WO2006038753A1 - Sonde de papillomavirus humain, et puce a adn pourvue de cette sonde - Google Patents

Sonde de papillomavirus humain, et puce a adn pourvue de cette sonde Download PDF

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WO2006038753A1
WO2006038753A1 PCT/KR2005/000775 KR2005000775W WO2006038753A1 WO 2006038753 A1 WO2006038753 A1 WO 2006038753A1 KR 2005000775 W KR2005000775 W KR 2005000775W WO 2006038753 A1 WO2006038753 A1 WO 2006038753A1
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hpv
gene
dna
seq
genes
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PCT/KR2005/000775
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English (en)
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Woo-Chul Moon
Myung-Ryurl Oh
Su-Bin Yim
Tae-Han Eum
Eun-Hae Jung
Jung-Eun Ko
Jae-Han Bae
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Goodgene Inc.
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Priority to JP2007534506A priority Critical patent/JP4977612B2/ja
Priority to US11/664,549 priority patent/US7670774B2/en
Priority to EP05789712A priority patent/EP1802756A4/fr
Priority to CN2005800335409A priority patent/CN101035896B/zh
Publication of WO2006038753A1 publication Critical patent/WO2006038753A1/fr

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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
    • C12Q1/708—Specific hybridization probes for papilloma
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • the present invention relates to probes which binds complementarily to the nucleic acid of human papillomavirus (HPV) which is major cause of cervical cancer and which is the most common sexually transmitted disease, a DNA chip or microarray comprising the probe, a diagnosis kit for analyzing genotypes of HPV using them and a method for detecting presence or absence of HPV infection and analyzing genotypes thereof.
  • HPV human papillomavirus
  • HPV Human papillomavirus
  • STD sexually transmitted disease
  • HPV is the most common sexually transmitted disease (STD). It is reported that 50% or more of all adult women is infected with HPV at least one during their whole life.
  • HPV is infected to the human epithelial cell, and induces hyperproliferation. Usually, such hyperproliferation is a benign tumor such as simple skin wart, condyloma accuminata around external genital organ or anus and the like.
  • HPV can be cause of inducing cancer, and actually nearly all cervical cancer, majority of head and neck tumor and numerous anal cancers are induced by HPV (Howley PM. Virology. VoI 2, 1996, 2045-2109; Murinoz N et al. , NEngl JMed, 2003, 348:518-27).
  • HPV can be classified into following two types. One is a type that invades skin; the other is an anogenital type that invades boundary of skin and mucous membrane of external genital organ or anus. Depending on base sequence of genome, namely phylogenic tree or genotype), HPV can be classified specifically into approximately 120 types.
  • High-risk type of HPV includes 22 types of HPV, that is HPV-16, HPV-18, HPV-26, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-53, HPV-56, HPV-57, HPV-58, HPV-59, HPV-61, HPV-67, HPV-68, HPV-70, HPV-73, And HPV-82.
  • HPV-16 the most common high-risk type of HPV is HPV-16, and next is HPV-18, HPV-45, HPV-31, HPV-33, HPV-52 and HPV- 58, although there is a difference in worldwide.
  • Low-risk types of HPV includes approximately 20 types of HPV such as HPV ⁇ 2a, HPV-3, HPV-6, HPV- 10, HPV-Il, HPV-32, HPV-34, HPV-40, HPV-42, HPV-43, HPV-44, HPV-54, HPV- 55, HPV-61, HPV-66, HPV-69, HPV-70, HPV-72, HPV-81, and HPV-CP6108 (Murinoz N et al., NEngl JMed, 2003, 348:518-27).
  • Genome structure of HPV can be divided roughly into early transcription region E (early gene region), late transcription region L (late gene region), and non-expression region LCR (long control region). Genome structure of HPV affects outbreak type, risk and prognosis of HPV greatly. Particularly, E6 and E7 genes of the E region are integrated in the genome of infected cell, maintained and expressed, thereby played an important role to induce cancer. High-risk types of HPV such as HPV-16, HPV-18 and the like react with the most important tumor suppressor genes in human such as p53, E6AP, retinoblastoma (Rb, P105RB), P107, and P130 and the like and inactivate the tumor suppressor genes.
  • the infected cell is transformed to cancer cell due to disorder of cell cycle regulation and apoptosis control mechanism.
  • low- risk types of HPV have low ability to react p53 or Rb tumor suppressor genes and inactivate the tumor suppressor genes, low-risk types of HPV is difficult to induce cervical cancer.
  • Ll the largest gene of HPV genes is Ll. Ll is present in similar preservative base sequence in most HPV types. Ll protein composes primarily of HPV capsid protein, and its antigenecity is the highest (Schneider A. Science 1993; 281:263-5; zur Hausen H. Seain Cancer Biol 1999; 9:405-11).
  • cervical cell transformed by HPV is advanced to, so called, carcinoma in situ via precancerous lesion or dysplasia, cervical intraepithelial neoplasma (CIN), or squamous intraepithelial lesion (SIL).
  • carcinoma in situ invades base layer under the epithelium cell, it becomes so called carcinoma or invasive carcinoma.
  • 90% or more of HPV infected women removes HPV by means of immune system in body naturally.
  • HPV is maintained in 10% of high-risk types of HPV infected women, and it induces cervical cancer.
  • About 8% of precancerous lesions are advanced to carcinoma in situ, and about 20% of carcinoma in situ is developed to cancer.
  • the high-risk types of HPV induce cervical cancer, and the frequency is estimated to about 0.16%. Since the outbreak of cervical cancer is needed so long time period, and is induced by stages, it is possible to treat or prevent cervical cancer by early examining precancerous lesions in the middle stage of the outbreak. That is, it is possible to block carcinogenesis by removing precancerous lesions with preservative medical operation. Recently, the clinical test regarding vaccine against Ll, the major antigen of HPV is in advancing. Also, an attempt to treat cervical precancerous lesions by using vaccines against E6 and E7 of HPV which are major causes inducing cervical cancer is advanced actively.
  • the gene chip (or DNA chip or DNA microarray) was developed in recent by means of the combination of molecular biology and electronics, which can examine from tens to tens of thousands of genes on only one microscopic slide simultaneously.
  • Such DNA chip is a new analytic system, which is applicable to analysis of gene expression, gene diagnosis, gene mutation diagnosis, drug screening and disease screening, and accurate diagnosis of bacteria and virus and the like. Accordingly, development of HPV DNA chip to detect rapidly and accurately high-risk types of HPV related to cervical cancer is attempted in worldwide.
  • Cervical screening is a conventional examination method used to screen primarily cervical cancer and prelesions thereof. Cervical screening is carried out by swabbing or scraping cervical cell with a tool of which a brush is attached to the tip of the tool, for example cotton stick, and then examining cytological type of the cell. Readout of the cervical screening is classified into normal, atypical squamous cell of unknown significance (ASCUS), low-risk type or low-grade squamous intraepithelial lesion (LSIL), high-risk type or high-grade squamous intraepithelial lesion (HSIL), carcinoma in situ or cancer.
  • the cervical screening is also referred to papanicolou smear examination (Pap smear) according to the inventor.
