KR20030003181A - A method for detecting a nucleic acid by a membrane chromatograph technique and a kit therefor - Google Patents

Abstract

PURPOSE: Provided are a method for detecting a nucleic acid by a membrane chromatograph technique and a kit therefor, thereby detecting a specific nucleic acid from a nucleic acid sample, particularly nucleic acid polymerization products using tagged probes. CONSTITUTION: The method for detecting a nucleic acid by a membrane chromatograph technique comprises the steps of: hybridizing a nucleic acid sample, which consists of nucleotide sequence capable of being specifically bound to a nucleic acid to be analyzed, with at least two kinds of tagged probes; binding the obtained nucleic acid-probe complex to coloring colloid particles; binding the obtained nucleic acid-probe-colloid complex to a complementary material on a membrane; and analyzing a coloring band on the membrane.

Description

A method for detecting a nucleic acid by a membrane chromatograph technique and a kit therefor}

The present invention relates to a nucleic acid detection method by a membrane chromatographic technique, more specifically a method for detecting a specific nucleic acid by a membrane chromatographic technique in a nucleic acid sample, in particular, the result of the nucleic acid polymerization reaction using labeled probes And a kit therefor.

In general, techniques for detecting a particular nucleic acid from a nucleic acid sample isolated from a bacterium or a cell or a result of amplification by a nucleic acid polymerization reaction include nucleic acid fragments having complementary base sequences that specifically bind to a part of the nucleic acid to be analyzed. By using a nucleic acid probe (probe) to determine whether the presence of an assay nucleic acid by binding between them, a method for hybridizing with the nucleic acid to be analyzed by attaching a specific nucleic acid probe to the surface of the microwell plate, and Reverse dot hybridization is used to fix specific nucleic acid probes to a membrane and inject amplified nucleic acids into nucleic acid hybridization. However, these methods have a variety of difficulties in practice.

First, the technique using a microwell plate involves immobilizing avidin on the surface of the microwell plate, and adding a biotin-attached specific nucleic acid probe to the avidin-fixed microwell plate, thereby allowing biotin and avidin to be separated. A specific binding reaction is performed, and the resultant nucleic acid polymerization reaction product, which is denatured by heating or the like, is injected into a microwell plate to which the nucleic acid probe is fixed to allow a specific nucleic acid hybridization reaction between the nucleic acid probe and the nucleic acid polymerization reaction product. As a method of confirming the result of the binding reaction through an enzymatic method, this method confirms the reaction result by the enzyme measurement method, which takes a long experiment time of 2 hours or more and has a disadvantage in that the process is complicated.

The second technique, which uses a membrane, involves attaching a nucleic acid probe that specifically binds to the nucleic acid to be analyzed on the membrane surface, and then denatures the labeled nucleic acid sample, together with the nucleic acid binding solution, by physical or chemical methods. After the injection into the prepared membrane and the nucleic acid hybridization reaction by keeping the temperature constant, the labeling material is read to confirm the binding of the nucleic acid on the membrane. The test procedure is complicated and it takes at least 2 hours. .

As such, conventional nucleic acid detection methods have a problem that the test process is complicated and takes a long time.

Accordingly, the present inventors can quickly and easily analyze nucleic acid samples, and in the search for a nucleic acid detection method having excellent accuracy of results, by applying an immune membrane chromatographic technique used for the analysis of a specific protein using an antibody in a nucleic acid detection process. The present invention has been completed.

Accordingly, the present invention provides a nucleic acid analysis method that can accurately and easily detect specific nucleic acids in a sample in a short time compared to the conventional nucleic acid detection method, and can detect several kinds of nucleic acids at once. have.

In particular, the present invention provides a method for detecting amplified nucleic acid in a nucleic acid sample as a process for identifying a nucleic acid for a result obtained after performing a nucleic acid polymerization reaction.

The present invention also provides a kit for nucleic acid analysis for easily carrying out such a nucleic acid detection method.

1 schematically depicts a kit for nucleic acid analysis for carrying out the method of the present invention.

