WO2020218772A1

WO2020218772A1 - Hpv detection kit and hpv detection method using same

Info

Publication number: WO2020218772A1
Application number: PCT/KR2020/005059
Authority: WO
Inventors: 박영석; 이찬효; 황병오; 장시운
Original assignee: 주식회사 팍스젠바이오
Priority date: 2019-04-24
Filing date: 2020-04-16
Publication date: 2020-10-29
Also published as: KR102239254B1; KR20200124441A

Abstract

The present invention relates to a Human Papillomavirus (HPV) detection kit and an HPV detection method using same, and, particularly, relates to: a kit for detecting HPV, comprising (a) a primer, which comprises a binding site complementary binding to an HPV-specific gene and a nucleic acid oligomer that is non-complementary to an HPV-specific gene and (b) a probe capable of identifying the amplified product; and an HPV detection method and an HPV infection diagnosis method which use same. The present invention can diagnose HPV infection by using only a small amount of target genes, and amplifies and diagnoses a high-risk genotype HPV-specific gene, and thus can rapidly and accurately enable the qualitative and molecular diagnosis of HPV.

Description

HPV detection kit and HPV detection method using the same

The present invention relates to a kit for detecting HPV and a method for detecting HPV using the same, and specifically, (a) contains a complementary binding site that binds to a HPV (Human Papillomavirus)-specific gene and a non-complementary nucleic acid oligomer with an HPV-specific gene. The present invention relates to a kit for detecting HPV including a primer, and (b) a probe capable of identifying the amplified product, a method of detecting HPV using the same, and a method of diagnosing HPV infection.

HPV (human papillomavirus) is one of the most commonly transmitted sexually transmitted infectious diseases. In addition, HPV causes a variety of diseases, from benign diseases such as skin warts to cervical cancer. Cervical cancer worldwide accounts for 15% of female cancers, and in Korea, it is the 6th among female cancers, still showing a high incidence of 10.6%, and the resulting mortality rate reaches 60%.

HPV cannot be grown in tissue culture or experimental animals, but recently, it is possible to identify and identify various subtypes of HPV using PCR as a molecular biological method. The PCR method is fast and highly sensitive, so it can detect even trace amounts of HPV, but it has to go through an electrophoresis process, showing problems in safety and speed. As an alternative, real-time PCR, which is currently most actively developed, requires expensive equipment, which has a disadvantage of low economic efficiency.

Worldwide, the infection rate of HPV, which causes cervical cancer, reaches 11.4%, and the main pathogen is HPV, and 118 types of HPVs are known to date, of which about 10 are classified as high-risk genotypes that cause cancer. Until now, persistent infection of the high-risk genotype HPV is known as the greatest risk factor.

The Pap smear test, which is widely used as a screening test for cervical cancer, is an inexpensive and effective method that has greatly contributed to the reduction of the prevalence and mortality of cervical cancer. However, the Pap smear test has an unavoidable false negative rate due to its own limitations, and also has a blind spot in which it cannot diagnose HPV infection, which is the most important factor in the occurrence of cancer.

When Pap smear and HPV genotyping were performed at the same time, the importance of HPV test was recognized due to the results of the study showing that the predictive value of cervical cancer detection was higher than that of Pap smear or HPV genotyping alone.

Diagnosis tests for human papillomavirus infection using nucleic acid amplification technology are currently provided by Roche and QIAGEN, and as domestic companies, Bioneer Co., Ltd., Panazine Co., and Seegene Co., Ltd. are provided among domestic companies. Are doing.

However, since the kit used for the test uses a fluorescent material, it is expensive, and requires an expensive reaction facility for temperature control and an expensive fluorescence detector for analysis, so there is a great economic limitation for its use. The real-time PCR method requires expensive equipment, and the economic feasibility is greatly reduced, which is a big obstacle to improving public health.

Under this technical background, the inventors of the present application can quickly and accurately and qualitatively diagnose HPV infection by detecting a high-risk genotype HPV-specific gene, and a kit capable of diagnosing HPV infection with only a small amount of sample genes. It was confirmed that a detection method and a diagnostic method for HPV infection using the same can be provided, and the present invention was completed.

