KR20140027736A - Dna chip for determining genomic types of human papillomavirus, kit comprising the same and determining method of genomic types of human papillomavirus using the same - Google Patents

Abstract

The present invention relates to a DNA chip for genotyping a human papilloma virus, a kit including the same, and a method for genotyping the human papilloma virus using the same. In the present invention, oligonucleotides complementarily bind to a tumorigenic HPV type of HPV nucleic acids and can be used for a novel type of a specific primer which can specifically amplify the HPV type of nucleic acids. Especially, the oligonucleotides used in producing the DNA chips according to the present invention can be used in detecting or diagnosing the presence of HPV nucleic acids and a type thereof in a sample, thereby capable of being used for an early diagnosis of a cervical cancer caused by HPV. As a HPV infection can be early diagnosed by a gene approach method using a genome according to the present invention, an accurate diagnosis can be continuously executed in the long-term, which is expected to have an effect in preventing cervical cancer. In addition, the HPV diagnosis can not only be performed at relatively low costs but also inform subjects who are being examined of up-to-date results thereby giving reliability and satisfaction, meanwhile playing a key part in promoting public health against diseases such as cervical cancer, which is becoming a major problem at a national level.

Description

TECHNICAL FIELD [0001] The present invention relates to a DNA chip for analyzing HPV genotypes, a kit containing the same, and a method for analyzing HPV genotypes using the same. BACKGROUND ART < RTI ID = 0.0 &

The present invention relates to a DNA chip for analyzing a genotype of HPV, a diagnostic kit comprising the DNA chip, and a method for analyzing genotypes of HPV using the same.

Cervical Cancer (Cervical Cancer) is a malignant tumor of the uterine cervix that accounts for more than 95% of the total uterine cancer incidence, followed by breast cancer, which is followed by breast cancer. Are reported.

In Korea, it is confirmed that about 5,000 new cases occur in one year by referring to the statistics of cancer registration of the Ministry of Health and Welfare in 2000. It accounted for 10.6% of all female cancers, accounting for the third most common cause of cancer (15.7%) and breast cancer (15.1%). In particular, the infection rate of young women in their 20s and 30s has recently increased to 32% of all cervical cancer patients. For this reason, periodic HPV testing is essential in adult women and HPV testing is basically included in sexually transmitted disease testing (Centers for Disease Control and Prevention National Center for HIV / AIDS, Viral Hepatitis, STD, and TB. Prevention Division of STD Prevention. Sexually Transmitted Disease Surveillance 2008. Division of STD Prevention. 2009: November; Tchernev G. Sexually transmitted papillomavirus infections: epidemiology pathogenesis, clinic, morphology, An Bras Dermatol, 2009; 84 (4): 377-89).

HPV is a clearly proven virus that can cause tumors in areas of human infections and cause cancer. HPV causes hyperproliferation after infection with human contact epithelial cells. The overgrowth is mostly benign tumors such as simple warts of the skin, external genitalia, perianal gonad or condyloma accuminata. However, HPV may be the cause of carcinogenesis, and virtually all uterine cervical or cervical cancers, as well as oral, parietal, and laryngeal cancer, and multiple anal cancers are caused by HPV Has been confirmed. HPV is of great importance in that it can induce cancer and kill life. On the other hand, HPV tests can be used to diagnose cancer and precancerous lesions such as cervix and anus. The actual HPV test has been shown to be superior to the Papa nicolaou cell test (Pap smear), which is the standard test for early detection of cervical cancer, and is therefore recognized as a cervical cancer screening test in many countries including the US FDA Parkin M, F. Bray F, J. Ferlay J and P. Pisani (Eds.), N. Engl J Med, 2003, 348: 518-27; P. Global cancer statistics, 2002. CA Cancer J. Clin. 2005; National Network of STD / HIV Prevention Training Center, Genital human papillomavirus infection). For these reasons, the market for HPV-related fields is very large and the economic value of HPV tests is reported to be very high.

Cervical cancer has been reported to be closely related to human papillomavirus (HPV). After HPV infected cervical epithelial basal cells, HPV infiltrated the cervical epithelial basal cells through a prolonged global phase such as low-grade SIL, high grade SIL, and squamous intraepithelial lesion (SIL) Cancer develops. Since there are pre-cancer stage lesions before cancer development, it is possible to prevent cervical cancer effectively by treating these lesions early and effectively.

Human Papillomavirus (HPV) is an 8-kb circular double-stranded DNA virus. All types of HPV viruses have open reading frames (ORFs), a DNA part that synthesizes proteins of similar nature. It is divided into an early gene and a late gene. The initial gene of about 4.5 kb induces or suppresses the action of DNA to form a protein that causes viral DNA replication (E1), malignant transformation of the host cell (E2) (E4), activation of EGF (epidermal growth factor) and CSF (colony stimulator factor) receptors (E5), permanent survival of cells and activation of cancer genes and malignant transformation by the deactivation mechanism of cancer inhibitors . In particular, E6 and E7, the oncogenic proteins expressed after HPV infects host epithelial cells, bind to p53 and pRB, the tumor suppressor proteins of the host cell, respectively, to inhibit the function of these tumor suppressor proteins, , Which has been shown to develop into tumorigenesis upon transformation of infected cells. On the other hand, a late gene of 2.5 kb has a size of 1 kb, which is a non-coding region that regulates transcription and translation of the DNA responsible for the synthesis of the viral coat protein (L1) and the epidermal protein (L2) Of the LCR (long control region).

