WO2020218772A1 - Kit de détection de vph et procédé de détection de vph l'utilisant - Google Patents

Kit de détection de vph et procédé de détection de vph l'utilisant Download PDF

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WO2020218772A1
WO2020218772A1 PCT/KR2020/005059 KR2020005059W WO2020218772A1 WO 2020218772 A1 WO2020218772 A1 WO 2020218772A1 KR 2020005059 W KR2020005059 W KR 2020005059W WO 2020218772 A1 WO2020218772 A1 WO 2020218772A1
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hpv
nucleic acid
complementary
acid oligomer
kit
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PCT/KR2020/005059
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English (en)
Korean (ko)
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박영석
이찬효
황병오
장시운
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주식회사 팍스젠바이오
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/131—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Definitions

  • 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
  • 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.
  • real-time PCR which is currently most actively developed, requires expensive equipment, which has a disadvantage of low economic efficiency.
  • HPV high-risk genotype
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • HPV Human Papillomavirus
  • 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.
  • HPV human papillomavirus
  • 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.
  • HPV human papillomavirus
  • 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.
  • FIG. 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.
  • ULFA side flow assay
  • FIG. 3 shows the results of amplifying and detecting HPV genotypes using a kit according to an embodiment of the present invention.
  • FIG. 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.
  • FIG. 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.
  • 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. .
  • 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.
  • 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.
  • HPV Human Papillomavirus
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • 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,
  • a primer including a binding site complementary to a plurality of HPV genotypes may be used in a plurality of panels of the kit.
  • 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.
  • 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.
  • 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 selected from the group consisting of L1, E6 and E7, and L1 and/or E6 and E7 genes Can be selected from the group consisting of L1, E6 and E7, and L1 and/or E6 and E7 genes Can be selected from the group consisting of L1, E6 and E7, and L1 and/or E6 and E7 genes Can be selected from the group consisting of L1, E6 and E7, and L1 and/or E6 and E7 genes Can be selected from the group consisting of L1, E6 and E7, and L1 and/or E6 and E7 genes Can be selected from the group consisting of L1, E6 and E7, and L1 and/or E6 and E7 genes Can be selected from the group consisting of L1, E6 and E7, and L1 and/or E6 and
  • 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.
  • '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.
  • 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.
  • 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.
  • 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.
  • the primer may be selected from the group consisting of SEQ ID NOs: 1 to 10 and SEQ ID NOs: 21 to 30.
  • the kit may include two panels, and panel 1 includes the primers of SEQ ID NOs: 1 to 10.
  • panel 2 may include the primers of SEQ ID NOs: 21 to 30.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the binding agent may be streptavidin, but is not limited thereto.
  • 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.
  • particles conjugated to biotin for example, gold particles according to the tendency of nanoparticles.
  • the kit according to the present invention includes (b) a membrane to which a probe complementarily binding to the nucleic acid oligomer is immobilized.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HPV human papillomavirus
  • 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.
  • the kit according to the present invention 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 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).
  • the ULFA device 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un kit de détection de papillomavirus humain (VPH) ainsi qu'un procédé de détection de VPH l'utilisant et, en particulier, (a) une amorce qui comprend une liaison complémentaire de site de liaison à un gène spécifique du VPH et un oligomère d'acide nucléique qui n'est pas complémentaire d'un gène spécifique du VPH et (b) une sonde apte à identifier le produit amplifié ; et un procédé de détection de VPH et un procédé de diagnostic d'infection par le VPH qui utilisent ce dernier. La présente invention peut diagnostiquer une infection par le VPH en utilisant uniquement une petite quantité de gènes cibles et amplifie et diagnostique un gène spécifique du VPH de génotype à haut risque et peut ainsi permettre le diagnostic qualitatif et moléculaire du VPH rapidement et avec précision.
PCT/KR2020/005059 2019-04-24 2020-04-16 Kit de détection de vph et procédé de détection de vph l'utilisant WO2020218772A1 (fr)

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KR1020190047768A KR102239254B1 (ko) 2019-04-24 2019-04-24 Hpv 검출용 키트 및 이를 이용한 hpv 검출방법
KR10-2019-0047768 2019-04-24

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KR20230150077A (ko) * 2022-04-21 2023-10-30 주식회사 팍스젠바이오 성 매개 감염증 유발 병원체 검출용 키트 및 이를 이용한 성 매개 감염증 유발 병원체의 검출방법

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KR20060015668A (ko) * 2006-01-11 2006-02-17 다이아프로브 (주) 멤브레인 측면 흐름 디엔에이 칩을 이용한 인체 유해미생물의 검출 방법과 이를 위한 검사 키트
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