FIG. 2 is a diagram schematically illustrating an example of the present invention applied to Example 1. FIG.

Figure 3 is an electrophoretic photograph of the result obtained by the polymerization reaction of Mycobacterium tuberculosis nucleic acid in Example 1 [lane 1 shows the criteria for DNA size, lanes 2-9 are serial dilution of the nucleic acid amplification results of the obtained Mycobacterium tuberculosis Lanes 10 and 11 indicate no nucleic acid of Mycobacterium tuberculosis as a negative control.

Figure 4 is a membrane strip photograph obtained by performing the method of the present invention on the result of the polymerization reaction of Mycobacterium tuberculosis nucleic acid in Example 1 [Strips 1-8 shows the results of nucleic acid analysis of the serially diluted Mycobacterium tuberculosis nucleic acid, strips 9 and 10 Represents a result of nucleic acid analysis for the case where there is no result of nucleic acid polymerization reaction as a negative sample.

FIG. 5 is a diagram schematically illustrating an example of the present invention applied to Example 2. FIG.

FIG. 6 is an electrophoretic photograph of the result obtained by polymerization of human papilloma virus (HPV) nucleic acid in Example 2 [lane 1 shows the criteria of DNA size, lane 2 obtained human papilloma virus (HPV) type 16 and 18 nucleic acid amplification products, lane 3 represents HPV type 16 nucleic acid amplification products, lane 4 represents HPV type 18 nucleic acid amplification products, and lanes 5 and 6 represent negative control nucleic acid amplification products of normal DNA.

FIG. 7 is a membrane strip photograph obtained by performing the method of the present invention on the result of the polymerization reaction of HPV nucleic acid in Example 2. [Strip 1 shows nucleic acid analysis results for HPV types 16 and 18, and strip 2 shows Nucleic acid analysis results for HPV type 16, strip 3 shows nucleic acid analysis results for HPV type 18, and strips 4 and 5 show nucleic acid analysis results for normal DNA as negative samples.

<Description of Symbols for Main Parts of Drawings>

10 hybridization vessel 20 membrane strip

1 sample pad 2 colloid pad

3: reaction pad 4: reading area

5: control part 6: absorption pad

The present invention relates to a nucleic acid detection method by a membrane chromatograph technique, and more particularly, to label a specific nucleic acid in a nucleic acid sample, in particular, a nucleic acid polymerization reaction product using labeled probes. It relates to a method for detecting by chromatographic techniques and a kit for nucleic acid analysis for the same.

Specifically, the nucleic acid detection method of the present invention is characterized by including the following steps:

(1) two or more probes consisting of a nucleic acid sequence capable of specifically binding to a portion of the nucleic acid (s) to be analyzed in the sample and having a label attached thereto, wherein Labels are different kinds of substances that can be distinguished by analysis) and hybridization,

(2) allowing the obtained nucleic acid-probe complex to bind to the chromogenic colloidal particles immobilized on the surface of a complementary material which can specifically bind only to one of the labels used in step (1). ,

(3) thereafter, the nucleic acid sample is moved by chromatographic techniques on the membrane fixed in the form of a band at a predetermined interval on the surface, which can bind specifically to each of the remaining labels. Allowing the nucleic acid-probe-colloid complex obtained as a result of step (2) in the sample to bind to the complementary material of the membrane; and

(4) confirming the formation of a color band on the membrane.

The nucleic acid detection method of the present invention is useful for detecting a desired nucleic acid from a nucleic acid sample isolated and synthesized from bacteria or cells, and particularly for detecting a desired nucleic acid from a nucleic acid sample amplified by a method such as a nucleic acid polymerization reaction. Can be.

In the case of detecting a desired nucleic acid from the result of the nucleic acid polymerization reaction by the method of the present invention, one of the labels to be attached is previously attached to a nucleic acid primer used for the nucleic acid polymerization reaction instead of the nucleic acid probe. More advantageous.