The information described in the background section is only for improving the understanding of the background of the present invention, and thus does not include information on the prior art known to those of ordinary skill in the art to which the present invention belongs. I can.

발명의 요약Summary of the invention

It is an object of the present invention to recognize the problems of the prior art mentioned above, and to diagnose HPV infection using the kit for detecting HPV, a method for detecting HPV infection, and a method for diagnosing HPV infection. It is to provide information provision method and diagnosis method.

In order to achieve the above object, the present invention includes (a) a primer containing a complementary binding site that binds to a HPV (Human Papillomavirus) specific gene and a non-complementary nucleic acid oligomer with an HPV specific gene; (b) It provides a kit for detecting HPV comprising a membrane to which a probe complementarily binding to the nucleic acid oligomer is immobilized.

The present invention also provides a step of amplifying a nucleic acid extracted from a specimen with a primer containing a complementary binding site that binds to a human papillomavirus (HPV)-specific gene and a nucleic acid oligomer that is non-complementary with an HPV-specific gene; And it provides a method for detecting HPV in vitro using the kit, comprising the step of loading the amplification product onto a membrane on which a probe complementarily binding to the nucleic acid oligomer is immobilized.

The present invention further provides a step of amplifying a nucleic acid extracted from a specimen with a primer containing a complementary binding site that binds to a human papillomavirus (HPV)-specific gene and a nucleic acid oligomer that is non-complementary with an HPV-specific gene; And loading the amplification product onto a membrane on which a probe complementarily binding to the nucleic acid oligomer is immobilized. It provides an information providing method and a diagnostic method for diagnosis of HPV infection.

1 is a diagram illustrating a process of manufacturing a primer and a probe included in the kit according to an embodiment of the present invention.

2 is a schematic diagram of a side flow assay (ULFA) reaction for confirming an amplification product in a kit according to an embodiment of the present invention.

3 shows the results of amplifying and detecting HPV genotypes using a kit according to an embodiment of the present invention.

4 is a diagram illustrating a position of a probe fixed to a kit according to an embodiment of the present invention and an HPV type applied thereto.

5 is a diagram illustrating a test result according to a negative or positive determination criterion for HPV through a kit according to an embodiment of the present invention.

발명의 상세한 설명 및 바람직한 구현예Detailed description and preferred embodiments of the invention

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

The inventors of the present application include HPV16, 18, 31, 33, 35, 39, 45, 52, 58,59 and HPV 6, 11, 51, 53, 56, 66, 68, 69, 70, 73 genotype-specific genes and HPV PaxView HPV20 Genotyping MPCR-ULFA Kit comprising a primer containing a complementary binding site to bind and an HPV-specific gene and a non-complementary nucleic acid oligomer, and a membrane to which a probe complementarily binding to the nucleic acid oligomer is immobilized. . These kits are molecular diagnostic kits that quickly and accurately qualitatively identify the genotype of HPV, amplifying the gene using a gene extracted from a human cervical smear as a template, and developing nanogold particles in the ULFA to appear in the form of a band. Then, HPV16, 18, 31, 33, 35, 39, 45, 52, 58,59 and 6, 11, 51, 53, 56, 66, 68, 69, 70, 73 genotypes can be identified to diagnose infection. Confirmed that there is.

Accordingly, in one aspect, the present invention includes: (a) a primer containing a complementary binding site that binds to a HPV (Human Papillomavirus) specific gene and a nucleic acid oligomer that is non-complementary with an HPV specific gene; (b) It relates to a kit for detecting HPV comprising a membrane to which a probe complementarily binding to the nucleic acid oligomer is immobilized.

The kit according to the present invention can occupy the market and provide a platform for diagnosing new HPV-related diseases in a way that complements the disadvantages of other companies' HPV20 genotyping kits and accepts the advantages as much as possible.