  • Pap smear examination has been used from 1940s and played an important role to reduce significantly mortality due to cervical cancer.
  • Pap smear has disadvantage that false negative rate is high of 30 ⁇ 40%.
  • Such high false negative rate is due to sampling error of sampler or readout error of inspector. Therefore, to avoid sampling error and reduce readout error, liquid based cytology examination, which is also referred to Thin Prep, is attempted in recent. Nevertheless, the false negative rate is still high. Accordingly, in recent ten years an attempt to recognize high-risk types of HPV and predict outbreak risk of cervical cancer by examining presence or absence of HPV infection and genotypes thereof has been accomplished. It is believed that HPV examination has higher screening accuracy than that of Pap smear.
  • HPV examination can diagnose nearly all high-risk types of prelesions, and one HPV examination has higher accuracy than that of two Pap smear or colposcopic examinations.
  • a combination of HPV examination and Pap smear can improve both screening sensitivity and specificity, since the combinational examination can solve the problems of Pap examination.
  • the combinational examination has an advantage that time interval of screening examination can be extended from 1 year (for only Pap smear) to 3-4 years.
  • FDA recommends the combinational examination of Pap smear and HPV examination (Ledger WJ et al., Am J Obstet Gynecol, 2000, 182: 860-5; Wright TC Jr et al., JAMA, 2001, 287:2120-9; Wright TC Jr et al. , New Engl J Med, 2003, 348:6-7; Sherman ME et al., J Natl Cancer Inst, 2003, 95; 46- 52).
  • examination of HPV genes can be divided into two methods. One is a method which examines presence or absence of HPV infection and rough type of HPV. The other is a genotyping method which examines presence or absence of HPV infection and rough type of HPV.
  • PCR based method is carried out by amplifying major virus capsid Ll gene of which its base sequence in genome of external genital organ type HPV is preserved most or E6 and E7 genes using consensus primer and confirming the results by means of electrophoresis and the like.
  • this method can confirm only presence or absence of HPV infection, but cannot examine the genotype or even high-risk type or low-risk type of HPV which is presented.
  • a representative hybridization based method is a hybrid II capture analytic method (Digene Diagnostics, Inc. USA).
  • hybrid capture method is carried our by extracting HPV DNA from specimen with hybrid capture technology, hybridizing the HPV DNA with high-risk HPV probe cocktail and low-risk HPV probe cocktail and diagnosing presence or absence of HPV infection.
  • hybrid capture method has disadvantages that it is not possible to analyzing accurate genotype of HPV and lower sensitivity of examination due to no amplification.
  • Similar method includes a method carrying out by PCR using consensus primer, hybridization with high-risk HPV probe cocktail and observation of enzyme immune response. This method is relatively simple and is useful for recognizing high-risk type of HPV, but cannot recognize low-risk types of HPV and accurate type of HPV (Kornegay JR et al. , / Clin Microbiol, 2001, 39:3530-6).
  • the most standard method which has been used to recognize presence or absence of HPV infection as well as genotypes thereof, is sequencing after PCR.
  • the method is carried out by amplifying the region which base sequence is preserved in Ll gene and E6/E7 genes of external genital organ and base sequence is different depending on each type; and sequencing the base sequence directly or after cloning.
  • the method is the most golden standard test.
  • sequencing method has disadvantage that it can examine only one specimen by one or two examination; a plenty of time and cost are needed; it is labor-intensive. Accordingly, it is difficult to apply the sequencing method to clinical test.
  • cloning is necessary to recognize complex infection of at least one types of HPV, actually such procedures are impossible. Accordingly, following methods are attempted in place of sequencing method.
  • First method is a method carrying out several PCR using genotype specific primer according to each HPV types. This method is to recognize various sizes of PCR products by means of electrophoresis or southern blotting for each HPV genotypes. The method is simple, but labor- intensive and inefficient. Also, the method has disadvantage that it can recognize minority of HPV types.
  • Second method is restriction fragment length polymorphism after PCR (PCR-RFLP).
  • PCR-RFLP restriction fragment length polymorphism after PCR
  • This method is carried out by amplifying Ll gene or E6 and E7 genes with PCR using consensus primer; digesting the PCR products with restriction enzyme; and determining the length of the products by means of electrophoresis.
  • the method is conventional and labor-intensive. Also, the method has disadvantage that it can recognize minority of HPV types (Vermon SD et al. J Clin Microbiol, 2000, 38:651-5).
  • Third method is a reverse hybridization line blot detection method, which is used during recent 6-7 years.
  • the method is carried out by preparing nylon membrane strip which genotype specific oligonucleotide probes for each types of HPV are attached thereon; positioning the consensus primer PCR products of HPV; and determining the types exhibiting the strongest response by hybridization.
  • the method is used in the name of PGMY-Iine blot assay or SFPlO line probe assay and the like (Gravitt PE et al., J Clin Microbiol, 1998, 36:3020-7; van Doom L et al. , / Clin Microbiol, 2002, 40:979-983). It is reported that these methods can recognize 25-27 types of HPV in maximum.
  • oligonucleotide microarray or DNA chip has been developed and used.
  • the DNA chip uses procedures of the reverse hybridization method above, except that microscopic glass slide is used in place of nylon membrane strip (Cho NH et al. Am J Obstet Gynecol, 2003, 188:56-62).
  • the DNA chip can be automated readily compared to reverse hybridization method above.
  • the DNA chip has same disadvantages as those of the reverse hybridization method.
  • the DNA chip can not recognize all genotypes of external genital organ HPV, and can recognize only 22 types; since target region for determination is localized to 50 bases in certain region of Ll gene, PCR amplification can not be carried out efficiently; to recognize gene variants is difficult; and it can not analyze E6 and E7 genes.
  • DNA chip is suitable.
  • no DNA chip has been commercialized which satisfies the conditions above.
  • the present invention is accomplished to solve the problems above, and provide a probe capable of diagnosing genotype of sexual HPV infection with high selectivity and sensitivity automatically and correctly, which is a major cause of cervical cancer and which is one of the commonest reasons of sexually transmitted diseases.
  • An aspect of the present invention provides oligonucleotide microarray chip (DNA chip) for probing sexual HPV or analyzing genotype thereof.
  • Another aspect of the present invention provides all in one kit for probing sexual HPV or analyzing genotype thereof, comprising the probe above, all reagents related to the HPV DNA chip, control specimen and the like.
  • Another aspect of the present invention provides the probe analyzing genotype of HPV and a method for probing sexual HPV or analyzing genotype thereof using oligonucleotide microarray (oligo DNA chip) comprising the probe.
  • oligonucleotide microarray oligo DNA chip
  • a probe for analyzing HPV, DNA, kit and analyzing method of the present invention is accomplished with 9 steps as follows• 1. Preparation of standard and control specimen
  • Oligonucleotide primers for amplifying E6/E7 genes, Ll gene of HPV and human beta-globulin gene were selected and designed, and suitable PCR conditions were established. PCR conditions were established for single PCR, duplex PCR, and triplex PCR respectively. In addition, PCR for E6/E7 genes, Ll gene of HPV and human beta-globulin gene were carried out using the DNA isolated in the step 2 as a template (example 3).