Accordingly, the present invention also relates to a method for detecting a desired amplified nucleic acid from a nucleic acid polymerization reaction product, comprising the following steps:

(1) A nucleic acid amplification sample produced by a nucleic acid polymerization reaction using a labeled primer is composed of a nucleic acid sequence capable of specifically binding to a portion of the nucleic acid (s) to be analyzed, and the label is attached. Hybridizing with two or more probes, wherein the primers and the labels attached to the probes are different kinds of materials distinguishable by analysis,

(2) allowing the obtained nucleic acid-probe complex to bind to the chromogenic colloidal particles immobilized on the surface thereof, with a complementary substance capable of specifically binding only to a label attached to the primer;

(3) thereafter, the nucleic acid sample is moved by chromatographic techniques on the membrane fixed in the form of a band at a predetermined interval on the surface, which can bind specifically to each of the remaining labels. (2) allowing the obtained nucleic acid-probe-colloid complex to bind with the complementary material of the membrane;

(4) confirming the formation of a color band on the membrane.

The present invention further provides a nucleic acid analysis kit for performing the nucleic acid detection method, specifically, a reaction vessel including a nucleic acid sample and two or more probes to which a label is attached; And a sample pad for adding a sample, a colloid pad containing chromogenic colloid particles fixed on the surface thereof by a complementary material specifically binding to any one of the above labels, and a complementary material specifically binding to the remaining labels. (S) consisting of a membrane pad consisting of a reaction pad comprising a reading site fixed in the form of a band at regular intervals and an absorbent pad for continuously moving the sample and removing unreacted sample components. It provides a kit for nucleic acid analysis.

Kits of the present invention are largely a reaction vessel for the hybridization reaction between the nucleic acid sample and the labeled probe; And a membrane strip in which nucleic acid adsorption occurs by a binding reaction between a label and a complementary substance thereto, wherein the reaction vessel and the membrane strip are installed in the same system or continuously in separate systems for continuous analysis. Each of the sample pads, colloid pads, reaction pads, and absorbent pads in the membrane strip are arranged partially overlapping so that the sample is continuously moved by capillary action.

An example of a kit for nucleic acid analysis for carrying out the method of the present invention is shown in FIG. 1.

Hereinafter, the present invention will be described in more detail with reference to a process for detecting nucleic acid in a sample using the nucleic acid analysis kit of the present invention.

Nucleic acid detection method of the present invention using a nucleic acid probe (or primer) with a label attached to the nucleic acid (s) to be analyzed two or more labels, one of the labels and complementary material having a specific binding to Of the colloidal particle-nucleic acid-membrane which binds the nucleic acid to the chromogenic colloidal particles by the primary binding reaction of the compound and then binds the nucleic acid to the membrane by the secondary binding reaction of the complementary material having a specific binding ability to the remaining labels. It is characterized by confirming the presence or absence of the analysis nucleic acid in the sample by showing the coloring effect through the sandwich-type binding.

Therefore, in the present invention, various kinds of labeling substances are used together with various kinds of nucleic acid probes as described above. The probes used in the present invention are each labeled with a different kind of labeling substance, and these probes are composed of base sequences capable of specifically binding to a part of the nucleic acid to be analyzed, resulting in hybridization with the probes. As a result, nucleic acids are multilabeled with several kinds of labeling substances.

In the case of using the nucleic acid polymerization reaction product as the assay sample, the labeling step can be simplified by using a nucleic acid primer with a labeling substance attached thereto during the nucleic acid polymerization reaction. In this case as well, nucleic acid primers and probes should be labeled with different kinds of labeling substances.

Probes used in the present invention are designed to bind to different sites of the nucleic acid according to the purpose of nucleic acid analysis, in the case of simply confirming the amplification of the nucleic acid, each nucleic acid by distinguishing the internal sequence difference of the nucleic acid to be analyzed If you want to Typing of is designed to bind to the same site or overlap of nucleic acids.

In the present invention, the most important thing is the appropriate selection of the labeling substance attached to the nucleic acid probe (or primer). Probe used in the present invention is selected in consideration of requirements such as reactivity, availability and price among those having a complementary corresponding material that can be attached to the oligonucleotide, and specifically binds to the oligonucleotide Different kinds of labeling materials must be used depending on the type of (or primer) to allow the nucleic acid of interest to be labeled with various kinds of materials.

