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

[Example]

Example 1. Microparticle-based visual detection method of amplified DNA: construction of a biosensor

The principle of the microparticle-based visual detection system (STAT-DNA) of amplified DNA according to the present invention is shown in FIG. 1 . The final optimized protocol proceeded with the following steps.

Nucleic acids amplified with forward/reverse primers were labeled with other molecules such as biotin and digoxigenin at the 5' and 3' ends, respectively. After amplification via RT-PCR with biotinylated forward primer and reverse primer labeled with digoxigenin, 2 μl of streptavidin-coated magnetic bead suspension (1 μm size, 10 mg/ml Dynabead) MyOne Streptavidin C1, Life Technologies, Grand Island, NY, USA) and 1 μl of anti-digoxigenin polystyrene particles (anti-digoxigenin alpha donor beads sized 250-350 nm, PerkinElmer, Waltham, MA, 5 mg/ml in PBS, pH 7.2, USA) to the PCR tube containing the amplified DNA, then generate visual aggregates and add 60 μl Low Ionic Strength Solution (LISS) buffer (BLISS, Ortho-Clinical Diagnostics) Thus, positive (+) results and negative (-) results were clearly distinguished.

Particle aggregation was visualized within seconds of adding the beads in the presence of amplified HCV cDNA. As a result, as shown in FIG. 2, aggregation of particles in the absence of a target showed a negative result, and streptavidin-coated blue polystyrene particles (0.3-0.39 μm, When the same experiment was additionally performed with other microparticles such as SPHERO, Spherotech, Lake Forest, IL, USA), positive and negative aggregation results were clearly observed with the naked eye depending on the presence or absence of amplified HCV cDNA. Could.

Mixtures due to aggregation were observed visually or with another optical device such as a smartphone, and a smartphone was used to objectively distinguish between positive and negative results.

Direct visual detection of HCV amplicons has been optimized, and for HCV RNA detection and quantification, a comparison of assay sensitivity and detection systems was performed using the existing real-time PCR test, the cobas HCV test on the Roche cobas 6800 system (Roche Molecular Diagnostics International, Rotkreuz, Switzerland) was evaluated.

[optimization]

For particle concentration optimization, assays were performed with various particle concentrations ranging from 0.5 μl to 2 μl, and all experiments were performed at least three times.

Example 2. cDNA synthesis and RT-PCR after HCV RNA extraction

2-1. sample collection

Serum samples were collected from HCV-infected patients at Asan Hospital in Seoul from May 2018 to October 2020. After routine HCV RNA testing with the Roche cobas 6800 system (Roche Molecular Diagnostics), residual or residual serum samples were each aliquoted and stored at -80 °C, and the experiment was approved by the ethics review committee of Asan Hospital, Seoul (IRB No. 2017-). 1742).

2-2. RNA extraction

50 μl of HCV RNA was extracted from clinical samples using the QIAamp MinElute Viurs Spin Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. The nucleic acids were then stored at -80 °C.

2-3. cDNA synthesis

HCV cDNA synthesis was performed using the Roche Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) according to the manufacturer's protocol.

That is, 5 μl of the isolated RNA sample was mixed with 0.5 μl of Transcriptor Reverse Transcriptase (Roche), 2 μl of random hexamer (20 pmol), 2 μl of deoxynucleotide mixture, 0.5 μl of Protector RNase inhibitor, 6 It was mixed with 20 μl of a reverse transcription (RT) reaction mixture containing μl of nuclease-free water (Roche), and 4 μl of a 5x reaction mixture of the above substances. The samples were then incubated in an FX thermocycler (Bio-Rad, California, USA) using the cDNA synthesis protocol (25 °C for 10 min, then 55 °C for 30 min, then 85 °C for 5 min. ). cDNA samples were stored at -80 °C until PCR analysis.