Among the molecular diagnostic products, products that use polymerase chain reaction (PCR) can be classified into two broad categories. One is conventional PCR, which extracts a nucleic acid from a sample, amplifies the target nucleic acid, and then confirms the result through electrophoresis. Using this method, you can buy a kit at an inexpensive price, but electrophoresis takes a lot of labor and time. Real-time PCR, which is used instead of electrophoresis, can monitor the amplification of nucleic acids of pathogens in real time, but the cost of the kit is expensive.

Accordingly, while maintaining user convenience, as the development of inexpensive molecular diagnostic products is required, the inventors of the present application have developed an in vitro diagnostic kit for qualitative analysis.

The kit according to the present invention comprises a primer comprising a complementary binding site and a nucleic acid oligomer that binds to an HPV-specific gene, and the primers are HPV genotypes 16, 18, 31, 33, 35, 39, 45, 52, 58 , 59, 6, 11, 51, 53, 56, 66, 68, 69, 70, and 73 include a nucleic acid oligomer containing a gene according to HPV and a non-complementary base thereto.

In one embodiment, the HPV-specific gene is HPV genotype 16, 18, 31, 33, 35, 39, 45, 52, 58, 59, 6, 11, 51, 53, 56, 66, 68, 69, With one or more selected from the group consisting of 70 and 73, a primer including a binding site complementary to a plurality of HPV genotypes may be used in a plurality of panels of the kit. For example, the kit may include two panels, and panel 1 contains primers for amplifying 10 types of nucleic acids of HPV16, 18, 31, 33, 35, 39, 45, 52, 58, 59. It may include, and the second panel may include primers for amplifying 10 kinds of nucleic acids of HPV6, 11, 51, 53, 56, 59, 66, 68, 69, 70, 73. Through this, HPV genotypes 16, 18, 31, 33, 35, 39, 45, 52, 58, 59 and 6, 11, 51, 53, 56, 66, 68, 69, 70, 73 can be simultaneously detected. .

The HPV-specific gene may be one or more or two or more genes specifically present in HPV, for example, may be selected from the group consisting of L1, E6 and E7, and L1 and/or E6 and E7 genes Can be

Accordingly, the primer includes a complementary binding site that specifically binds to the L1 and/or E6 and E7 genes, which are specific genes of each genotype of HPV, and is non-complementary to the genes present in each genotype of HPV. It may contain a nucleic acid oligomer containing a base. According to the present invention, it is possible to simultaneously identify and diagnose whether or not 20 genotypes of HPV are infected.

In the present specification,'primer' refers to a single-stranded oligonucleotide that can serve as an initiation point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerases) in a suitable temperature and buffer. Means.

The primer according to the present invention may include a fragment of a specific gene of HPV of 20 genotypes. The fragment may mean any site of a specific gene of HPV of 20 genotypes, and the fragment may have any length, for example, the L1 and/or E6 and E7 genes of HPV, or any of these. It may mean a site having a length of 5 bp to 50 bp, specifically 10 bp to 30 bp for the site of.

In the present specification, "complementary binding" means that a primer is hybridized with a corresponding nucleic acid strand under a polymerization reaction, and a duplex structure may be formed. Complementary bonds may be formed even when complementarity between nucleotide sequences forming a pair constitutes a Watson-Crick bond, or some non-Watson-Crick bonds (Non-Watson-Crick base pair) exist.

The primer is a forward primer, and may include, for example, a complementary binding site that specifically binds to the L1 and/or E6 and E7 genes, which are specific genes of HPV of 20 genotypes. When the forward L1 and/or E6 and E7 genes are used as a template, a sequence complementary to the L1 and/or E6 and E7 genes may be specifically amplified by binding to a complementary strand thereto.

The primer includes a nucleic acid oligomer containing an HPV-specific gene or a fragment thereof and a non-complementary base. When amplifying the target genes L1 and/or E6 and E7 genes using PCR, it is related to the nucleic acid oligomer containing the L1 and/or E6 and E7 genes and a non-complementary base, that is, the L1 and/or E6 and E7 genes. The missing gene may include about 20-60 bp, specifically 20-40 bp, preferably about 20-30 bp. By reacting such a nucleic acid oligomer with a probe synthesized complementarily on a membrane, the result, that is, whether a nucleic acid is amplified can be easily confirmed.