To date, more than 120 different types of HPV have been identified, of which about 36 HPV types (HPV-2, -3, -6, -10, -11, -13, -16, -18 , -26, -30, -31, -32, -33, -34, -35, -39, -40, -42, -43, -44, -45, -51, -52, 54, -55, -56, -57, -58, -59, -61, -66, -67, -68, -69, -70 and -73) are so-called oncogenic HPV types that cause cervical cancer It turned out.

Among them, HPV-16, -18, -26, -30, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, , HPV-2, HPV-1, HPV-2, HPV-1, HPV-2, HPV-1, HPV-1, HPV-2, and HPV- HPV types such as 3, -6, -7, -10, -11, -13, -32, -34, -40, -42, -43, -44, Low-risk HPV due to poor prognosis (Garrenstroom et al., J. Gynecol. Cancer 4: 73-78, 1994).

Another important fact about HPV is that as the vaccine is developed, it is becoming possible to prevent viral and cancer by viruses. There are two types of HPV vaccines currently on the market. One is Gardasil (Merck & Co. Inc., Whitehouse Station, NJ, USA), a quadrivalent vaccine designed to prevent four types of HPV 16, 18, 6 and 11. The other is Cervarix (GlaxoSmithKline Biologicals, Rixensart, Belgium), a diabetic vaccine designed to prevent two types of HPV types 16 and 18. These vaccines are most effective for adolescent women before having sex and are less effective for women who have previously been infected with HPV 16 or HPV 18. This is controversial for adult women, however, HPV vaccine may be adaptable if the HPV type has been infected and the type is not type 16 or 18. Therefore, it is becoming increasingly important to know the type of infection as well as the presence or absence of HPV (Selva L, Gonzalez-Bosquet E, Rodriguez-Plataa MT, Esteva C, Sunol M and Munoz-Almagro C. Detection of human paclitaxel, papillomavirus infection in women attending a colposcopy clinic. 2009; 64: 416-421; Reynales-Shigematsu LM, Rodrigues ER, Lazcano-Ponce E. Costeffectiveness analysis of a quadrivalent human papilloma virus vaccine in Mexico Arch Med Res 2009 Aug; 40 (6): 503-13).

Papanicolaou (Pap) Smear has been performed as a standard test since the 1940s with the primary diagnosis of cervical cancer and its precancerous lesions. Cytologic examination is based on the cytologic pattern of cervical cytology, which is used to diagnose cervical cancer, which contributes greatly to the reduction of mortality from cervical cancer. However, there is a disadvantage in that the accuracy of the test is reduced depending on the skill of the tester (Menezes et al., Acta Cytol. 45: 919-926, 2001). In addition, the use of colposcopy can diagnose up to 70% of HPV infections, but requires trained specialists and expensive equipment, and can not classify HPV genotypes (Reid et al., Clin Obstet Gynecol. 32: 157-179, 1989).

Since the HPV hybrid capture method, which detects the liquid phase complementarity between HPV DNA and RNA probe of cervical cytology by antibody enzyme development, was developed in 1994, it has been used as an adjuvant screening or tracking tool for cervical cancer and its precursor lesion It has been used as a method of screening, and recently received approval from the US FDA for primary screening of cervical cancer.

As described above, there are various analytical methods for diagnosing cervical cancer including hybrid capture. However, as can be seen from the fact that about 500,000 to 500,000 deaths occur every year due to cervical cancer in the world, comprehensive early detection of cervical cancer and its precursor lesion Development of a general method for detection or diagnosis is urgent.

Recently, in the detection and diagnosis of various types of HPV infection, polymerase chain reaction (PCR) technique capable of rapidly and simply amplifying a DNA fragment of a specific nucleotide sequence has become popular, and all types of HPV There has been a vigorous effort to search for a comprehensive detection of a target.

In addition, PCR-RFLP (Restriction Fragment Length Polymorphism) using restriction enzyme after amplifying L1 site by PCR (polymerase chain reaction) is advantageous to obtain simple and easy result, (Lungu et al., JAMA 267: 2493-2496, 1992).

The PCR technique involves the step of denaturation, annealing and polymerization of a complementary nucleic acid fragment sequence (primer) that specifically binds to the nucleotide sequence of the target pathogen, (US Pat. Nos. 4,683,195 and 683,202), which is one of the representative techniques used for diagnosing various types of HPV infection.

Unlike the cytological test of Pap smear, the PCR technique can be objectively large-scaled in terms of the practicality of clinical diagnosis.In terms of measurement principle, the test cost, experimental procedure, and sensitivity are higher than those of cytology or liquid hybrid capture. ) And relative specificity. However, there is a lack of a general primer suitable for early detection and diagnosis of HPV infection. Therefore, it is difficult to clinically apply HPV infection using PCR.

Currently, the most common common primers widely used for clinical diagnosis and diagnosis of HPV infection in the world are MY09 / MY11 primer set mainly used in North America, South America and Asia, and GP5 ⁺ / GP6 ⁺ primer set and SPF1 / SPF2 primer set. In addition, there are recently developed and used FAP59 / FAP64 primer sets and PGMY09 / PGMY11 primer sets. There are a number of common primer sets for detection / diagnosis of many HPVs used for some research purposes. However, In comparison, it is found that HPV detection and diagnosis are restricted to very limited types of HPV types, and that detection sensitivity is greatly reduced depending on the type of HPV. In addition, the efficiency of PCR amplification depends on the HPV genotype and the accuracy of the test may be a problem. In addition, all of the DNA chips approved by the KFDA have to be subjected to hybridization after individual screening primers and internal control primers are amplified and mixed in the analysis, resulting in a costly increase (Qu et al., J. Clin J. Microbiol. 34: 2095-2100, 1996; Gravitt et al., J. Clin. Microbiol. 38: 357-361).