2-4. RT-PCR

Primer of reverse transcription-polymerase chain reaction (RT-PCR) for simple visual detection of HCV mRNA is 5'-untranslated region (5'-UTR) of HCV (Chen, 2016 # 637) was modified using a pair of primers specifically designed specifically for

In this case, the primer sequences used are shown in Table 1 below.

Primer Sequence Location PCR target size forward primer 5'-biotin-TGCACGGTCTACGAGAC-3'
(SEQ ID NO: 1) 322-339 ^† 157 Bp Reverse primer 5'-Dig_N/CGACCGGGTCCTTTCTTGGAT-3'
(SEQ ID NO: 2) 182-203

The forward primer was biotinylated at the 5' end and the reverse primer was labeled with digoxigenin at the 5' end. They were prepared for capture with streptavidin-labeled magnetic beads (Dynabead MyOne Streptavidin C1) and anti-digoxigenin antibody coated polystyrene particles (anti-digoxigenin alpha donor beads, PerkinElmer), respectively. .

RT-PCR analysis was performed using the QIAGEN Taq PCR Master Mix Kit (Qiagen) according to the instruction manual. That is, 5 μl of HCV cDNA was amplified in a reaction volume of 25 μl containing 12.5 μl of QIAGEN Taq PCR Master Mix (Qiagen); 1 μl of 10 μmol/L biotinylated forward primer; 1 μl of 10 μmol/L of digoxigenin-labeled reverse primer and distilled water (DW). Thermal cycling was performed using an Applied Biosystems 7500 instrument [AB Sciex, Foster City, CA (formerly Applied Biosystems)] under the following conditions: 94 °C, 5 min; Final extension (40 cycles) at 95° C. for 30 seconds, 56° C. for 30 seconds, 72° C. for 30 seconds, and 72° C. for 10 minutes. Then, HCV PCR products were detected using 2% agarose gel electrophoresis.

[Experimental example]

Experimental Example 1. Analysis Optimization: Confirmation of Mobility Differences between Amplicon Capture and Non-Amplicon Magnetic Beads

The concentration of microparticles (anti-digoxigenin antibody coated microparticles, streptavidin coated microparticles) was optimized to produce a clear visualization of amplicon aggregation, and the optimal concentration of microparticles was 2 μl streptavidin magnetic Beads (S-b)/blue polystyrene particles (Blue-b)) were determined to be 10 mg/ml and 1 μl of anti-digoxigenin alpha particles (alpha-b) 5 mg/ml. The results for nucleic acid detection through aggregation at the concentration and content of these microparticles are shown in FIG. 3 . 3 shows an example of increasing reproducibility by using a mixer (HulaMixer Sample Mixer, Thermo Fisher Scientific) for about 10 seconds because the tapping method may be different for each experimenter. A distinction was made between positive specimens.

To avoid several optimizations such as conjugation to microparticles, commercially available antibody-coated or conjugated microparticles were selected for reproducibility in the present invention. Among several commercially available anti-digoxigenin particles, anti-digoxigenin polystyrene particles (anti-digoxigenin alpha donor beads with a size of 250-350 nm, PerkinElmer, Waltham, Mass. , USA, 5 mg/ml in PBS, pH 7.2).

In the previously reported study, lateral flow type molecular diagnostic technology, the minimum amount of capture reagent to generate an intense red line was 20 μg for both streptavidin-coated microparticles and anti-digoxigenin antibody-coated microparticles, whereas In the present invention, an amount as small as 5-10 μg could be used. In addition, the dispensing system for dispensing the lateral flow dipstick, membrane paper, and line required for the existing lateral flow type molecular diagnosis technology was not required in the present invention, and the protein blocker, stabilizer, and membrane Fixation of the capture reagent was not required. The detection process to which the existing lateral flow type technology is applied requires additional steps such as an additional hybridization step including heating, but this additional process was not required in the present invention.

The stability or shelf life of DNA sensors based on the existing lateral flow method was limited, and complex setup and detection mechanisms including centrifugal microfluidic platforms, fluorescence scanning or UV light sources were required, but were not required in the present invention.