  • oligonucleotide probes capable of analyzing Ll and E6/E6 of external genital organ HPV and human beta-globulin respectively on the DNA chip by hybridization reaction (example 6).
  • Grid was contrived to array or spot the probes designed in the step 5. And then, the probes mixed with suitable buffer were arrayed or spotted on a glass slide for microscope. After appropriate treatments for stabilization and quality control, the slides were stored for future analysis (example 7).
  • HPV E6/E7 and HPV Ll genes and beta-globulin gene were amplified by multiplex PCR using standard specimens produced by various combinations and concentrations of one, two or three clones of each HPV type established in the step 4. Suitable conditions were established by positioning the PCR products on the DNA chip, carrying out hybridization reaction several times, and then analyzing by fluorescent scanner (example 8).
  • the present invention provides diagnosis kit (all in one kit) using the DAN chip, comprising 1) tools for sampling clinical specimens such as cervical swab and the like and reagents for extracting DNA from the specimen, 2) reagents for PCR amplifying E6/E7 genes and Ll gene of HPV, and beta-globulin gene, 3) plasmid DNA clones to be uesd as positive control in amplification of HPV genes, 4) oligo DNA chip for analyzing genotypes of HPV, 5) reaction liquid for hybridization reaction using the DNA chip and washing liquid to be used after the hybridization reaction.
  • FIG. 1 is an electrophoretic photograph of E6 and E7 genes of HPV, obtained by electrophoresis that is carried out on 2% agarose gel.
  • the E6 and E7 genes were obtained by isolating genome DNA from positive control cell line and then amplifying E6 and E7 genes by PCR using the genome DNA as a template (Lane KM): 100 bp DNA size marker, lane 2(N/C): negative control, lane 3 (HPV-16): HPV-16 infected positive control cell line (Caski, ATCC CRL-1550), lane 4 (HPV-18): HPV-18 infected positive control cell line (HeLa, ATCC CCL-2), lane 5 (HPV-35): HPV-35 infected positive control cell line (ATCC 40331)).
  • FIG. 2 is an electrophoretic photograph of Ll gene of HPV obtained by electrophoresis which is carried out on 2% agarose gel.
  • the E6 and E7 genes were obtained by isolating genome DNA from positive control cell line and then amplifying Ll gene by PCR using the genome DNA as a template (Lane KM): 100 bp DNA size marker, lane 2(N/C): negative control, lane 3 (HPV-16): HPV-16 infected positive control cell line (Caski, ATCC CRL- 1550), lane 4 (HPV-18): HPV-18 infected positive control cell line (HeLa, ATCC CCL-2), lane 5 (HPV-35): HPV-35 infected positive control cell line (ATCC 40331)).
  • FIG. 3 is an electrophoretic photograph of E6/E7 genes of HPV and human beta-globulin (HBB) genes obtained by electrophoresis which is carried out on 2% agarose gel.
  • the E6/E7 genes and HBB genes were obtained by isolating genome DNA from positive control cell line and then amplifying the E6/E7 and HBB genes by duplex PCR using the genome DNA as a template (Lane KM): 100 bp DNA size marker, lane 2(N/C): negative control, lane 3 (HPV-16): HPV-16 infected positive control cell line (Caski, ATCC CRL-1550), lane 4 (HPV-18): HPV-18 infected positive control cell line (HeLa, ATCC CCL-2), lane 5 (HPV-35): HPV-35 infected positive control cell line (ATCC 40331)).
  • FIG. 4 is an electrophoretic photograph of Ll gene of HPV and human beta-globulin (HBB) genes obtained by electrophoresis which is carried out on 2% agarose gel.
  • the Ll genes and HBB genes were obtained by isolating genome DNA from positive control cell line and then amplifying the Ll and HBB genes by duplex PCR using the genome DNA as a template (Lane KM)-' 100 bp DNA size marker, lane 2 (HPV-16): HPV-16 infected positive control cell line (Caski, ATCC CRL-1550), lane 3 (HPV-18): HPV-18 infected positive control cell line (HeLa, ATCC CCL-2)).
  • FIG. 5 is an electrophoretic photograph of Ll gene and E6/E7 genes of HPV and human beta-globulin (HBB) gene obtained by electrophoresis which is carried out on 2% agarose gel.
  • the Ll and E6/E7 genes and HBB genes were obtained by isolating genome DNA from positive control cell line and then amplifying the Ll, E6/E7 and HBB genes by triplex PCR using the genome DNA as a template (Lane KM): 100 bp DNA size marker, lane 2 (HPV-16): HPV-16 infected positive control cell line (Caski, ATCC CRL- 1550), lane 3 (HPV-18): HPV-18 infected positive control cell line (HeLa, ATCC CCL-2)).
  • FIG. 6 is an electrophoretic peak pattern showing that the results of amplified E6/E7 genes analyzed with ABI prism 377 automated sequencer.
  • the amplified E6/E7 genes were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and human beta-globulin (HBB) gene using the DNA extracted from Caski, namely HPV-16 infected cervical cancer cell line. As shown in FIG.6, the genotype of the HPV is HPV-16.
  • FIG. 7 is an electrophoretic peak pattern showing that the results of amplified E6/E7 genes analyzed with ABI prism 377 automated sequencer.
  • the amplified E6/E7 genes were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from HeLa, namely HPV-18 infected cervical cancer cell line. As shown in FIG. 7, the genotype of the HPV is HPV-18.
  • FIG. 8 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from Caski, namely HPV-16 infected cervical cancer cell line. As shown in FIG. 8, the genotype of the HPV is HPV-16.
  • FIG. 9 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from HeLa, namely HPV-18 infected cervical cancer cell line. As shown in FIG. 9, the genotype of the HPV is HPV-18.
  • FIG. 10 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of korean women.
  • the genotype of the HPV is HPV-31 which is a kind of high-risk type.
  • FIG. 11 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of korean women.
  • the genotype of the HPV is HPV-35 which is a kind of high-risk type.
  • FIG. 12 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of korean women.
  • the genotype of the HPV is HPV-39 which is a kind of high-risk type.
  • FIG. 13 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of Korean women.
  • the genotype of the HPV is HPV-67 which is a kind of high-risk type.
  • FIG. 14 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of Korean women.
  • the genotype of the HPV is HPV-56 which is a kind of high-risk type.
  • FIG. 15 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing only the positive reactive change in cervical screening.
  • the genotype of the HPV is HPV- 6b which is a kind of low-risk type.
  • FIG. 16 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing only the positive reactive change in cervical screening.
  • the genotype of the HPV is HPV- 11 which is a kind of low-risk type.
  • FIG. 17 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing only atypical squamous cell of unknown significance (ASCUS) in cervical screening.
  • ASCUS atypical squamous cell of unknown significance
  • the genotype of the HPV is HPV-58.
  • FIG. 18 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing microinavasive sqamous carcinoma in cervical screening and biopsy.
  • the genotype of the HPV is HPV-16.
  • FIG. 19 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing high-grade squamous intraepithelial lesion in cervical screening and biopsy.
  • the genotype of the HPV is HPV-31.
  • FIG. 20 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening and biopsy.