Therefore, current detection systems for the prevention of cervical cancer due to the detection of HPV virus have a great limitation in practical clinical use despite its value and necessity. In particular, the newly identified and classified HPV types are associated with HPV types that are closely associated with cervical cancer, and there is no clue as to whether HPV types are high risk.

Korean Patent Registration No. 10-0786637 Korean Patent Publication No. 10-2011-0126076

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above problems of the prior art, and an object of the present invention is to provide a DNA chip for HPV genotyping analysis for detection / diagnosis of tumorigenic HPV DNA.

Another object of the present invention is to provide a primer set for amplifying the type-specific E6 / E7 gene of HPV.

Another object of the present invention is to provide a diagnostic kit comprising a DNA chip for HPV genotyping and a primer for amplifying the HPV type E6 / E7 gene.

Another object of the present invention is to provide a method for analyzing HPV genotypes using the DNA chip and the primer for analyzing HPV genotypes of the present invention.

In order to solve the above-mentioned problems, the present invention provides a sample human papillomavirus (hereinafter, referred to as " human papilloma virus ") comprising an oligonucleotide selected from the group consisting of the nucleotide sequence of SEQ ID Nos. 1 to 29 or a complementary base sequence thereof HPV) genotyping DNA chip.

HPV-16, HPV-18, HPV-26, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-6 and HPV-11 as low-risk HPV and HPV-53 and HPV-56, HPV-58, HPV-59, HPV-66, HPV-67, HPV- For the analysis of HPV genotypes including HPV-34, HPV-40, HPV-43, HPV-44, HPV-54, HPV-55 and HPV-61.

The oligonucleotide is complementary to the type-specific E6 / E7 gene region of HPV.

The DNA chip may include an oligonucleotide having a nucleotide sequence of SEQ ID NO: 30 or a complementary base sequence thereof complementarily binding to human GAPDH gene as a positive control.

Preferably, the DNA chip is divided into eight wells capable of accumulating oligonucleotides.

The concentration of the oligonucleotide is preferably 50 pmol or more.

Preferably, the oligonucleotide is integrated on a support coated with a superaldehyde group by bonding an amine-modified dideoxythymidine of C6 as a linker.

The support is preferably selected from the group consisting of a glass slide, a paper, a nitrocellulose membrane, a microplate well, plastic, silicon, DVD and beads.

The sample may be a cervix or vaginal swab; Tissue of the cervix; Tissue of male penis; Pee; Anus, rectum, pharynx, oral or head and neck swabs.

The sample is preferably selected from the group consisting of cancer of male genitalia, cancer of male urinary tract, anal cancer, head and neck cancer, and precancerous cells thereof.

Also, the present invention provides a primer for amplifying an HPV E6 / E7 gene, wherein the primer is selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 31-88.

In addition, the present invention provides a kit for genotyping human papillomavirus (HPV) comprising the DNA chip of the present invention, the primer of the present invention and a labeling means for detecting the amplified DNA.

The primer may further comprise a primer for amplifying human GAPDH gene having the nucleotide sequence of SEQ ID NOS: 89 and 90.

The labeling means may be selected from the group consisting of Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM, FITC, Fluor X, Calcium red 610, Quasar 670, Biotin, Au, Ag, and polystyrene, such as Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor Orange 546 Is preferably selected from the group consisting of

(A) amplifying a target gene of the test sample by multiplex PCR using a primer selected from the group consisting of SEQ ID NOS: 31 to 88; (b) hybridizing the amplified PCR product to the DNA chip according to claim 1 or 2; And (c) detecting the hybridized product obtained by the hybridization. The present invention also provides a method for genotyping HPV of a human papilloma virus (HPV).

The DNA chip may further comprise one or more oligonucleotides selected from the group consisting of Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM, FITC, Fluor X, A group consisting of ROX, Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor Orange 546, Calfluor red 610, Quasar 670 and Biotin , ≪ / RTI >

The analysis method may further include a step of analyzing the plasmid vectors into which the E6 / E7 gene of the HPV type has been inserted as a positive control clone.

Wherein the specimen is selected from the group consisting of swabs of the cervix or vagina; Tissue of the cervix; Tissue of male penis; Pee; Anus, rectum, pharynx, oral or head and neck swabs.

The oligonucleotide synthesized in the present invention can be used as a novel type-specific primer that can complementarily bind HPV type HPV type of tumorigenic HPV type and specifically amplify the HPV type nucleic acid.

In particular, the oligonucleotide used in the production of the DNA chip according to the present invention can be utilized for the detection / diagnosis of the presence and type of HPV nucleic acid in the sample, and thus can be used for early screening of cervical cancer caused by HPV .

Since the HPV infection can be diagnosed prematurely through the gene approach using the genome according to the present invention, accurate and long-term accurate screening can be carried out, which is expected to prevent the cervical cancer.

In addition, HPV diagnosis can be performed at a relatively low cost, and it is possible to provide advanced results to the examinee at an early stage, thereby giving credibility and satisfaction. Also, I think you can take charge of.