For example, the primer may be selected from the group consisting of SEQ ID NOs: 1 to 10 and SEQ ID NOs: 21 to 30. In the case of using a primer including a complementary binding site to a plurality of HPV genotypes in a plurality of panels of the kit, the kit may include two panels, and panel 1 includes the primers of SEQ ID NOs: 1 to 10. Can be, and panel 2 may include the primers of SEQ ID NOs: 21 to 30.

In order to react with the probe synthesized complementarily on the membrane, such a nucleic acid oligomer is used to bind with a probe synthesized complementarily with a non-complementary base portion and a non-complementary base portion of the 20 genotype HPV in order to react with the probe. It may further include a non-nucleotide spacer between bases. The non-nucleotide spacer may be a C3, C6, C9 or C12 aliphatic spacer, and the number of C refers to the number of carbon atoms in the non-nucleotide spacer structure. Such non-nucleotide spacers may be alkyl, akenyl, or akynyl groups.

In one embodiment, a non-nucleotide spacer, such as a C3 spacer (propyl spacer), between the HPV-specific gene or fragment thereof and a base for binding to a non-complementary base moiety and a probe synthesized complementarily on the membrane When the gene is amplified using Taq polymerase by positioning the gene, the complementary amplification does not invade the base portion for binding to the probe and is not amplified, so that a partial single-stranded nucleic acid product can be secured. To be able to bind to the probe.

The primer may further include a primer including a marker. The primer containing the marker may be a reverse primer, and the reverse primer may bind to the template strand when using the L1 and/or E6 and E7 genes as a template to amplify the template. For example, the primers are SEQ ID NOs: 11 to It may be selected from the group consisting of 20 and SEQ ID NOs: 31 to 40. In the case of using a primer containing a complementary binding site to a plurality of HPV genotypes in a plurality of panels of the kit, the kit may include two panels, and panel 1 includes the primers of SEQ ID NOs: 11 to 20. Can be, and panel 2 may include the primers of SEQ ID NOs: 31 to 40.

In one embodiment, the marker included in the primer is biotin, Cy5, Cy3, FITC, EDANS (5-(2'-aminoethyl) amino-1-naphthalene sulfate), tetramethylrhodamine (TMR), tetramethyl It may be rhodamine isocyanate (TMRITC), x-rhodamine, DIG, or an antibody or nanoparticles bound thereto, but is not limited thereto.

Here, by reacting with a binding agent that induces color development of the marker, it is possible to check whether color development or fluorescence is expressed, and to detect amplification of the target nucleic acid. In this case, the binding agent may be streptavidin, but is not limited thereto.

In a specific example, biotin is attached to the reverse primer and amplified, thereby binding to streptavidine in the membrane, and particles conjugated to biotin, for example, gold particles according to the tendency of nanoparticles. In this case, it is possible to confirm by color development, and in the case of fluorescence, by fluorescence in various wavelength bands, it is possible to distinguish between several to dozens of target nucleic acids according to the type of probe attached to the membrane.

The kit according to the present invention includes (b) a membrane to which a probe complementarily binding to the nucleic acid oligomer is immobilized. Through this, when the product amplified by the primer is injected into the side of the membrane, the amplified product moves, and the probe fixed to the membrane and the nucleic acid oligomer included in the amplified product may undergo a complementary hybridization reaction.

The "membrane" may be made of any of a variety of materials. Naturally occurring materials, such as polysaccharides (eg, cellulosic materials, paper, cellulose acetates, and cellulose derivatives such as nitrocellulose), eg, natural, synthetic, or synthetically modified; Polyether sulfone; Polyethylene; nylon; Polyvinylidene fluoride (PVDF); Polyester; Polypropylene; Silica; Inorganic materials uniformly dispersed in a porous polymer matrix with polymers such as vinyl chloride, vinyl chloride-propylene copolymer and vinyl chloride-vinyl acetate copolymer, e.g. inactivated alumina, diatomaceous earth, MgSO4, or other inorganic finely divided materials ; Naturally occurring (eg cotton) and synthetic (eg nylon or rayon) fabrics; Porous gels such as silica gel, agarose, dextran and gelatin; Polymeric films such as polyacrylamide; It can be formed from materials such as. Preferably the membrane may comprise a polymeric material such as nitrocellulose, polyethersulfone, polyethylene, nylon, polyvinylidene fluoride, polyester and polypropylene.