Examples of suitable labeling materials for use in the present invention include Biotin, Fluorescein, Digoxigenin and the like.

In addition, the complementary material to be immobilized on the surface of the chromogenic colloidal particles or the membrane should be selected as a corresponding material having specific binding ability only according to the type of labeling material attached to the nucleic acid probe (or primer).

For example, in the case where biotin is attached as a labeling substance to a nucleic acid probe (or primer), complementary substances attached to the surface of the chromogenic colloidal particles or the membrane include streptoavidine or avidin [Methods in Enzymology, Green. N. M., Vol. XVIII, p148 (1970)], antifluorescein antibody as complementary material for fluorescein and antidigoxigenin antibody as complementary material for digoxigenin. digoxigenin antibody).

In general, the selection of the labeling material and its complementary material should be such that the binding specificity between them and the reaction affinity are so strong that they provide detectable sensitivity even when present in extremely low concentrations. In addition, any material may be used in the method of the present invention as long as it meets the above requirements.

In the method of the present invention, step (1) is a process of multiple labeling nucleic acids by hybridization reaction between a nucleic acid sample and a labeled probe, wherein the temperature and time used for these reactions are determined by the sequence configuration of the nucleic acid and probe to be analyzed, For example, it is appropriately selected in consideration of the GC ratio and the like, but in general, the labeled probes are inserted into a nucleic acid sample and thermally denatured at a temperature of about 90 ° C to 96 ° C for 3 to 10 minutes, and then 45 ° to 65 ° C The reaction is allowed to take place between nucleic acid and probe for 3 to 10 minutes at temperature. In the case of carrying out the present invention on the resultant after the nucleic acid polymerization reaction, the hybridization reaction may be performed as it is in an amplification vessel without using a separate vessel.

After the nucleic acid-probe hybridization reaction of step (1), the obtained result is applied through the sample pad 1 to the membrane strip 20 provided in the same system as the reaction vessel 10 or in a separate system, and this sample The sample absorbed in the pad 1 is then moved to the colloidal pad 2 located at the top of the sample pad 1 by capillary action.

The colloidal pad 2 includes chromogenic colloidal particles having a complementary substance having a specific binding ability only to one of the labeled substances labeled on the nucleic acid sample on the surface thereof, thereby binding the nucleic acid by a binding reaction between the label and the complementary substance thereto. A colloidal particle complex is formed.

In the case where a nucleic acid amplification product is used as the assay sample, it is preferable to attach the complementary binding material for the label attached to the nucleic acid primer to the colloidal particle.

In this case, the colloidal particles to be used have a color development, which can be observed by the naked eye upon deposition. The colloidal particles have excellent color stability, are easily polymerized with a polymer, and can be moved by a capillary effect on the membrane. As such, colloidal particles or plastic colloidal particles of metal materials such as gold, silver, and iron having a particle diameter of 5 to 50 nm may be used, and the sodium citrate method [Immunocytochemistry, RM Albrecht et al., A Practical Approach: 151-176 (1993) ); Techniques in Immunocytochemistry, J. Roth, Vol. 2, 217-284 (1983), it is preferable to use gold colloidal particles having a particle diameter of about 30 nm.

The nucleic acid-colloidal particle complex obtained as a result of step (2) is sequentially moved by the capillary phenomenon onto the reaction pad 3 in which the complementary substance (s) to the label are fixed in the form of a band.

Reaction pad (3) is fixed in the form of a band of the complementary material (s) having a specific binding force to the remaining labels other than the label that reacts with the colloid particles in step (2), so that the sample moves the membrane surface In the process, the nucleic acid-colloidal particle complex containing the label is captured on the surface of the membrane by binding with a complementary material, and a color signal is observed in the form of colored lines by the deposition of chromogenic colloidal particles in the captured complex.