  • the genotype of the HPV is HPV-18.
  • FIG. 21 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening and biopsy.
  • the genotype of the HPV is HPV-58 which is a kind of high-risk type.
  • FIG. 22 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening and biopsy.
  • the genotype of the HPV is HPV-34 which is a kind of high-risk type.
  • FIG. 23 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene was obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing carcinoma in situ in cervical biopsy.
  • the genotype of the HPV is HPV-68.
  • FIG. 24 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing low-grade squamous intraepithelial lesion (LSIL) in cervical screening.
  • LSIL low-grade squamous intraepithelial lesion
  • FIG. 25 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing LSIL in cervical screening.
  • the genotype of the HPV is HPV-35.
  • FIG. 26 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL and then complex infection of HPV-18 and HPV-70 in cervical screening.
  • FIG. 27 is an electrophoretic peak pattern showing that the results of amplified Ll gene analyzed with ABI prism 377 automated sequencer.
  • the amplified Ll gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing microinavasive sqamous carcinoma and then complex infection of HPV-16 and HPV-52 in cervical screening.
  • FIG. 28 is a schematic diagram illustrating types and positions positioned on the DNA chip according to an embodiment of the present invention.
  • FIG. 29 is a front photograph of the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • FIG. 30 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from Caski, namely cervical cancer cell line.
  • the genotype of the HPV is HPV-16.
  • FIG. 31 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from HeLa, namely cervical cancer cell line.
  • the genotype of the HPV is HPV-18.
  • FIG. 32 is a photograph showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene and E6/E7 genes of HPV, and HBB gene using the DNA extracted from K562, namely non-infected negative control cell line.
  • FIG. 33 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of Korean women.
  • the genotype of the HPV is HPV-16 which is a kind of high-risk type.
  • FIG. 34 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of Korean women.
  • the genotype of the HPV is HPV-18 which is a kind of high-risk type.
  • FIG. 35 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of Korean women.
  • the genotype of the HPV is HPV-31 which is a kind of high-risk type.
  • FIG. 36 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of Korean women.
  • the genotype of the HPV is HPV-35 which is a kind of high-risk type.
  • FIG. 37 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of Korean women.
  • the genotype of the HPV is HPV-39 which is a kind of high-risk type.
  • FIG. 38 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of Korean women.
  • the genotype of the HPV is HPV-67 which is a kind of high-risk type.
  • FIG. 39 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical cancer cell finely dissected in cervical cancer tissue of Korean women.
  • the genotype of the HPV is HPV-56 which is a kind of high-risk type.
  • FIG. 40 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing only positive reactive change in cervical screening.
  • the genotype of the HPV is HPV-6b which is a kind of low-risk type.
  • FIG. 41 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing only positive reactive change in cervical screening.
  • the genotype of the HPV is HPV-Il which is a kind of low-risk type.
  • FIG. 42 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing atypical squamous cell of unknown significance (ASCUS) in cervical screening.
  • ASCUS atypical squamous cell of unknown significance
  • the genotype of the HPV is HPV-58 which is a kind of high-risk type.
  • FIG. 43 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing microinavasive sqamous carcinoma in cervical screening and biopsy.
  • the genotype of the HPV is HPV-16 which is a kind of high-risk type.
  • FIG. 44 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing high-grade squamous intraepithelial lesion in cervical screening.
  • the genotype of the HPV is HPV-31 which is a kind of high-risk type.
  • FIG. 45 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening.
  • the genotype of the HPV is HPV-18 which is a kind of high-risk type.
  • FIG. 46 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening.
  • the genotype of the HPV is HPV-58 which is a kind of high-risk type.
  • FIG. 47 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening.
  • the genotype of the HPV is HPV-34 which is a kind of high-risk type.
  • FIG. 48 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing carcinoma in situ in cervical screening.
  • the genotype of the HPV is HPV-68 which is a kind of high-risk type.
  • FIG. 49 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing low-grade squamous intraepithelial lesion (LSIL) in cervical screening.
  • LSIL low-grade squamous intraepithelial lesion
  • the genotype of the HPV is HPV-56 which is a kind of high-risk type.
  • FIG. 50 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing LSIL in cervical screening.
  • the genotype of the HPV is HPV-35 which is a kind of high-risk type.
  • FIG. 51 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening. As shown in FIG. 51, the complex infection of HPV-18 and HPV-70 is found.
  • FIG. 52 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening. As shown in FIG. 52, the complex infection of HPV-16 and HPV-52 is found.
  • FIG. 53 is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing normal evaluation in cervical screening. As shown in FIG. 53, there is not any HPV infection. [Best Mode]
  • Human cervical cancer cell lines which are confirmed that presence or absence of HPV and type thereof are confirmed, and has been used widely in study of HPV genotype.
  • the human cervical cancer cell lines were purchased from ATCC (Manassas, VA20108, USA) and Korea Cell Line Bank (KCLBXSeoul University Medical center cancer institute, KOREA), and used after monolayer culturing.
  • the specifications of the cell lines are summarized in Table 1.
  • Second specimens were obtained from the cervical tissues of women who were diagnosed certainly as cervical cancer or carcinoma in situ lesion and were underwent an operation. After formalin fixation of the specimens, tissues which were stored in paraffin-embedded status were cut into 6-10 microsections at 10 ⁇ m of thickness, attached to microscopic slide and microdissected only cancer cells. Among 68 cervical cancer lesions, 61 cases were cervical squamous cell carcinoma, and 7 cases were cervical intraepithelial neoplasma, CIN. Average age distribution the women was 32-69 years old, and average age was 49 years old.
  • Cervical cells were sampled by inserting Pap brush (Sanga Medical; songpaku Seoul Korea) or cotton stick into cervix, and rolling the brush or stick to gather well cervical cell. And then, the tip of the Pap brush or the cotton stick was introduced in the tube with screw cap containing sterilized transferring buffer and store. 3 ml of CytoLyt Solution (CYTYC Corporation, MA 01719, USA) was primarily used as transferring buffer. Table 1 Specification of control cell line
  • DNA is isolated from the various specimens sampled in the example with suitable methods as follows:
  • DNA was concentrated and purificated with Accuprep Genomic DNA extraction kit (Cat. No. K- 3032, Bioneer co., Ltd. Korea). The procedures are as follows: 1.5 ml of cervical cell smear specimens was centri-precipitated at 12,000 rpm for 2 min, added 1.5 ml of phosphate buffered solution (PBS), washed, add suitable amount of proteinase K and cell lysis buffer, and cultured at 60 ° C for 20 min. After completion of reaction, the reaction liquid was passed through DNA binding column by centrifugal machine, and washed with washing buffer 1 and 2 by centrifugal machine to harvest DNA.
  • PBS phosphate buffered solution
  • E6/E7 genes and Ll gene of HPV were amplified were PCR, and human beta-globulin gene as an internal control was amplified were PCR.
  • oligonucleotide primers were selected and designed.
  • the primers consist of HPCF/ HPCR primer that can detect E6/E7 genes of HPV, MYIl and GP6-1 primer (SEQ ID NO. 1) that can detect Ll gene and HBBF/HBBR primer of human beta-globulin gene that is used to confirm efficiency of DNA extraction and PCR.