The smartphone-based visual detection of PCR amplicons was performed without the aid of electric power, motor drive or centrifugal force.

First, the difference in particle aggregation between the 'positive' magnetic beads that capture the target amplicon and the 'negative' magnetic beads that do not capture the target amplicon was observed within a few minutes.

After adding microparticles to the PCR reaction tube, particle aggregation was monitored visually as well as by smartphone. This direct visual observation was confirmed with a smartphone, and as shown in FIG. 2 , the positive sample showed characteristic aggregation and precipitation, and the negative control remained a cloudy solution without aggregation and precipitation. In addition, as a result of observing particle aggregation at 400x and 1000x magnifications, as shown in FIG. 4a, aggregation was observed when streptavidin-coated magnetic beads and anti-digoxigenin antibody-coated alpha donor beads were added in the presence of the target amplicon. Formation was confirmed, and when the target amplicon was not present, it was confirmed that aggregation was not formed as shown in FIG. 4B .

Experimental Example 2. Reproducibility and detection limit

The precision and limit of detection (LOD) of the method according to the present invention were determined by serial dilutions of HCV cDNA to concentrations of 10 ¹ , 10 ² , 10 ³ and 10 ⁴ IU/ml. Reproducible results of the microparticle-based visual detection biosensor of amplified cDNA according to the present invention were obtained by performing two runs for 5 days with serially diluted samples. The detection limit of the biosensor was determined by comparing it with the results obtained by the agarose gel method of serially diluted PCR amplicons.

As a result, as shown in FIG. 5, streptavidin-coated blue polystyrene particles (A)/magnetic beads (B) and anti-digoxige using a microparticle-based visual detection biosensor of amplified cDNA according to the present invention When the aggregation of the amplified cDNA was confirmed after the addition of the nin antibody-coated alpha donor beads, aggregation did not occur in NTC (no template control), but it was observed that aggregation was clearly seen even at ~10 ¹ IU/ml. became The biosensor was similar to conventional PCR or real-time PCR, and analysis with a low HCV RNA detection limit of less than 15 IU/ml was recommended.

Experimental Example 3. Sensitivity, Analysis Specificity

To evaluate the assay specificity of this technique, nucleic acids from several virus strains other than HCV virus were used.

The microparticle-based visual detection biosensor for diagnosing amplified HCV nucleic acids according to the present invention did not detect the nucleic acids of the following 8 viruses; Human rhinovirus (HRV), influenza A, B, norovirus, human matapenumovirus, parainfluenza virus type 3 (PIV3), CoV- 229E, adenovirus. DNA biosensor results were consistent with real-time PCR results. Each virus used for comparison and real-time PCR results (CT (Cycle Threshold)) values are shown in Table 2 below.

Research sample number date Viruses used for comparison CT value AMC-0001 190403 HRV 33.59 AMC-0002 190403 Flu B 30.16 AMC-0003 190403 Flu A (H3) 32.08 AMC-0015 190408 Norovirus 23.95 AMC-0021 190411 hMPV 23.17 AMC-0022 190411 PIV 3 18.08 AMC-0033 190418 CoV-229E 19.87 AMC-0058 ADV 20.03

As such, it was found that the microparticle-based visual detection biosensor of amplified cDNA according to the present invention has analysis specificity.

In addition, in order to confirm the sensitivity of the biosensor according to the present invention, electrophoresis results with actual clinical samples (HCV positive 4, 5, 6; negative N4, N5, N6) and streptavidin coating by the method according to the present invention As a result of comparing the aggregation results when the microparticles (S-b) and anti-digoxigenin antibody-coated alpha donor beads (alpha-b) were added, as shown in FIG. 6, HCV positive (4, 5, 6) Aggregation was observed in the sample.