In addition, when there is no analysis nucleic acid in the sample, since the hybridization with the probe is not performed, neither the binding with the colloidal particles nor the membrane binding can occur, so that no color band is formed.

Therefore, the nucleic acid detection method by the membrane chromatographic technique of the present invention can be said to be particularly suitable for qualitative analysis to determine the presence or absence of the analysis nucleic acid.

In the production of the reaction pad 3, when it is desired to simultaneously detect several kinds of nucleic acids in the analysis process, it is arranged in the form of a band at intervals to distinguish the various complementary substances in the reading site (4), A control site 5 is placed on top of the reading site 4 to confirm the fluid flow and reactivity of the sample.

Unreacted sample components that are not bound while moving the membrane through the reaction pad 3 are then absorbed and removed by the absorbent pad 6 located at the top of the membrane.

The membrane used in the present invention is a nucleic acid complex can move on the surface by a capillary phenomenon caused by the lateral flow of the fluid, it is possible to easily fix the desired binding material to the surface, for example with a reaction pad Nitrocellulose-based membranes and polyvinyl ester-based synthetic polymers are used as sample pads.

The present invention utilizes chromogenic particles and membrane chromatographic techniques that can be easily observed with the naked eye, and the result of the analysis can be easily confirmed within a short time, and only nucleic acid primers and probes corresponding to the nucleic acid sequences to be analyzed in the same system are used. By replacing them, the nucleic acid sequence can be analyzed easily and quickly.

Hereinafter, the present invention will be described in more detail with examples. However, the following examples are provided to illustrate the process of carrying out the method of the present invention specifically, it should not be understood that the present invention is limited only to these examples.

Example 1

Nucleic acid detection test for Mycobacterium tuberculosis nucleic acid polymerization reaction product

Purely cultured Mycobacterium Tuberculosis was washed several times with distilled water, heat-denatured at 95 ° C. for 10 minutes to extract nucleic acid, which was used as a template, and the following two PCR primers (genset oligo, USA), TBP1 and Nucleic acid polymerization was carried out by conventional procedures using TBP2 as described below:

TBP1 5 'gAT gCA CCg TCg AAC ggC TgA T

TBP2 5 'TCg CTg AAC Cgg ATC gAT gTg T

Specifically, 10 μl of the extracted DNA, 1 μl of primer mix (10 pmol per primer), 0.5 μl of dNTP mix (10 mM), 3 μl of reaction buffer, 0.2 μl of Taq polymerase (5 units / μl), and 15.3 of distilled water 30 μl of the PCR reaction solution was prepared by adding μl, and placed in a nucleic acid polymerization vessel, and subjected to a pre-denature process for 3 minutes at 95 ° C., followed by denature and 70 at 95 ° C. for 40 seconds. A total of 40 amplification processes (Annealing-Extension) for 1 minute at ℃ and carried out a post-extension process for 5 minutes at 72 ℃ to perform the polymerization of Mycobacterium tuberculosis nucleic acid. The polymerization reaction result of Mycobacterium tuberculosis nucleic acid can be confirmed through the electrophoretic photograph of FIG. 3.

In addition, biotin and fluorescein were attached to two probes (genset oligo, USA) capable of binding different portions of Mycobacterium tuberculosis nucleic acid, respectively, to prepare two nucleic acid probes labeled as follows, TBP3 and TBP4. :

TBP3 5 'B-gCC CgC Agg ACC ACg ATC gC (B = biotin labeling)

TBP4 5 'F-gCC gAg gAC CAT gga ggT gg (F = fluorescein label)

20 pL of the resulting nucleic acid polymerization reaction mixture was mixed with 30 pmol of each of the two labeled nucleic acid probes, and thermally denatured at 95 ° C. for 3 minutes using a polymerization reaction equipment (Termal cycler, Takara, Japan). After the reaction, nucleic acid-probe hybridization was performed at 65 ° C for 3 minutes.

The nucleic acid sample containing the obtained nucleic acid-probe complex was added to the sample pad 1, and the membrane strip 20 was formed of the colloid pad 2, the reaction pad 3, and the absorption pad 6 by the capillary phenomenon. ) To move on.