  • GP6-1 primer is newly designed and the other two primers are selected from known primers.
  • PCR of E6/E7 genes of HPV amplifies products of about 225 bp length
  • PCR of Ll gene of HPV amplifies products of 182 bp length
  • PCR of beta- globulin gene amplifies products of 182 bp length.
  • the base sequences of PCR primer for each genes are summarized in Table 2, and conditions of PCR are as follows:
  • PCR E6/E7 genes and HPV Ll gene of HPV, and human beta-globulin were carried out by using DNA isolated in the example 2 as an template.
  • the procedures of PCR are as follows:
  • a PCR reaction composition for detecting HPV infection was prepared by, as described in table 2, adding 1 ⁇ i (10 pmoles/ ⁇ O of each of MY11/GP6-1, HPCF/HPCR and HBBF/HBBR primer to 15 ⁇ i of SuperTaq plus pre- mix (lOxbuffer 2.5/z ⁇ , 10 mM MgC12 3.75 ⁇ i, 10 mM dNTP 0.5 / /4, Taq polymerase 0.5 ⁇ i) purchased from Super Bio Co., Ltd, (Seoul, Korea), further adding 4.0 ⁇ i (150ng/' ⁇ i) of template DNA of the specimen and finally adding distilled water to adjust the volume of total reaction 1iquid to 30 ⁇ i.
  • PCR of human beta-globulin was carried out by predenaturing the reaction liquid containing HBB primer at 95°C for 5 min, repeating 40 cycles at 95 ° C for 30 sec, at 50 ° C for 30 sec, and at 72 ° C for 30 sec, and extending at 72 ° C at 5 min.
  • PCR of E6/E7 of HPV was carried out by predenaturing the reaction liquid containing HPCF/ HPCR primer at 95°C for 5 min, repeating 40 cycles at 95"C for 30 sec, at 56 ° C for 30 sec, and at 72TC for 30 sec, and extending at 72 ° C at 5 min.
  • PCR of Ll of HPV was carried out by predenaturing the reaction liquid containing MYIl and GP6-1 primer at 95 ° C for 5 min, repeating 40 cycles at 95 ° C for 30 sec, at 56 ° C for 30 sec, and at 72 ° C for 30 sec, and extending at 72°C at 5 min. 2.
  • Duplex PCR
  • the primer combination of duplex PCR for confirming HPV infection consists of (1) the combination of HPCF/HPCR primer that detects E6/E7 gene of HPV and HBBF/HBBR primer that detects beta-globulin gene and (2) the combination of MY11/GP6-1 primer which detects Ll gene of HPV and beta-globulin primer.
  • Duplex PCR of HPV E6/E7 and HBB genes were carried out as follows: A PCR reaction composition for detecting HPV infection was prepared by adding 1 ⁇ l (10 pmoles/ ⁇ ) of each of HPCF/HPCR and HBBF/HBBR primer to 15 ⁇ l of SuperTaq plus pre-mix, further adding 4.0 ⁇ l (150ng/ / ⁇ ) of template DNA of the specimen and finally adding distilled water to adjust the volume of total reaction liquid to 30 ⁇ l. PCR was carried out by predenaturing the reaction liquid at 95°C for 5 min, repeating 30 cycles at 95 ° C for 1 min, at 72°C for 1 min, and at 72°C for 1 min, and extending at 72 ° C at 5 min.
  • Duplex PCR of HPV Ll and HBB genes were carried out as follows: A PCR reaction composition for detecting HPV infection was prepared by adding 1 ⁇ l (10 pmoles// ⁇ ) of each of MYll/GPG-1 and HBBF/HBBR primer to 15 ⁇ l of SuperTaq plus pre-mix, further adding 4.0 ⁇ l (150 ng/ ' ⁇ l) of template DNA of the specimen and finally adding distilled water to adjust the volume of total reaction liquid to 30 ⁇ l.
  • PCR was carried out by predenaturing the reaction liquid at 95 ° C for 5 min, repeating 10 cycles at 95 ° C for 1 min, at 72 ° C for 1 min, and at 72°C for 1 min, repeating again 30 cycles at 95 ° C for 1 min, at 50°C for 1 min, and at 72°C for 1 min and extending at 72°C at 5 min.
  • a PCR reaction composition for detecting HPV infection was prepared by adding 1 ⁇ l (10 pmoles// ⁇ ) of each of MYIl/GPG-I, HPCF/HPCR and HBBF/HBBR primer to 15 ⁇ l of SuperTaq plus pre-mix, further adding 4.0 ⁇ l (150 ng/ ⁇ i) of template DNA of the specimen and finally adding distilled water eo adjust the volume of total reaction liquid to 30 ⁇ l.
  • PCR was carried out by predenaturing the reaction liquid at 95°C for 5 min, repeating 10 cycles at 95°C for 1 min, at 72°C for 1 min, and at 72°C for 1 min, repeating again 30 cycles at 95 ° C for 1 min, at 50"C for 1 min, and at 72°C for 1 min and extending at 72°C at 5 min.
  • FIG. 1 to FIG. 5 The results of the experiments are represented in FIG. 1 to FIG. 5. As shown in FIGS. 1 to 5, the conditions for single, duplex and triplex PCR of HPV were established suitably, and also PCR of both cervical swab specimen and paraffin-embedded cervical cancer tissue was carried out well.
  • EXAMPLE 4 Sequencing analysis and database built-up After the PCR of the example 3, automated sequencing analysis of the PCR products were carried out to analyze base sequence of HPV-E6/E7 and HPV Ll, and database was built by sorting out the analyzed information. In addition, clinical DNA specimens which their HPV genotype is confirmed were stored, and then used for analyzing degree of accuracy of the DNA chip of the present invention
  • the concentration of the PCR products is adjusted suitably. For example, if the length is 100-200 bp, 1-3 ng/ ⁇ l is needed, and if the length is 200-500 bp, 3-10 ng/ ⁇ l is needed.
  • Cycle sequencing is carried out with GeneAmp 2700 (PE Biosystems, USA) on the mixture obtained in (2) at 96 ° C for 10 sec, at 50 ° C for 5 sec, and at 60 ° C for 6 min at 25 cycles.
  • HPV was found in all 68 cases of total 68 cases. Found types were all, so called, high-risk types. Among them, HPV 16 type was 33 cases; 58 type was 12 cases; 31 type was 11 cases; 18 type and 35 type were 4 cases, respectively; 33 type was 3 cases, and these 7 types hold 99%. Complex infection could not be found by PCR-sequencing.
  • HPV infection was found in 1,013 cases among 4,898 cases of cervical cell specimens sampled from Korean general adult women, and the frequency was 20.6%.
  • HPV types include 35 types, among them 15 types were high-risk type; 11 types were low-risk type; 4 types were middle-risk type; and 5 cases were unknown.
  • High-risk types, which were found, were 838 cases (82.7%), and the frequency was 17.1% as a whole.