The primers according to the present invention can be used for multiplex-PCR, and it is possible to diagnose infection by simultaneously detecting 20 genotypes of HPV. That is, if multiple PCs are performed with probes with several to dozens of additionally different oligomer sequences, and then a probe capable of complementarily reacting with it is used to react with the bound oligomer probe, one With the lateral flow membrane, several to dozens of amplified products can be identified.

Therefore, it can be used jointly with a variety of multiple PCs, enabling mass production in the production of side-flow membranes. This is to check the results of various items using one side flow membrane, and because the amplification products can be identified using the same membrane regardless of the type of PCR amplification products, cost reduction and quality control of product production are easy. To increase the productivity of the product.

A probe that complementarily binds to the nucleic acid oligomer may include, for example, one or more sequences selected from the group consisting of SEQ ID NOs: 44 to 55.

In some cases, the probe may further include a nucleic acid oligomer for immobilization on the membrane, and may further include an oligomer of 20-60 bp, specifically 20-40 bp, having a repeating nucleotide sequence.

Optionally, the kit according to the present invention may optionally further comprise an internal control primer. These internal control primers are used to check whether a false negative problem, that is, whether the PCR reaction has been properly performed, and genes that are normally expressed can be arbitrarily selected regardless of the presence or absence of HPV in the sample.

The kit according to the present invention may optionally contain reagents necessary for carrying out a nucleic acid amplification PCR reaction such as polymerase, buffer, and deoxyribonucleotide-5-triphosphate. The kit according to the present invention may also further comprise various polynucleotide molecules, various buffers and reagents.

The optimal amount of reagents, buffers or reactants used for a specific reaction in the kit can be determined by a person skilled in the art, and contains a complementary binding site that specifically binds to the aforementioned HPV-specific gene and a non-complementary base thereto. A primer (forward) containing a nucleic acid oligomer and/or (ii) a labeled primer containing a marker (reverse direction), and the membranes on which the probe is immobilized are prepared in separate packaging or compartments each containing Can be.

In another aspect, the present invention comprises the steps of amplifying a nucleic acid extracted from a specimen with a primer containing a complementary binding site that binds to a human papillomavirus (HPV)-specific gene and a nucleic acid oligomer that is non-complementary with an HPV-specific gene; And loading the amplification product onto a membrane on which a probe complementarily binding to the nucleic acid oligomer is immobilized. The method relates to a method of detecting HPV in vitro using the kit.

In another aspect, the present invention comprises the steps of amplifying a nucleic acid extracted from a specimen with a primer containing a complementary binding site that binds to a human papillomavirus (HPV)-specific gene and a nucleic acid oligomer that is non-complementary with an HPV-specific gene; And loading the amplification product onto a membrane on which a probe complementarily binding to the nucleic acid oligomer is immobilized. The method relates to a method for providing information for diagnosis of HPV infection.

In another aspect, the present invention comprises the steps of amplifying a nucleic acid extracted from a specimen with a primer containing a complementary binding site that binds to a human papillomavirus (HPV)-specific gene and a nucleic acid oligomer that is non-complementary with an HPV-specific gene; And loading the amplification product onto a membrane on which a probe complementarily binding to the nucleic acid oligomer is immobilized, using the kit to diagnose HPV infection.

The description of the configuration related to the kit used in performing the method according to the present invention can be equally applied to the method.

In one embodiment, the specimen may contain a wide range of all biological body fluids obtained from body fluids, cell lines, tissue culture, etc. derived from a patient suspected of having HPV or an individual or individual to be diagnosed, for example, cervical and vaginal swabs. , Cervical tissue, male genital tissue, urine, anus, rectum, pharynx, oral cavity, and head and neck, but are not limited thereto.