The surface of the colloidal pad 2 includes gold colloidal particles (Sigma, USA) having a particle size of 30 nm in which avidin (Sigma, USA) is immobilized on the surface as a corresponding substance for biotin, which is one of the labels. As the nucleic acid sample injected by 1) passes through the colloid pad 2, the nucleic acid-probe complex in the sample is combined with the gold colloidal particles to form the nucleic acid-probe-gold colloid complex.

The sample component is then passed through a read site 4 in the reaction pad 3 immobilized on the surface in the form of a band as an anti-fluorescein antibody (Fitzgerald, USA) as a counterpart to the remaining label fluorescein. The nucleic acid-probe-gold colloid complex in the sample is captured by forming a specific binding with the anti fluorescein antibody in the reading site (4) and concentrated in the form of a band on the membrane, and the remaining components with the flow of the fluid. It continues to move and is absorbed and removed by the absorption pad 6.

As a result, the complex concentrated at the reading site 4 could be observed as a clear red band that can be visually distinguished as shown in FIG.

From these results, it was confirmed that Mycobacterium tuberculosis nucleic acid to be analyzed exists in the nucleic acid sample, and thus, a large amount of the desired nucleic acid was produced by the nucleic acid polymerization reaction.

On the top of the readout site 4, a control site 5 in which a biotin-HRP conjugate (Biotin-HRP conjugate) was immobilized was prepared to check the fluid flow and reactivity of the sample component in the membrane chromatographic process.

Figure 2 is a schematic diagram illustrating the nucleic acid detection process for the result of the Mycobacterium tuberculosis nucleic acid polymerization reaction of the present embodiment.

Example 2

Nucleic Acid Detection Tests for Human Papilloma Virus (HPV) Nucleic Acid Polymerization Outputs

SiHa cell lines (HPVtype 16, KCLB 30035, Human squamous carcinoma, cervix) and HeLa cell lines (HPV type 18, KCLB 10002, Human epithelial carcinoma, cervix), which are known to contain nucleic acids of human papillomavirus, were purchased from the Korean Cell Line Bank. Several times, and heat-denatured at 95 ° C. for 10 minutes to extract nucleic acids, which were described below using the following two PCR primers (genset oligo, USA), GP5 and GP6, which were used as templates and had biotin attached as a label. The nucleic acid polymerization reaction was carried out by conventional procedures as follows:

GP5 5 'B-TTT gTT ACT gTg gTA gAT ACT AC (B = biotin labeling)

GP6 5 'CTT ATA CTA AAT gTC AAA TAA AAA g

Specifically, 10 μl of the extracted DNA, 1 μl of primer mix (10 pmol per primer), 0.5 μl of dNTP mix (10 mM), 3 μl of reaction buffer, 0.2 μl of Taq polymerase (5 units / μl), and 15.3 of distilled water 30 μl of the PCR reaction solution was prepared by adding μl, and placed in a nucleic acid polymerization vessel, and then subjected to a pre-denature process for 3 minutes at 95 ° C., followed by denature, 55 ° C. at 95 ° C. for 55 seconds. 40 cycles of annealing for 40 seconds at 40 ° C. and 40 seconds for extension at 72 ° C. followed by 3 minutes of post-extension at 72 ° C. for the polymerization of HPV nucleic acid. The reaction was carried out. The result of polymerization of the human papilloma virus nucleic acid can be confirmed through the electrophoretic photograph of FIG. 6.

In addition, digoxigenin and fluorescein were attached to two probes (genset oligo, USA) capable of binding to type 16 and type 18 of human papilloma virus nucleic acids, respectively, as described below. And PVP18 were produced:

PVP16 5'D-gTC ATT ATg TgC TgC CAT ATC TAC TTC AGA (D = digoxigenin label)

PVP18 5'F-TgC TTC TAC ACA gTC TCC TgT ACC Tgg gCA (F = fluorescein label)

20 pL of the resulting nucleic acid polymerization reaction mixture was mixed with 30 pmol of two labeled nucleic acid probes, which were then thermally denatured at 95 ° C. for 3 minutes using equipment for polymerization reaction, and then 3 minutes at 60 ° C. Nucleic acid-probe hybridization was performed.