  • the results tended to high-risk type of HPV. The reason is why all women received the HPV test for early medical examination of cervical cancer and majority in these women were high-risk type having cervical lesion. It is believed that this conditions corresponds well with the study which primary purpose is to recognize accurately high-risk type of HPV.
  • the appearance frequency was HPV-16, HPV-58, HPV-31, HPV-52, HPV-33, HPV-53, HPV-35, and HPV-18 order.
  • HPV-16 was most common. This appearance order is quite different that of America and Europe. In America and Europe, the appearance frequency is HPV-16, HPV-18, HPV-45, HPV-52, HPV-31, HPV-33 and HPV-58 order (Murinoz N et al., NEngl JMed , 2003, 348:518-27).
  • peak in the electrophoretic peak pattern was overlapped. It is a phenomenon that appears when analyzing several different template DNA, namely when various types of HPV are mixed. In this case, only portion thereof can be confirmed by blast search. It means that PCR products are cloned and numerous clones should be analyzed by sequencing. In this case, the DNA chip, which can analyze complex HPV infection, is very useful. Actually, for the above case, the presence of the complex infection of HPV-18 and HPV-70 (see FIG. 51) and the complex infection of HPV-16 and HPV-52 (see FIG. 52) was found by the DNA chip of the present invention.
  • EXAMPLE 5 Establishment of clones for HPV analysis After the PCR of the example 4, PCR products of which their HPV type was confirmed by sequencing method were cloned by using plasmid vector and E.coli. Thereafter, these clones were used as standard and control specimens when establishing reaction conditions of DNA chip of the present invention.
  • the cloning method is as follows:
  • Buffer stored at -20 ° C were melted, mixed by shaking the tube slightly with fingers, centrifuged at low speed, mixed with insert DNA to be cloned in the ratio described in Table 5 and introduced into 0.5 ml tube, thereby preparing ligation reaction.
  • Table 5
  • the example explains the design process of oligonucleotide probes to be positioned on the DNA chip. It is the core of the present invention.
  • Probes were designed to make oligonucleotide probes as genotype specific probe, which can bind specifically to DNA of Ll gene and E6/E7 genes, according to the object of the present invention.
  • HPV database of National Center for Biotechnology Information (NCIB) in USA (2) HPV database of LOS ALAMOS in USA, and (3) database of 35 types of HPV found in the cervix of Korean women in the example 4, base sequences of genome DNA obtained from total 76 types of HPV including HPV-Ia, -2a, -3, -4, -5, -6b, -7, -8, -9, -10, -11, -12, -13, -15, -16, -16r, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -35h, -36, -37, -38, -39, -40, -41, -42, -44, -45, -47, -48, -49,
  • TM established. After executing the computer program DNASTAR (MegAlign 5, DNASTAR Inc.) on the obtained DNA sequences (pairwise alignment and multiple sequence alignment) with ClustalW method, phylogenetic tree was drawn up. After screening genotype specific base sequence for each group, genotype specific probes were designed with computer program, primer premier 5 (PREMIER Biosoft International Co.). In this case, setting 20 Ap and 18 Ap bb of oligonucleotide of probe length, 110 genotype specific probes were designed primarily.
  • the DNA chip and diagnosis kit for diagnosing genotypes of HPV are characterized in that the analysis targets of the DNA probes are total 40 types of Ll genes including 8 high-risk HPV E6/E7 genes, 20 high-risk HPV Ll genes, 17 low-risk HPV Ll genes, and 3 middle-risk HPV Ll genes, wherein high-risk HPV types include HPV-l ⁇ f HPV-18, HPV-31, HPV-33, HPV-35, HPV-52, HPV-58, HPV-67, HPV-26, HPV-30, HPV-34, HPV-39, HPV-45, HPV-51, HPV-53, HPV-56, HPV-57, HPV-66, HPV-68, and HPV-70; low-risk HPV types include HPV-6, HPV-7, HPV-IO, HPV-Il, HPV- 27, HPV-32, HPV-40, HPV-42, HPV-44, HPV-54, HPV-55, HPV-59,
  • probes were selected according to its ability to genotype specifically bind to HPV-39, HPV-45, HPV-51, HPV-56, HPV-59, HPV-61, HPV- 68, HPV-70, HPV-73, HPV-74, HPV-6, HPV-7, HPV-Il, HPV-32, HPV-34, HPV-40, HPV-42, HPV-44, HPV-55 and HPV-66.
  • SEQ ID Nos. and types are summarized in Table 6 and 7.
  • probes mixed with suitable buffer were spotted to microscopic glass slide. And then, the slide was stabilized with suitable treatments, quality controlled and stored until examination.
  • the process for producing DNA chip is as follows: Preparation of order grid to be positioned on the DNA chip
  • grouping grid was prepared.
  • the order of the grip was shown in FIG. 28.
  • 8 types of E6/E7 probes of HPV high-risk types and 20 types of Ll probes of HPV high-risk types were spotted on the left
  • Ll probes of HPV middle-risk types were spotted on the center
  • 17 types of Ll probes of HPV low-risk types were spotted on the right.
  • corner marker and oligonucleotide probes which are specific for human beta-globulin gene to confirm suitability of DNA isolation and PCR amplification were spotted on the top of left (2 probes), the top and bottom of center (2 probes) and the bottom of right (1 probe).
  • actin In addition to the human beta-globulin gene, actin, glyceraldehydes- 3-phosphate dehydrogenase gene and the like can be used as standard marker probe.
  • Each oligonucleotide probe was spotted with arrayer. In this case, same probes were spotted in duplicate in order that each genotypes of HPV is exhibited two times in minimum and four times in maximum.
  • the reasons that oligonucleotide probes of 8 HPV E6/E7 gene were added are as follows. At First, The 8 genotypes exists at most high frequency in worldwide including Korean. At Second, the 8 types of HPV are representative high-risk type and are closely related to outbreak of cervical cancer. At Third, it is important to recognize accurate genotype of E6/E7 in case of administrating vaccines against high-risk types of HPV.
  • grid illustrated in FIG. 28 exists repetitively on the 6 to 8 compartments divided on the one DNA chip (See FIG. 29). Accordingly, the DNA chip can analyze 6 to 8 different specimens on the only one chip, and is very useful for saving time, labor and cost.
  • Oligonucleotides which amine is attached to C6 moiety, synthesized according to the example were purified with HPLC, and solublized in third distilled water to adjust final concentration of 200
  • Such prepared oligonucleotide probes were mixed with spotting solution, micro spotting solution Plus (Telechem, TC-MSP, USA) to adjust concentration of 38 pmole/ ⁇ . That is, 7.7' ⁇ l of 200 pmole/ ⁇ i oligonucleotide probe was mixed with 32.4 ⁇ £ of spotting solution to make final volume of 40 ⁇ i.
  • the resulting mixtures were dispended into 96-wel master plate in order.
  • Arrayer such as GMS 417 arrayer (Pin-Ring Type, Affymetrix, USA)M" MGII (Biorobotics Inc, MA01801, USA), or equivalents, is preferable.