It may further include the step of extracting the nucleic acid from the specimen, and extraction of the nucleic acid may be performed using, for example, various kits or extraction reagents commercially supplied.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

1. Making the kit

The kit according to the present invention (hereinafter, also referred to as PaxView HPV 20 Genotyping MPCR-ULFA Kit) includes two kits. One is a kit for amplifying nucleic acids (hereinafter, Component 1. PaxView HPV 20 Genotyping MPCR Kit), and the other is a kit (hereinafter, Component 2. PaxView HPV 20 Genotyping MPCR Kit) that identifies amplification products. The primers included in the primer mixture, which is a component of the PaxView HPV 20 Genotyping MPCR-ULFA Kit, is a forward primer (Forward primer = SN) in addition to the site for 20 types of HPV and the site for internal control composed of two panels according to Table 1. ) Is a universal region, which is a non-complementary nucleic acid oligomer, and a biotin sequence is tagged to a reverse primer (ASN) (Fig. 1).

After PCR, the amplification product is loaded into the ULFA device of the PaxView ULFA Kit, and after binding with the gold-streptavidin conjugate (Gold-streptavidin conjugate=Gold-SV, present on the glass fiber pad), NC ( nitrocellulose), a probe adsorbed on the membrane (probe = oligo complementary to the universal region) is combined to develop color at the labeled site of each pathogen. The principle of color development is based on the reaction method between biotin and streptavidin tagged to each primer, and can be used to confirm DNA reactions in addition to antigen-antibody reactions, which are widely used. Streptavidin, similar to avidin, can form a complex with biotin, resulting in a visually readable color development by gold particles conjugated to biotin (FIG. 2).

After color development, the ULFA device is visually read. It can be confirmed that the predicted band pattern and the actual band pattern are identical because the position of the color line is different according to the result determination described in the method of use of the kit.

The HPV diagnostic kit (HPV20 Genotyping Diagnostic Kit) according to the present invention is a new HPV diagnostic platform. It is a rapid and accurate qualitative molecular diagnostic kit. Amplifies a gene using a gene extracted from a sample as a template, and develops it in ULFA. When the particles appear in the form of bands, it is possible to diagnose infection of 20 types of HPV.

2. HPV diagnosis

(1) Manufacture of Mixture

1) Preparation of PCR master mix: It was prepared as follows.

2) The prepared PCR master mix was dispensed into a PCR tube.

3) DNA extracted from the sample or 5 µl of positive control or negative control was put into the same PCR tube.

(2) Polymerase chain reaction (PCR)

1) Put the PCR tube above into the thermal cycler.

2) The thermal cycler was set up and the equipment was operated under the following conditions.

(3) Deploy to ULFA

1) 5 µl of the amplification product for which the PCR reaction was completed was loaded into the sample inlet of ULFA. One probe can identify and detect two genotypes of HPV. For example, the Universal probe (Uprobe) 06 can identify and detect HPV genotype 16 and HPV genotype 06.

2) 10 μl of Running Buffer was loaded into the sample inlet of the same ULFA, and the PCR amplified product was developed for 10 minutes.

3) 10 µl of Washing Buffer was loaded into the sample inlet of the same ULFA and developed for 10 minutes to clean up the Nitrocellulose membrane.

4) The amplification result was confirmed by the presence or absence of a red line formed in the number indicated on the device (with line: positive, no line: negative).

(4) result judgment

In the ULFA device, the result of the band in which the reaction occurred by binding to the PCR amplification product was visually checked to determine negative and positive (see FIGS. 3 and 5).

The technology applied to the PaxView HPV20 Genotyping MPCR-ULFA Kit is composed of two panels, and the PCR product produced after performing 10 types of multiplex PCR at the same time is detected by ULFA (universal lateral flow assay). Two probes are immobilized, and a primer-specific HPV type having a universal region including a nucleic acid oligomer complementary to each probe can be detected. One set of 12 universal probes (Uprobe) can detect both 20 types of HPV consisting of two panels, internal control and hybridization control.