The nucleic acid sample containing the obtained nucleic acid-probe complex was added to the sample pad 1 as in Example 1, and the sample was collected by colloidal pad 2, reaction pad 3 and absorption pad 6 by capillary action. ) To move on a membrane strip (20).

The colloidal pad 2 contains gold colloidal particles in which avidin is immobilized on the surface of the colloidal pad as a corresponding substance for biotin labeled in the primer, and the nucleic acid sample injected by the sample pad 1 is colloided. In the course of passing through the pad 2, the nucleic acid-probe complex in the sample is combined with the gold colloidal particles to form the nucleic acid-probe-gold colloidal complex.

Subsequently, the sample components are anti-digoxigenin antibodies (Roche Molecular Biochemicals, Germany) and anti-fluorescein antibodies (Fitzgerald, USA) at different intervals as counterparts for the remaining labels digoxigenin and fluorescein. Passed through the read site 4 in the reaction pad 3 immobilized on the surface in the form of a band, and the nucleic acid-probe-gold colloid complex in the sample was subjected to anti-digoxigenin antibody and anti fluorescein in the read site 4. By specific binding with the antibody, it is captured and concentrated in the form of a band on the membrane, and the remaining components continue to move with the flow of fluid and are absorbed and removed by the absorbent pad 6.

As a result, the complex concentrated at the reading site 4 could be observed as two clear red bands that can be visually distinguished as shown in FIG.

From these results, it was confirmed that both kinds of human papillomavirus nucleic acids to be analyzed exist in the nucleic acid sample, and therefore, the two kinds of human papillomavirus nucleic acids, type 16 and type 18, were prepared in a large amount by the nucleic acid polymerization reaction. .

As in Example 1, a control site 5 having a biotin-HRP conjugate (HRP) fixed thereon is prepared on the top of the readout site 4 to check the fluid flow and reactivity of the sample component in the membrane chromatographic process. It was made.

5 is a schematic diagram illustrating the nucleic acid detection process for the result of the HPV nucleic acid polymerization reaction of the present embodiment.

The nucleic acid detection method of the present invention can accurately and easily analyze specific nucleic acids in a sample in a short time, compared to conventional nucleic acid analysis methods such as microwell plate nucleic acid hybridization or membrane nucleic acid hybridization.

In addition, using the nucleic acid analysis kit of the present invention, anyone can easily confirm the analysis result with the naked eye, without complicated separate analysis process or equipment, unlike in the conventional analysis method, and it is possible to perform the nucleic acid analysis test more easily. Since it is possible to detect different kinds of nucleic acids at the same time, a wide range of gene-related industries and research fields that require nucleic acid analysis tests such as amplification confirmation tests of nucleic acids by nucleic acid polymerization reactions or detection tests of specific nucleic acids in samples extracted from bacteria or cells. It is expected to be useful in.

Claims (21)