  • the DNA chip produced by spotting the probes to glass slide as described above was placed into glass jar which humidity is maintained to 80%, and reacted at room temperature for 15 min. After completion of the reaction, the immobilized slide was placed into dry oven, baked at 120°C for 1 hour and 30 min, washed with sodium dodecyl sulfate (SDS) solution for 2 min twice, dipped into third distilled water at 95 ° C for 3 min to denature immobilized oligonucleotide probes, and washed again with third distilled water for 1 min.
  • SDS sodium dodecyl sulfate
  • the slide was reduced in blocking solution (1 g of NaBH4, 300ml of phosphate buffered solution (PBS), 100ml of ethanol) for 15 min, washed in 0.2% sodium dodecyl sulfate solution for 2 min twice, washed in third distilled water for 2 min twice, centrifuged at 800 rpm for 1 min and 30 sec to remove moisture, placed into slide box and stored in desiccator.
  • blocking solution (1 g of NaBH4, 300ml of phosphate buffered solution (PBS), 100ml of ethanol) for 15 min, washed in 0.2% sodium dodecyl sulfate solution for 2 min twice, washed in third distilled water for 2 min twice, centrifuged at 800 rpm for 1 min and 30 sec to remove moisture, placed into slide box and stored in desiccator.
  • PBS phosphate buffered solution
  • the resulting DNA chip of the present invention was quality controlled by means of the method such as the following example 8.
  • EXAMPLE 8 Hybridization reaction on the DNA chip and establishment of analytic conditions
  • HPV E6/E7 and HPV Ll genes and betaglobulin gene were amplified by multiplex PCR using 100 artificial standard specimens produced by various combinations and concentrations of one, two or three clones of each HPV type established in the example 5. Suitable conditions were established by positioning the PCR products on the DNA chip, carrying out hybridization reaction more than three times, and then analyzing by fluorescent scanner. The procedures are as follow:
  • PCR of E6/E7 and Ll genes of HPV, and human betaglobulin gene was carried out by using the procedures described in the example 3, with provided that for a reverse primer among the combination of primers, namely GP6-1, HPCR and HBBR, oligonucleotides labeled by Cy-5 fluorescent was used.
  • Cy-3 biotin-binding material, 5-2'-(aminoethyl)amino-l-naphalene sulfate (EDANS), tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC), ⁇ -rhodamine and TEXAS RED can be used as the labeling marker.
  • EDANS 5-2'-(aminoethyl)amino-l-naphalene sulfate
  • TMR tetramethylrhodamine
  • TRITC tetramethylrhodamine isocyanate
  • ⁇ -rhodamine and TEXAS RED can be used as the labeling marker.
  • Hybridization reaction was carried out by positioning HPV PCR products amplified by PCR on the slide substrate which various oligonucleotide probes were immobilized thereon. lOO ⁇ i perfusion 8 wells chamber (Schleicher & Schuell BioScience, German) was used as hybridization reaction chamber. 10 ⁇ i of each amplified products of E6/E7 genes, HBB gene and Ll gene were mixed, and tertiary distilled water was added to the mixture to make 50 ⁇ i of final volume. After denaturing at 95°C for 5 min, the mixture was stood in ice for 3 min immediately.
  • hybridization reaction liquid (2 ml of 20 X SSC, 1.7 ml of 90% glycerol, and 6.3 ml of 50 mM PBS was mixed and adjusted final volume to 10 ml) to make 100 ⁇ i of final volume, reacting it with probes immobilized on slide at 45 ° C for 30 min.
  • FIGS. 30 to 32 is a photography of DNA microarray hybridization reaction results using 30 pairs of HPV type probes synthesized with Caski, HeLa and K-562, respectively.
  • circles represent a position depending on the kind of probe.
  • cross-hybridization among the different probes was not occurred, and hybridization reaction was occurred in HPV-16 and HPV-18 specifically, respectively.
  • FIGS. 33 to 52 is a photography of DNA microarray hybridization reaction results using 48 pairs of oligonucleotide probes spotted on the DNA chip of the present invention respectively.
  • circles represent a position depending on the kind of probe.
  • the results of hybridization reaction caused by the plasmid DNA amplification products illustrates that there was no cross-hybridization, and type-specific expression was occurred in only inherent probes, respectively.
  • each HPV type probes in the DNA chip produced in the present invention were bound to certain type of HPV DNA specifically and there was no cross-hybridization.
  • complex infected specimens mixed with at least one type of HPV were diagnosed accurately. That is, for diagnosing genotype of single or complex infection HPV, the DNA chip of the present invention exhibited 100% of sensitivity and 100% of specificity.
  • the reproducibility of the DNA chip was 100%.
  • the 48 pairs of the probes synthesized in the present invention can analyze accurately numerous combinations of HPV types that were not considered in the examples of DNA microarray hybridization reaction.
  • FIG. 51 is a photography taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening.
  • the complex infection of HPV-18 and HPV-70 was found. In this case, only HPV- 70 was found in the first direct sequencing assay, and HPV-18 was found additionally in the second sequencing assay after cloning.
  • the DNA chip for analyzing genotypes of HPV of the present invention can analyze complex infection of HPV-18 and HPV-70 readily and rapidly in only one assay.
  • FIG. 52 is case proving that the presence of cervical carcinoma in situ was confirmed by biopsy afterward, and is a photograph taken by fluorescent scanner showing that the results of amplified Ll gene, E6/E7 genes and HBB gene analyzed with the DNA chip for diagnosing genotype of HPV according to an embodiment of the present invention.
  • the amplified Ll gene, E6/E7 genes and HBB gene were obtained by triplex PCR amplifying Ll gene, E6/E7 genes and HBB gene using the DNA extracted from cervical swab specimen of Korean women showing HSIL in cervical screening. As shown in FIG. 52, the complex infection of HPV-16 and HPV-52 was found.
  • the DNA chip for analyzing genotypes of HPV of the present invention can analyze complex infection of HPV-18 and HPV-70 readily and rapidly in only one assay.
  • the DNA chip produced in the present invention can discriminate accurately each type of HPV from cervical swab specimens in clinical test.
  • Each HPV type probes were bound to certain type of HPV DNA specifically and there was no cross- hybridization.
  • complex infected specimens mixed with at least one type of HPV which were difficult in diagnosis by direct sequencing and were able to recognizing by many sequencing assays after cloning, were diagnosed accurately. That is, for diagnosing genotype of single or complex infection HPV, the DNA chip of the present invention exhibited 100% of sensitivity and 100% of specificity.
  • the reproducibility of the DNA chip was 100%.
  • EXAMPLE 10 Correlation analysis with clinical data after analyzing clinical specimens on the DNA chip
  • HPV types were analyzed by the DNA chip of the present invention and sequencing method in paraffin-embedded 68 cervical cancer tissues and 49 normal cervix tissue specimens.
  • the DNA chip can predict conditions of cervical cancer, and particularly is useful for selective analysis of cervical cancer and cervix carcinoma in situ.
  • the analytic study using the DNA chip of the present invention and sequencing method was performed in concurrent with cervical colposcopy, cervical scanning, cervical smear screening and cervical biopsy on cervical swab specimens obtained from 20 adult women who went to domestic obstetrics via cooperation of Korean Genecologic Cancer Foundation (KGCF), and the results of the study were analyzed.