According to the present invention, it is possible to diagnose HPV infection with only a small amount of sample genes, and by amplifying and diagnosing a high-risk genotype HPV-specific gene, it is possible to quickly and accurately qualitative molecular diagnosis for HPV. In addition, since the result is confirmed through a side flow assay after gene amplification, it is economical because expensive equipment as in real-time PCR is not required, and an agarose gel for electrophoresis to identify amplified genes as in conventional PCR. Since the manufacturing process is not required, results can be confirmed in a shorter time than PCR-electrophoresis.

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Electronic file attached.

Claims

(a) a primer containing a complementary binding site that binds to a HPV (Human Papillomavirus)-specific gene and a nucleic acid oligomer that is non-complementary with an HPV-specific gene;

(b) HPV detection kit comprising a membrane to which a probe complementarily binding to the nucleic acid oligomer is immobilized.
The method of claim 1, wherein the kit comprises HPV genotype 16, 18, 31, 33, 35, 39, 45, 52, 58, 59, 6, 11, 51, 53, 56, 66, 68, 69, 70 and 73. Kit, characterized in that for simultaneously diagnosing at least one HPV selected from the group consisting of.
The kit according to claim 1, wherein the HPV-specific gene is selected from the group consisting of L1, E6 and E7.
The kit according to claim 1, wherein the nucleic acid oligomer has a length of 20-60bp.
The kit according to claim 1, comprising a non-nucleotide spacer between the complementary binding site and a nucleic acid oligomer containing a non-complementary base.
The kit according to claim 1, wherein the primers are selected from the group consisting of SEQ ID NOs: 1 to 10 and SEQ ID NOs: 21 to 30.
The kit according to claim 1, further comprising a primer comprising a marker.
The kit according to claim 7, wherein the primers are selected from the group consisting of SEQ ID NOs: 11 to 20 and SEQ ID NOs: 31 to 40.
The kit according to claim 1, wherein when the product amplified by the primer is injected into the side of the membrane, the amplified product moves, and the probe fixed to the membrane and the nucleic acid oligomer contained in the amplified product undergo a complementary hybridization reaction. .
The kit according to claim 1, wherein the probe complementarily binding to the nucleic acid oligomer comprises one or more sequences selected from the group consisting of SEQ ID NOs: 44 to 55.
The method of claim 8, wherein the marker is biotin, Cy5, Cy3, FITC, EDANS (5-(2'-aminoethyl) amino-1-naphthalene sulfate), tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate ( TMRITC), x-rhodamine, DIG or antibody or a kit, characterized in that the nanoparticles bound thereto.
The kit according to claim 8, wherein color development is induced by the reaction of the marker and the binder.
The kit according to claim 12, wherein the binding agent is streptavidin.
Amplifying the nucleic acid extracted from the body with a primer containing a complementary binding site that binds to a human papillomavirus (HPV)-specific gene and a nucleic acid oligomer that is non-complementary with an HPV-specific gene; And

A method for detecting HPV in vitro using the kit according to any one of claims 1 to 13, comprising loading the amplification product onto a membrane on which a probe complementarily binding to the nucleic acid oligomer is immobilized. .
Amplifying the nucleic acid extracted from the sample with a primer containing a complementary binding site that binds to a human papillomavirus (HPV)-specific gene and a nucleic acid oligomer that is non-complementary with an HPV-specific gene; And

For diagnosis of HPV infection by using the kit according to any one of claims 1 to 13, comprising the step of loading the amplification product onto a membrane on which a probe complementarily binding to the nucleic acid oligomer is immobilized. How to provide information.
Amplifying the nucleic acid extracted from the sample with a primer containing a complementary binding site that binds to a human papillomavirus (HPV)-specific gene and a nucleic acid oligomer that is non-complementary with an HPV-specific gene; And

A method for diagnosing HPV infection using the kit according to any one of claims 1 to 13, comprising loading the amplification product onto a membrane in which a probe complementarily binding to the nucleic acid oligomer is immobilized.