In addition, when compared with the conventional nucleic acid amplification detection method, as shown in FIG. 7 , in the case of using the conventional nucleic acid amplification detection method, no band was observed in the electrophoresis result in PCR 40 cycles (arrow in the upper figure of FIG. 7 ) ), while it was confirmed that the band was observed only in the electrophoresis result at 45 cycles, when the microparticle-based nucleic acid amplification product detection method according to the present invention was used, it showed a clear aggregation (positive) reaction even when PCR 40 cycles were performed. It was confirmed (bottom drawing of FIG. 7).

Therefore, from the above results, it was confirmed that the amplification product of the target nucleic acid can be detected with high sensitivity when using the microparticle-based nucleic acid amplification product detection method according to the present invention.

Experimental Example 4. Comparison with clinical samples

The microparticle-based visual detection biosensor of amplified cDNA according to the present invention was compared with the results of the Roche Cobas 6800 HCV test (Roche HPV; Roche Molecular Diagnostics, Pleasanton, CA, USA). Of the 54 frozen samples, 32 were confirmed positive for HCV by the biosensor compared to the Roche HCV kit and 22 were confirmed negative. All were 100% consistent with the existing results.

Experimental Example 5. Advantages of Visual Detection System

The microparticle-based visual detection biosensor of amplified cDNA according to the present invention was one of the simplest visual detection systems among DNA sensors, especially compared to the lateral flow-based detection. It was also robust and sensitive compared to clinical grade HCV real-time PCR. The microparticle-based visual detection biosensor of amplified cDNA according to the present invention was able to detect HCV amplicons within minutes without multiple incubation, adsorption/elution and washing steps with high sensitivity compared to conventional real-time PCR.

Improving the number of people diagnosed with HCV infection is critical to eliminating viral hepatitis, and alternatives to conventional HCV testing, including POC, are urgently needed for HCV screening and diagnosis.

Microparticle-based visual detection of amplified cDNA according to the present invention There are several advantages in identifying amplified DNA through the biosensor, the system being a special sophisticated instrument, immobilization technology, and transformation technology such as surface plasmon resonance. (Qiu, 2020 #688), electrochemical transformation (Abad-Valle, 2005 #689), and surface-enhanced Raman scattering (SERS)-based biosensor (Wang, 2019 #686) were not required. In addition, because only the microparticles are visible, no color or signal generation process is required, and no driving force or microfluidic actuation is required due to the PCR reaction tube in which the visualization is generated. A major limitation of PASS-DNA (Yang, 2019 #608) in previous studies was the need for a separator for detection.

Gold nanoparticles (AuNPs) have surface plasmon resonance (SPR)-like properties and undergo visible color and spectral changes (Ghasemi, 2018 #699). Therefore, gold nanoparticle aggregation based on charge-based aggregation induced by pH, ionic strength, catalytic DNA circuitry, or charge-dependent manner, whether cross-linked, non-cross-linked or unmodified, has been widely used in colorimetric assays for nucleic acid detection (Ghasemi, et al. 2018 #699; Mohammed, 2019 #697; Ravan, 2020 #698; Shawky, 2017 #696). However, in order for the colloidal solution to change color from red to blue, elaborate efforts are required to induce aggregation of gold nanoparticles.

In general, primer-dimers can produce problematic false positives, unless additional specific detection designs, such as probe-based detection, are considered, especially in real-time PCR formats. However, the microparticle-based visual detection biosensor of amplified cDNA according to the present invention minimizes the effect of the primer-dimer because the particle cannot be closed with a very short sequence such as a primer-dimer.

Magnetic nanoparticle-based capture has been widely used in DNA sensors due to its excellent dispersion, low cost, easy separation, purification of buffer systems, and signal detection (Tang, 2020 # 701). However, their signal detection required additional signal development processes for fluorescence, electrochemical or chemiluminescence (Zhuang, 2019 # 702; Cortina, 2016 # 704; Ali, 2016 # 703).

The microparticle-based visual detection method of amplified cDNA according to the present invention did not use the properties of magnetic nanoparticles for magnetically controllable aggregation, purification and separation. The role of magnetic particles is only visible to the naked eye when they aggregate. This is the same as the role of the streptavidin coated blue particles in the above method.