  • KGCF Korean Genecologic Cancer Foundation
  • the correlation of the results obtained by cervical screening and biopsy of volunteers with the results obtained by HPV DNA chip assay and sequencing method was shown in Table 8.
  • Oligonucleotide probes, DNA chip and diagnosis kit comprising the same, and method for analyzing genotype of HPV using the same can diagnose accurately 40 types of HPV which are found in cervix and are major cause of various conditions such as cervical caner, genital wart and the like and predict the risk thereof.
  • the accuracy of diagnosis can be improved significantly, since diagnosis according to the present invention is carried out by analyzing Ll gene of HPV as well as E6/E7 genes of HPV to address the problems caused by analyzing the only Ll gene of HPV.
  • DNA chip and the diagnosis method of the present invention can analyze complex infection by various types of HPV, has approximately 100% of diagnosis sensitivity, diagnosis specificity and reproducibility. Finally, examination procedures and analysis of the result are very simple, and cost of examination is inexpensive.
  • the HPV genotype diagnosis DNA chip and the diagnosis kit using the same can analyze automatically presence or absence of HPV infection and genotype thereof rapidly, accurately and massively in specimens such as cervical cells, urine and the like.
  • the DNA chip can be used alone, or together with Pap cervical screening to screen cervical cancer and precancerous lesion, substituted for existing HPV examination and save examination time, labor and cost.
  • the DNA chip is useful for applying fitted type vaccines by analyzing accurate genotypes of E6/E7 on HPV infection. Accordingly, the present invention has very useful industrial availability due to contributing greatly improvements of national health and welfare by reducing incidence and mortality of cervical cancer.
  • SEQ ID Nos. 1 to 6 consist of primer base sequence for amplifying nucleic acid of HPV or beta-globulin
  • SEQ ID NO. 7 to 55 consist of probe base sequence of HPV or beta-globulin. The sequence listing is attached to the present application.

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Abstract

Selon l'invention, des sondes oligonucléotidiques servant à analyser 40 types de HPV ont été synthétisées, et des puces à ADN ont été produites au moyen de ces sondes oligonucléotidiques. La synthèse desdites sondes oligonucléotidiques est fondée sur des clones des gènes Ll et E6/E7 de 35 types de HPV obtenus à partir d'échantillons de cellules de col de l'utérus de 4898 coréennes adultes, et à partir d'échantillons tissulaires de 68 cas de cancer du col de l'utérus, et sur des informations issues de cas américains et européens. Les puces à ADN peuvent analyser 40 types de HPV trouvés dans des infections du col de l'utérus à diagnostic complexe par au moins un type de HPV, et présentent une excellente sensibilité et spécificité de diagnostic sur un type de gène de HPV qui peut atteindre 100 %, et peuvent être reproduites facilement. Les puces à ADN selon l'invention sont en outre meilleures que les procédés d'analyse traditionnels, et peu onéreuses puisqu'elles peuvent analyser de nombreux échantillons en très peu de temps. Ainsi, les puces à ADN selon l'invention peuvent servir à prédire le cancer du col de l'utérus et des lésions précancéreuses.
PCT/KR2005/000775 2004-10-04 2005-03-18 Sonde de papillomavirus humain, et puce a adn pourvue de cette sonde WO2006038753A1 (fr)

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JP2007534506A JP4977612B2 (ja) 2004-10-04 2005-03-18 ヒトパピローマウイルスのプローブおよびそれを使用するｄｎａチップ
US11/664,549 US7670774B2 (en) 2004-10-04 2005-03-18 Probe of human papillomavirus and DNA chip comprising the same
EP05789712A EP1802756A4 (fr) 2004-10-04 2005-03-18 Sonde de papillomavirus humain, et puce a adn pourvue de cette sonde
CN2005800335409A CN101035896B (zh) 2004-10-04 2005-03-18 人乳头瘤病毒探针和包含其的dna芯片

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EP2535411A1 (fr) * 2010-02-12 2012-12-19 M&D.Inc. Sonde pour le diagnostic des génotypes de papillomavirus humains et procédé d'analyse correspondant
WO2014134607A1 (fr) * 2013-03-01 2014-09-04 The Johns Hopkins University Méthode de double capture de séquences permettant la quantification d'adn transrénal hpv dans l'urine
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KR101459074B1 (ko) * 2014-01-09 2014-11-12 (주)다이오진 인유두종바이러스 유전자형 분석용 ｄｎａ 칩， 이를 포함하는 키트 및 이를 이용한 인유두종바이러스 유전자형 분석방법
CN104073573B (zh) * 2014-07-15 2016-04-27 江苏同科医药科技有限公司 一种人乳头瘤病毒核酸分型检测试剂盒
CN108676797B (zh) * 2018-06-07 2019-04-26 迈基诺（重庆）基因科技有限责任公司 用于检测人乳头瘤病毒的成套试剂与方法
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CN112662764A (zh) * 2020-03-17 2021-04-16 博尔诚（北京）科技有限公司 一种检测11种癌症的探针组合物
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US8017757B2 (en) * 2006-03-03 2011-09-13 Gyngene Bio Co., Ltd Kits and method for detecting human papilloma virus with oligo nucleotide bead array
JP2010517556A (ja) * 2007-02-09 2010-05-27 ヘルス プロテクション エージェンシー ヒトパピローマウイルスの検出
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WO2009057993A1 (fr) * 2007-11-01 2009-05-07 Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg Nouveau procédé de détection de papillomavirus humains du col de l'utérus
US9435001B2 (en) 2007-11-01 2016-09-06 Self-Screen B.V. Detection method for cervical HPVS
US20120004113A1 (en) * 2008-11-25 2012-01-05 Goodgene Inc. Dna chip, kit for detecting or genotyping bacteria causing sexually transmitted diseases, genotyping antibacterial drug resistance and detecting or genotyping method using the same
WO2010069939A1 (fr) * 2008-12-15 2010-06-24 Pathofinder Bv Dosage à plusieurs paramètres
EP2336369A1 (fr) * 2009-12-15 2011-06-22 Deutsches Krebsforschungszentrum Sondes pour le génotypage de l'HPV à bas risque
WO2011073183A1 (fr) * 2009-12-15 2011-06-23 Deutsches Krebsforschungszentrum Sondes pour le génotypage des hpv à bas risque
EP2535411A1 (fr) * 2010-02-12 2012-12-19 M&D.Inc. Sonde pour le diagnostic des génotypes de papillomavirus humains et procédé d'analyse correspondant
EP2535411A4 (fr) * 2010-02-12 2013-07-03 M & D Inc Sonde pour le diagnostic des génotypes de papillomavirus humains et procédé d'analyse correspondant
WO2014134607A1 (fr) * 2013-03-01 2014-09-04 The Johns Hopkins University Méthode de double capture de séquences permettant la quantification d'adn transrénal hpv dans l'urine
US9809864B2 (en) 2013-03-01 2017-11-07 The Johns Hopkins University Dual sequence-capture method for quantifying trans renal HPV DNA in urine
CN114164305A (zh) * 2021-12-30 2022-03-11 广州安必平医药科技股份有限公司 一种检测人乳头瘤病毒的引物和分型探针组合及其应用

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