Figure 1 shows the position of the conventional DNA chip gene sequence in the entire HPV gene sequence and the position of the gene sequence used in the DNA chip of the present invention.
2A to 2C show the results of immunohistochemical staining for the HPV-16 infected cell line Caski (ATCC CRL-1550), HPV-18 infected cell line HeLa (ATCC CCL-2) and HPV non- (KCLB-10243, Korean Cell Line Bank) as a target DNA, followed by DNA chip analysis.
FIGS. 3A to 3C are photographs showing the types of 10 cervical cancer-associated specimens and 5 normal specimens obtained by PCR using the primer set synthesized according to the present invention, respectively, and analyzing the types of DNAs by DNA chip analysis.

Hereinafter, the present invention will be described in detail.

The present invention provides a DNA chip for HPV genotype analysis of a specimen, which comprises an oligonucleotide selected from the group consisting of the nucleotide sequence of SEQ ID NOS: 1 to 29 or a complementary base sequence thereof do.

The DNA chip may include at least one oligonucleotide having the nucleotide sequence described above, and the oligonucleotide may be restricted to an appropriate number according to the genotype to be analyzed.

Preferably, the DNA chip is HPV-16, HPV-18, HPV-26, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV- HPV-6 and HPV-73 as low-risk HPV-6, and HPV-6 as low-risk HPV, HPV-52, HPV-53, HPV-56, HPV-58, HPV-59, HPV-66, HPV- For the analysis of HPV genotypes including HPV-11, HPV-34, HPV-40, HPV-43, HPV-44, HPV-54, HPV-55 and HPV-61.

The oligonucleotide is complementary to the type-specific E6 / E7 gene region of HPV.

A common oligonucleotide primer capable of hybridization or complementary binding to several types of HPV DNA present in a sample using conventional PCR techniques can be used to detect Not only is the number of types that can be diagnosed significantly limited, but sensitivity to HPV types is very low. As such, the existing PCR common primer for HPV detection / diagnosis was for oligonucleotides capable of complementarily binding to the L1 region in the HPV genome.

As described above, the E6 gene and the E7 gene constituting the HPV genome each have a specific nucleic acid sequence encoding a binding site with p53 or pRb, which is one of the tumor suppressor proteins of host cells, In closely related tumorigenic HPV types, such as high-risk HPV types, nucleic acid sequences encoding p53 or pRb binding sites are well conserved. In addition, since the HPV genomic E1 gene encodes a protein that initiates replication of the HPV genome, HPV is an essential sequence for growth in host cells.

Based on these findings, the present inventors have designed a type-specific primer capable of complementarily binding to the nucleic acid sequence of the tumorigenic HPV type and amplifying the nucleic acid sequence of the corresponding HPV type. Since the type-specific primers synthesized according to the present invention can specifically complement the E6 / E7 region in the HPV genome, the presence of HPV DNA in the sample can be confirmed by amplification of these genes in the sample.

Therefore, using the type-specific primers synthesized according to the present invention, it is possible to identify / detect the presence of the tumorigenic HPV type through a single PCR process, as well as accurately identify these types.

The DNA chip may include an oligonucleotide having a nucleotide sequence of SEQ ID NO: 30 or a complementary base sequence thereof complementarily binding to human GAPDH gene as a positive control.

Preferably, the DNA chip is divided into eight wells capable of accumulating oligonucleotides.

The concentration of each oligonucleotide to be integrated is preferably 50 pmol or more.

Preferably, the oligonucleotide is integrated on a support coated with a superaldehyde group by bonding an amine-modified dideoxythymidine of C6 as a linker.

The support is preferably selected from the group consisting of a glass slide, a paper, a nitrocellulose membrane, a microplate well, plastic, silicon, DVD and beads.

The sample may be a cervix or vaginal swab; Tissue of the cervix; Tissue of male penis; Pee; Anus, rectum, pharynx, oral or head and neck swabs.

The sample is preferably selected from the group consisting of cancer of male genitalia, cancer of male urinary tract, anal cancer, head and neck cancer, and precancerous cells thereof.

Also, the present invention provides a primer for amplifying an HPV E6 / E7 gene, wherein the primer is selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 31-88.

In addition, the present invention provides a kit for genotyping human papillomavirus (HPV) comprising the DNA chip of the present invention, the primer of the present invention and a labeling means for detecting the amplified DNA.

The primer may further comprise a primer for amplifying human GAPDH gene having the nucleotide sequence of SEQ ID NOS: 89 and 90.

The labeling means may be selected from the group consisting of Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM, FITC, Fluor X, Calcium red 610, Quasar 670, Biotin, Au, Ag, and polystyrene, such as Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor Orange 546 Is preferably selected from the group consisting of

(A) amplifying a target gene of the test sample by multiplex PCR using a primer selected from the group consisting of SEQ ID NOS: 31 to 88; (b) hybridizing the amplified PCR product to the DNA chip according to claim 1 or 2; And (c) detecting the hybridized product obtained by the hybridization. The present invention also provides a method for genotyping HPV of a human papilloma virus (HPV).

The DNA chip may further comprise one or more oligonucleotides selected from the group consisting of Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM, FITC, Fluor X, A group consisting of ROX, Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor Orange 546, Calfluor red 610, Quasar 670 and Biotin , ≪ / RTI >

The analysis method may further include a step of analyzing the plasmid vectors into which the E6 / E7 gene of the HPV type has been inserted as a positive control clone.

Wherein the specimen is selected from the group consisting of swabs of the cervix or vagina; Tissue of the cervix; Tissue of male penis; Pee; Anus, rectum, pharynx, oral or head and neck swabs.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the following examples are for the purpose of illustrating the present invention in detail, but the present invention is not limited by the following examples.