(1) two or more probes consisting of a nucleic acid sequence capable of specifically binding to a portion of the nucleic acid (s) to be analyzed in the sample and having a label attached thereto, wherein Hybridizing the labels with different kinds of substances distinguishable by analysis), (2) allowing the obtained nucleic acid-probe complex to bind to the chromogenic colloidal particles immobilized on the surface of a complementary material which can specifically bind only to one of the labels used in step (1). , (3) thereafter, the nucleic acid sample is moved by chromatographic techniques on the membrane fixed in the form of a band at a predetermined interval on the surface, which can bind specifically to each of the remaining labels. Allowing the nucleic acid-probe-colloid complex obtained as a result of step (2) in the sample to bind to the complementary material of the membrane; and (4) detecting the desired nucleic acid in the sample, characterized in that it comprises the step of forming a color band on the membrane. The method of claim 1, The chromogenic colloidal particles are metal colloidal particles or plastic colloidal particles. The method of claim 2, The chromogenic colloidal particles are gold colloidal particles. The method according to any one of claims 1 to 3, Wherein said labels are selected from the population of nucleic acid labels consisting of biotin, fluorescein and digoxigenin. The method of claim 4, wherein If the label is biotin, characterized in that avidin is used as a complementary substance that can specifically bind thereto. The method of claim 4, wherein If the label is fluorescein, characterized in that an anti-fluorescein antibody is used as a complementary substance that can specifically bind thereto. The method of claim 4, wherein In the case where the label is digoxigenin, an anti-digoxigenin antibody is used as a complementary substance that can specifically bind thereto. (1) A nucleic acid amplification sample produced by a nucleic acid polymerization reaction using a labeled primer is composed of a nucleic acid sequence capable of specifically binding to a portion of the nucleic acid (s) to be analyzed, and the label is attached. Hybridizing with two or more probes, wherein the primers and the labels attached to the probes are different kinds of materials distinguishable by analysis, (2) allowing the obtained nucleic acid-probe complex to bind to the chromogenic colloidal particles immobilized on the surface thereof, with a complementary substance capable of specifically binding only to a label attached to the primer; (3) thereafter, the nucleic acid sample is moved by chromatographic techniques on the membrane fixed in the form of a band at a predetermined interval on the surface, which can bind specifically to each of the remaining labels. Allowing the nucleic acid-probe-colloid complex obtained as a result of step (2) in the sample to bind to the complementary material of the membrane; and (4) detecting a desired amplified nucleic acid from the result of the nucleic acid polymerization reaction, characterized in that it comprises the step of forming a color band on the membrane. The method of claim 8, The chromogenic colloidal particles are metal colloidal particles or plastic colloidal particles. The method of claim 9, The chromogenic colloidal particles are gold colloidal particles. The method according to any one of claims 8 to 10, And the labels attached to the primers and probes are selected from a population of nucleic acid labels consisting of biotin, fluorescein and digoxigenin. The method of claim 11, If the label is biotin, characterized in that avidin is used as a complementary substance that can specifically bind thereto. The method of claim 11, If the label is fluorescein, characterized in that an anti-fluorescein antibody is used as a complementary substance that can specifically bind thereto. The method of claim 11, In the case where the label is digoxigenin, an anti-digoxigenin antibody is used as a complementary substance that can specifically bind thereto. A reaction vessel 10 comprising two or more probes to which a nucleic acid sample and a label are attached; And A sample pad (1) for sample addition, a colloid pad (2) containing chromogenic colloid particles having a complementary substance specifically bound to only one of the above labels, immobilized on the surface, specific for the remaining labels Reaction pads (3) comprising a reading site (4) fixed in the form of bands at regular intervals to which the binding complementary material (s) are bonded and absorbed to remove unreacted sample components. Kit for nucleic acid analysis, characterized in that consisting of a membrane strip (20) consisting of a pad (6). The method of claim 15, The reaction vessel 10 and the membrane strip 20 are installed in the same system or in separate systems, and the sample pad 1, the colloid pad 2, the reaction pad ( 3) and the absorbent pad (6) are nucleic acid analysis kits, characterized in that each of the samples are arranged so as to overlap in part so as to be continuously moved by the capillary phenomenon. The method of claim 16, Kit for nucleic acid analysis, characterized in that the chromogenic colloidal particles are gold colloidal particles. The method according to any one of claims 15 to 17, The nucleic acid analysis kit, characterized in that the label attached to the probe is selected from the group of nucleic acid label consisting of biotin, fluorescein and digoxigenin. The method of claim 18, If the label is biotin, nucleic acid analysis kit, characterized in that using avidin as a complementary material that can specifically bind thereto. The method of claim 18, When the label is fluorescein, nucleic acid analysis kit, characterized in that to use an anti-fluorescein antibody as a complementary substance that can specifically bind thereto. The method of claim 18, When the label is digoxigenin, a nucleic acid analysis kit comprising an anti-digoxigenin antibody as a complementary substance that can specifically bind thereto.