The simple, inexpensive, and no complex instrumentation required for molecular detection has facilitated the development of DNA biosensors for molecular POC in resource-constrained environments (Sayad, 2018 #681), and the lateral flow immunoassay (LFIA) has meet these needs. Although several studies combining PCR and LFIA to detect HCV have been published (Zhuang, 2019 # 702), the detection of DNA amplified by LFIA is a rather complicated setup and capture oligos (Liu, # 532; Gao, # 534; Abad-Valle, 2005 # 689) or antibodies (Rosa, #533), and sophisticated detection probes or sequence design (Kalogianni, 2006 # 679). In addition, a heating process is also required for hybridization with the target (Kalogianni, 2006 # 679). Detection of captured DNA requires signal detection steps, including fluorescence scanning (Dou, 2014 #682) or visual RT-LAMP-CRISPR detection under blue light illumination (Wang, 2021 #690).

To avoid several optimizations, such as conjugation to microparticles, or to eliminate unexpected variability during optimization, commercially available antibody-coated or conjugated microparticles are prepared by selecting biotin- or digoxigenin-labeled primers to capture them. did. Among several commercially available anti-digoxigenin particles, PerkinElmer alpha donor particles successfully produced aggregation of DNA amplicons with streptavidin coated microparticles. Anti-digoxigenin alpha donor particles were anti-digoxigenin latex-based particles with a size of 250-350 nm (Yu, 2015 # 683) and were originally used for homogeneous (no washing) bead-based sandwich immunoassays.

Visual detection of smartphone-based PCR amplicons was achieved for the first time without the aid of electric power, motor drive or centrifugal force (Gou, 2018 #685). The microparticle-based visual detection biosensor of amplified cDNA according to the present invention was also detected in smartphone-based detection, which was more identifiable and quantitative than visualizing aggregation with the naked eye. It was also expected that a fully integrated microparticle-based NAT with sample-in-answer-out function would be inexpensive, user-friendly, fast and robust, and would significantly improve the implementation of POCT in resource-limited settings.

For proof of concept, conventional two-step PCR for nucleic acid amplification was used. However, to overcome the time-consuming obstacle of RT-qPCR, isothermal DNA amplification techniques have been developed to provide molecular diagnosis of infectious death in situ. Isothermal amplification methods (e.g., recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA)) are also used according to the present invention. It is compatible with microparticle-based visual detection systems of amplified cDNA.

The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation <120> Microparticle-based detecting kit for nucleic acid and Method for detecting nucleic acid amplification product <130> MP20-224 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> HCV forward primer <400> 1 tgcacggtct acgagac 17 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HCV reverse primer <400> 2 cgaccgggtc ctttcttgga t 21

Claims (17)