[ Example ]

1. Human papilloma virus type analysis chip chip Production

A DNA chip for the diagnosis of HPV type has a total of 29 kinds of probes capable of discriminating 29 types of HPV types in high-specificity (Table 1). An internal control was used to immobilize a probe for the human GAPDH gene. The chip layout was devised so that there were 8 grids (high-throughput analysis) of 29 kinds of genes on one chip substrate, and 8-well hybridization reaction chambers were used to prepare 8 individual samples And to analyze them simultaneously. The probe stock stored at -70 ° C was thawed at room temperature, diluted to a final concentration of 100 M in deionized sterilized tertiary distilled water, and used as a working stock. Each of the probes diluted 50-fold with Walking Stack was mixed with 3X SSC spotting solution (500 mM NaCl, 3 mM sodium citrate, 1.5 MN, N, N-trimethylglycine, pH 6.8) at a ratio of 1: 5-10 And the concentration of the probe to be dispensed in a final 96-well plate was adjusted to 20 - 30 pmole / uL. The plate was mounted on a Microssys 5100 microarrayer (Cartesian Technologies, Ann Arbor, MI, USA) from which aldehyde-, thioisocyanate-activated glass slides (CEL Associates Inc., Houston, , Epoxy-activated plastic chips, or probes on the Gold film surface were spotted in duplicate in duplicate. The average spot diameter was 80-140 micrometers and the spacing between spots was maintained at 350-500 micrometers to minimize the cross-talk effect between spots. Chip fabrication was performed in a class 10,000 room maintaining 75% humidity. The chip on which the probe was spotted was baked at 120 ° C for 1 hour, washed in 0.25% SDS (sodium dodecyl sulfate) solution for 3 minutes, and then washed again with sterilized tertiary distilled water. The chip was then blocked with a solution containing 0.2% sodium borohydride (NaBH ₄ ) to block the probe. Thereafter, it was washed twice with the third distilled water, and the water was removed and stored in a dessicator until the time of use.

SEQ ID NO: designation The sequence (5 '- > 3') Location (bp) One HPV-16 ATCATGCATGGAGATACACCTACATTGCATG 559-589 2 HPV-18 CAACATTGCAAGACATTGTATTGCATTTAGAG 606-637 3 HPV-26 CAGTGGAAAGGGTTGTGTACAAATTGTTGG 565-596 4 HPV-30 CGTCCACTGAGACAGCAGTATAATCATGC 624-655 5 HPV-31 CTGACCTCCACTGTTATGAGCAATTACCC 618-646 6 HPV-33 ACGTAGAGAAACTGCACTGTGACGTGTAAA 537-566 7 HPV-35 TGTATTACATGTCAAAAACCGCTGTGTCCAGTT 416-448 8 HPV-39 AAGAGAAACCCAAGTATAACATCAGATATGCG 565-596 9 HPV-45 TGCATTTGGAACCTCAGAATGAATTAGATCCT 624-655 10 HPV-51 ATGTACCACAATTAAAAGATGTAGTATTGCAT 570-601 11 HPV-52 ACCCCGACCTGTGACCCAAGTGTAAC 524-549 12 HPV-53 CACTTCCACAATATATTATAGAACTTATACCA 588-619 13 HPV-56 CTGCAAGACGTTGTATTAGAACTAACACCT 593-622 14 HPV-58 CCATGAGAGGAAACAACCCAACGCTAAG 572-599 15 HPV-59 AACAATGCATGGACCAAAAGCAACACTTTG 538-567 16 HPV-66 TACCAACGTTGCAAGAGGTTATATTAGAACTTG 588-618 17 HPV-67 AGTGTGTTGGAGACCTCAACGAACG 512-538 18 HPV-68 TGCAATGAAATAGAGCCGGTCGACCTTG 4428-4455 19 HPV-70 CGGTCGACCTTGTATGTCACGAGCAATTAGA 659-689 20 HPV-73 AGGCGATATAGACAATCAGTATATGGCACT 333-362 21 HPV-6 AGGTAAAACATATACTAACCAAGGCGCGG 448-476 22 HPV-11 TTACCTGTGTCACAAGCCGTTGTGTGAAA 413-441 23 HPV-34 TAGAAGATATAACCAATCAGTGTATGGACGG 332-362 24 HPV-40 CTGCACCCTGAACCTGTATGTCTAAACT 569-596 25 HPV-43 CATGGAAAAAAGCCGACGATCAGAGACTATG 591-616 26 HPV-44 GAAACAAATAAGTCAATTCTGGACGTGCTG 378-407 27 HPV-54 ACACAGGCATAAGGGTACTGCAGGAACTG 558-589 28 HPV-55 CGTTCGGCTGGTTGTGCAGTGCACAGGAAC 595-612 29 HPV-61 AACCATAAAGGACATAGTCCTTGAAGAGCG 535-564 30 Internal Control CTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACT

2. HPV E6 / E7  For specific gene amplification Primer  synthesis

In the present invention, a primer selects a target region so as to enable specific amplification by type, i.e., HPV type. In addition, multiple alignment was performed using the ClustalW method to select target specific site sequences if necessary, thereby selecting specific primer target sites. Primer premier version 5 (Premier Biosoft International, Palo Alto, Calif., USA), DNASIS MAX Version 2.7 (MiraiBio Group, South San Francisco, CA, USA) program was applied from the selected primer target sites The type-specific or gene-specific frit (Tm) was applied at 74-80 ° C, and the GC content at the 3 'end should not be high, so that no more than four bases in the primer sequence are complementary in succession The formation of hairpin secondary structure was prevented and the formation of primer dimer was prevented by not allowing three or more identical bases to be continuously located at the 3 'end. In addition, high specificity was ensured by not including single nucletide polymorphism (SNP) in the target site. In addition, it can be confirmed that the primer base sequence does not exhibit cross-reactive characteristics with other gene or pathogen base sequences constituting the target chip of the present invention, using a variety of base sequence search tools such as the US NCBI base sequence database Respectively. The primers used in the present invention were synthesized through the US IDT (Integrated DNA Technologies, Inc., San Jose, Calif., USA) (Table 2).

type SEQ ID NO: forward primer (5'-3 ') SEQ ID NO: reverse primer (5'-3 ') TSP-16 31 GAGCGACCCAGAAAGTTACCACAGTTATGCACAGAG 32 GCACACAATTCCTAGTGTGCCCATTAACAGGTC TSP-18 33 GAGGATCCAACACGGCGACCCTACAAGCTAC 34 TGCTGAGCTTTCTACTACTAGCTCAATTCTGGC TSP-26 35 GAGCTATGTGAAAGCTTGAATACTACTTTGC 36 GTGCAGCACACTGATGGCACACC TSP-30 37 CACCATCTTTGTGAGGTACAAGAAACATCGTTGC 38 GCACACAGGGGACACACTAGCTCCAG TSP-31 39 CCTGCAGAAAGACCTCGGAAATTGCATGAAC 40 GGCACACGATTCCAAATGAGCCCA TSP-33 41 GCAGTAAGGTACTGCACGACTATGTTTCAAGACACTG 42 TAGGTCACTTGCTGTACTGTTGACACATAAACGAACTG TSP-35 43 TTACAAACTGCATGATTTGTGCAACGAGGTAGAAG 44 GAACAGCCGGGGCACACTATTCCAAATG TSP-39 45 TAGCCTGTGTCTATTGCAGACGACCAC 46 GTTGCACACCACGGACACACAAATCCTAGTG TSP-45 47 TACAAGACGTATCTATTGCCTGTGTATATTGCAAAGC 48 ACGGACACACAAAGGACAAGGTGCTC TSP-51 49 CAAGAGGGAAAGACCACGAACGCTGCATG 50 TAACATCTGCTGTACAACGCGAAGGGTGTC TSP-52 51 CGGCCATGTTTGAGGATCCAGCAACAC 52 CAACAGCATTTGCTGTAGAGTACGAAGGTCCGTC TSP-53 53 GCACTGTGTACAACACCCAGGACATCC 54 CTGCACCAACGACTCACACCTACAACACTG TSP-56 55 CCACAATTCAACAATCCACAGGAACGTCCACG 56 CAGGTCCTCTTTGGTACTCTGAATGTCCAACTG TSP-58 57 GATTTGTGTCAGGCGTTGGAGACATCTGTGCATG 58 GTGCACAGCTAGGGCACACAATGGTACATG TSP-59 59 ATTTGAGCACAACATTGAATATTCCTCTGCATGATATTCGC 60 CAGCTGCTGTAAGGCTCGCAATCCGTC TSP-66 61 CCATATTCAGCAATACACAGGAACGTCCACGAAGC 62 CGTAGCTCCTCTTTGGTACTCTGAATGTCCAACTGC TSP-67 63 CAGACGAAAAACCACGCAACCTGCACGAATTGTG 64 CTATTCCTAGTGTGTTCATAAGCATCTGCTGGATTGTTCGG TSP-68 65 CATTGGACACCACATTGCATGACGTTACAATAGACTG 66 GTCCATAAACAGCAGTTCTACGTTCCGCAGG TSP-70 67 TAGACTGTGTCTATTGTAAAACACAGCTACAGCAAAC 68 GATGCACACCAGGGACACACAAATGACAGTG TSP-73 69 GTTTCCCAATTCAGAAGAACGACCATACAAGCTAC 70 GGCACACAATACCTAGTGTACCCATAAGCAACTC TSP-6 71 GGCATTATGGAAAGTGCAAATGCCTCCACGTCTGCAAC 72 GGTCTTCGGTGCGCAGATGGGACACACTATGTTTAG TSP-11 73 GCAGACGAGGCATTATGGAAAGTAAAGATGCCTCCACG 74 GTGTGCCCAGCAAAAGGTCTTGTAGTTGTCTGATGTCTC TSP-34 75 CAATCCTGAGGAACGGCCATACAAGCTACCAGC 76 CACCCATAAGCAGGTCTTCTAACACTAATAGGTCAGCG TSP-40 77 CCAGGACCCTGTATGAACTGTGTGACCAGTGC 78 CACTCTGTAGCTGCACAGTTGGGGCACACTATATGTAATGTG TSP-43 79 GACTGCACGTAGCTGCTCCCAAAACG 80 CCCAACAGCAGGTCTTCTAGCTTCTTGATG TSP-44 81 GCAAGCGGGGCATAATGGAAAGTGCAAATGC 82 CACACAGGACACAATATATCCAGTGAACCCAGCAG TSP-54 83 CCGAAACCGGTACATATAAAAGCGGTTGTAGAAAACAG 84 GTCGTGAAGCACAGGTGGGACACACTATTTGCAGTGC TSP-55 85 GCAAGCGGGGCATAATGGAAAGTGCAAATGC 86 CAGGACACAGTATTTCCAGTGAACCCAGCAGAAGCGTATG TSP-61 87 CGTAGGGTCAGCAAAGCACACTCATCTATGGGACCG 88 CAGCCAGGACACACTATGGACAAGTCGCCCAGCAG internal control 89 CCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCC 90 GCAGAATCCAGATGCTCAAGGCCCTTCATAATATCC

Primers used for the diagnosis of HPV type include primers labeled with fluorescent dye at the 5 'end or 3' end of the DNA sequence, or a general primer with no fluorescent dye labeled, and a fluorescent dye Both methods of labeling the amplified product by inducing labeled deoxyribonucleotide triphosphate to be inserted into the amplification product were confirmed. In addition, in order to diagnose HPV type, a primer labeled with a fluorescent dye is used in the secondary PCR step after the primary screening, or the fluorescent dye is labeled during the amplification reaction.

Fluorescent pigments used in the present invention include 6-carboxyfluorescein, Cy5, Cy3, Cy5.5, 6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein, Rhodamine Green , TAMRA NHS (N-hydroxysuccinimide) Ester, and Texas Red. The concentrations of the primers were determined by ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, Maine, USA) and resuspension with tertiary deionized sterile distilled water to a final concentration of 50 - 200 pmole / And stored frozen at -70 ° C.

3. Clinical sample collection and DNA  detach

For non-invasive diagnosis, efficient DNA isolation from cervical cytology, urine and other specimens was one of the prerequisites. The specimens were collected and transferred to the viral transport media. Bacterial genomic DNA was isolated from the specimen for the diagnosis of HPV type, and a commercially available DNA extraction kit (LaboPass Tissue kit, Kosomjin Tech, Seoul, Korea) was used. If other samples including the cervical cytology were low in viscosity and pipetting was possible, 0.3 mL of the sample was transferred to a 1.5 mL Eppendorf tube without using 2N sodium hydroxide (NaOH). In the case of highly viscous samples, 2N NaOH was added in the same amount as the sample to liquefy the sample. When adding NaOH, the sample should be poured with a tip, and the sample should be loosened so that 0.5 mL can be accurately taken with a pipette. The Eppendorf tube containing the sample was centrifuged at 12,000 rpm (revolutions per minute) for 1 minute using a 1524M high-speed centrifuge (Hanil Scientific., Korea). The supernatant was removed and the precipitate was dissolved in 300 μL of PBS, followed by centrifugation under the same conditions. To the precipitate was added 200 μL of a TG buffer solution to dissolve the sample, and 20 μL of protease K (100 mg / mL) was added to the precipitate, followed by vortexing with weak protease K (100 mg / mL) . Then, 400 μL of TB buffer solution was added to the reaction mixture, and the reaction mixture was mixed with stirring. The reaction was then loaded on top of a spin column and centrifuged at 12,000 rpm for 2 minutes. The collection tube was replaced with 500 μL of WB buffer to remove impurities from the membrane and centrifuged at 12,000 rpm for 2 minutes. Then, the upper part of the spin column was transferred to a new Eppendorf tube, and 70 μL of de-ionized sterilized tertiary distilled water was loaded for 2 minutes at room temperature and centrifuged at 12,000 rpm for 2 minutes to elute the DNA. .

4. Multiplex PCR Human papilloma virus target  Gene amplification

Prior to the amplification of the DNA chip, the target genes of the chip reaction were amplified by multiplex PCR using the viral DNA isolated from clinical specimens as a template. The amplified amplicon then forms a duplex with the DNA probes on the chip surface, thus confirming the result of HPV typing. The multiplex PCR according to a preferred embodiment of the present invention comprises a human GAPDH gene as an internal control.

The final concentration of the PCR buffer solution contained in the HPV 29 PCR multiplexed reaction mixture preferably described in this Example was 50 mM KCl, 3.5 mM MgCl ₂ , 10 mM Tris-HCl, pH 8.2, 2.5 units of Taq Polymerase, 300 μM dNTPs (Boehringer Mannheim, Mannheim, Germany) and 10 mg / mL bovine serum albumin. The sense primer of the individual genes included in the multiplex PCR for the diagnosis of HPV type can be synthesized by labeling Cy3 or Cy5 fluorescent dye at the 5'- or 3'-end, or without modification at both ends nascent oligonucleotides (Integrated DNA Technologies, Inc., Coralville, Iowa, USA).

In a multiplex PCR kit comprising a primer having no fluorescent pigment modification at the terminal according to a preferred embodiment of the present invention, the deoxycytosine triphosphate (Cy3-dCTP) labeled with Cy3 fluorescent dye constitutes an amplification product The sensitivity and specificity of the reaction can be improved. The PCR buffer used for the 29 multiplex multiplex PCR reaction using Cy3-dCTP was 50 mM KCl, 3.5 mM MgCl ₂ , 10 mM Tris-HCl, pH 8.2, 2.5 units of Taq polymerase, 300 μM dATP , 300 μM dGTP, 300 μM dTTP, 25 μM dCTP, 275 μM Cy3-dCTP (Fluorolink Cy3-dCTP, Amersham Pharmacia Biotech AB, Piscataway, NJ, USA) and 10 mg / mL bovine serum albumin To specifically amplify target genes.