A method for detecting a microparticle-based nucleic acid amplification product, comprising the steps of:
(a) synthesizing cDNA by isolating RNA from a sample collected from a subject;
(b) biotin, digoxigenin, antigen, target molecule and oligonucleotide each labeled with any one selected from the group consisting of a forward primer and a reverse primer, amplifying the cDNA using a primer set that specifically binds to a nucleic acid to be detected;
(c) streptavidin, avidin, an antibody capable of binding to the antigen bound to the primer through an antigen-antibody reaction to the amplified cDNA, and a target molecule bound to the primer specifically adding a microparticle coated with any one selected from the group consisting of an aptamer binding to and an oligonucleotide complementary to an oligonucleotide binding to the primer; and
(d) detecting aggregation of the nucleic acid amplification product captured by the microparticle.
However, the centrifugation step between steps (c) and (d) is not included.
The method of claim 1,
The primer set is a microparticle-based nucleic acid amplification product detection method, characterized in that it consists of a forward primer and a reverse primer each labeled with any one selected from the group consisting of biotin and digoxigenin.
The method of claim 1,
The microparticle-based nucleic acid amplification product detection method, characterized in that the microparticle is coated with streptavidin or anti-digoxigenin antibody.
According to claim 1,
The sample is a microparticle-based nucleic acid amplification product detection method, characterized in that at least one selected from the group consisting of whole blood, serum, plasma, urine, feces, and saliva.
According to claim 1,
In the step (b), the primer set is a microparticle-based nucleic acid amplification product detection method, characterized in that the size of the nucleic acid amplification product is 100 to 200 bp.
According to claim 1,
The amplification in step (b) is characterized in that it is made by one or more methods selected from the group consisting of polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), and isothermal amplification method, microparticles based nucleic acid amplification product detection method.
7. The method of claim 6,
The isothermal amplification method is selected from the group consisting of recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification (HDA). Microparticle-based nucleic acid amplification product detection method, characterized in that at least one that is.
According to claim 1,
The microparticle-based nucleic acid amplification product detection method, characterized in that the diameter of the microparticles in step (c) is 0.25 to 10 μm.
According to claim 1,
Microparticle-based nucleic acid amplification product detection method, characterized in that the microparticles in step (c) are magnetic beads or polystyrene particles.
According to claim 1,
The detection limit concentration of the cDNA is 1 to 15 IU / ml, characterized in that the microparticle-based nucleic acid amplification product detection method.
A method for providing information for diagnosing an infectious disease, comprising the steps of:
(a) synthesizing cDNA by isolating RNA from a sample collected from a subject;
(b) biotin, digoxigenin, antigen, target molecule and oligonucleotide each labeled with any one selected from the group consisting of a forward primer and a reverse primer, amplifying the cDNA using a primer set that specifically binds to a nucleic acid to be detected;
(c) streptavidin, avidin, an antibody capable of binding to the antigen bound to the primer through an antigen-antibody reaction to the amplified cDNA, and a target molecule bound to the primer specifically adding a microparticle coated with any one selected from the group consisting of an aptamer binding to and an oligonucleotide complementary to an oligonucleotide binding to the primer; and
(d) detecting aggregation of the nucleic acid amplification product captured by the microparticle.
However, the centrifugation step between steps (c) and (d) is not included.
12. The method of claim 11,
The infectious disease is hepatitis C, hepatitis B, influenza, human immunodeficiency virus (HIV) induced AIDS, tuberculosis, severe acute respiratory syndrome coronavirus (SARS-CoV) infection, and coronavirus infection-19 (COVID). -19), characterized in that at least one selected from the group consisting of, an information providing method for diagnosing an infectious disease.
It consists of a forward primer and a reverse primer each labeled with any one selected from the group consisting of biotin, digoxigenin, antigen, target molecule, and oligonucleotide. a primer set that specifically binds to a nucleic acid; and
Streptavidin, avidin, an antibody capable of binding to an antigen bound to the primer through an antigen-antibody reaction, an aptamer that specifically binds to a target molecule bound to the primer, and A kit for detecting nucleic acids based on microparticles, comprising microparticles coated with any one selected from the group consisting of oligonucleotides complementary to the oligonucleotides bound to the primers,
The kit, characterized in that it is used to detect aggregation of nucleic acids captured in microparticles without centrifugation.
14. The method of claim 13,
The primer set is a kit for detecting a microparticle-based nucleic acid, characterized in that it consists of a forward primer and a reverse primer each labeled with any one selected from the group consisting of biotin and digoxigenin.
14. The method of claim 13,
The microparticle is a kit for detecting a microparticle-based nucleic acid, characterized in that it is coated with a streptavidin or anti-digoxigenin antibody.
14. The method of claim 13,
The microparticle-based nucleic acid detection kit, characterized in that the diameter of the microparticle is 0.25 to 10 μm.
14. The method of claim 13,
The microparticles are magnetic beads or polystyrene particles, characterized in that the microparticle-based nucleic acid detection kit.

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