5. Human Papillomavirus Type Analysis Chip Hybridization Response and Results Analysis

A total of 30-60 μL of each of the 29 kinds of HPV multiplex PCR amplification products according to the preferred embodiment of the present invention were added to 1.5 mL Eppendorf tubes pre-added with deionized tertiary sterile distilled water of the same volume Lt; / RTI > The mixture was softly vortexed, denatured at 95 ° C for 5 minutes, stored on ice for 5 minutes, and spun down with a centrifuge. An 8-well hybridization chamber was placed on the chip surface and the well upper layer was covered with a well cover. Then, 60 to 80 μL of the hybridization reaction solution (3 × SSC, 0.1% SDS, 0.2 mg / mL bovine serum albumin, pH (pH)) preliminarily heated to 56 ° C. as the hybridization reaction temperature was added to the reaction mixture solution in the well 7) were added and mixed. Then, the reaction solution mixture was injected through the hole of the well cover, and care was taken so that no bubble would occur. After ligation of the chamber lid, hybridization reaction at 56 ° C for 30 min resulted in specific nucleotide complementary binding between the probe on the chip surface and the multiplex PCR reaction product. The well cover on the surface of the hybridized chip was removed and the chip was washed in washing buffer 1 (0.1X SSC, 0.05% SDS) solution for 2 minutes while stirring at 2,000 rpm and repeated. Then, washing was carried out with a washing solution 2 (2X SSC, 0.1% SDS) solution at 2,000 rpm for 2 minutes with stirring and repeated. Then, it was immersed in deionized tertiary sterilized distilled water, washed twice, and centrifuged at 1,000 rpm to dry the chip. The chip was read using a ScanArray Lite scanner (Packard Instrument Co., Meriden, CT, USA) and the mean fluorescence intensity of the positive control spots and standard The signal-to-noise (S / R) ratio was calculated by comparing the error with the values around the spot, and scoring was performed with a positive value when the value was 4 or more.

6. Probe  Test

Table 3 shows DNA chips for confirming the minimum detection limit of probes for 29 kinds of HPV (SEQ ID NOS: 1 to 29). Fluorescence signal intensity was measured and analyzed using Genepix 4100A (Epson, USA) for the analysis of fluorescence signals.

As shown in Table 3, the intensity of fluorescence signal between the probes was analyzed using Genepix 4100A (Epson, USA) software, and the sensitivity of S / N value was 11.25 ~ 13.32 as the minimum detection limit.

7. HPV -16, -18 infected cell lines and Noninflammatory  Cell line experiment

FIGS. 2A to 2C are photographs showing the results of PCR performed after performing PCR on Caski, HPV-18 infected cell line HeLa and HPV non-infected cell line K562 using the primers synthesized according to the present embodiment to be. The probes reacted only with the HPV-16 cell line Caski and the HPV-18 cell line, HeLa, in the chip hybridization reaction and the HPV-noninflammatory cell line with the probe in the chip hybridization reaction with K562 cells.

8. Clinical test

In this example, clinical samples were examined for HPV infection and cervical cancer according to conventional cytology. For this purpose, 15 clinical specimens submitted to the Ewon Medical Foundation were analyzed by colposcopy, cervicography, biopsy, and pap-smear, And other medical examinations. The results of the screening according to the cytotoxic assay according to this Example are shown in Table 4 below.

Subject No. Pathological grade One LSIL ^* 2 LSIL 3 LSIL 4 HSIL ^** 5 LSIL 6 HSIL 7 HSIL 8 SCC ^*** 9 SCC 10 HSIL 11 Squamous ^**** 12 Squamous 13 Squamous 14 Squamous 15 Squamous

^* : Low-grade SIL;

^** : High-grade SIL;

^*** : Squamous intraepithelial lesion

^**** : Squamous cell

As a result of the screening, as shown in Table 5, subjects 1, 2, 3 and 5 had low-grade SIL, subjects 4, 6, 7 and 10 had high-grade SIL, 8 and 9 were diagnosed as squamous intraepithelial lesions (SIL). The remaining subjects (subjects 11 to 15) were diagnosed as non-related cervical cancer patients having normal cells in the cervical cancer cell smear test according to this example.

In this example, the clinical samples were screened for the presence of HPV DNA. For this purpose, the DNA chip of the present invention, which is complementary to HPV DNA, was used. The results are shown in Table 5 and FIGS. 3A to 3O.

Subject No. DNA testing One positivity 2 positivity 3 positivity 4 positivity 5 positivity 6 positivity 7 positivity 8 positivity 9 positivity 10 positivity 11 voice 12 voice 13 voice 14 voice 15 voice

In the HPV DNA test according to the present embodiment, all of the samples obtained from the subjects (Subjects 1 to 10) related to cervical cancer were screened for HPV DNA and the subjects not related to cervical cancer (subjects 11 The HPV DNA was not selected in all of the samples obtained from the subjects 15 to 15.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. It is obvious that it is possible. It will be apparent, however, that such modifications and variations are all within the scope of the present invention.

<110> daiogene <120> DNA chip for determining genomic types of human papillomavirus,          kit of the same and determinig method of genomic types of          human papillomavirus using the same <160> 90 <170> Kopatentin 2.0 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> HPV-16 probe <400> 1 atcatgcatg gagatacacc tacattgcat g 31 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> HPV-18 probe <400> 2 caacattgca agacattgta ttgcatttag ag 32 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HPV-26 probe <400> 3 cagtggaaag ggttgtgtac aaattgttgg 30 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HPV-30 probe <400> 4 cgtccactga gacagcagta taatcatgc 29 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HPV-31 probe <400> 5 ctgacctcca ctgttatgag caattaccc 29 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HPV-33 probe <400> 6 acgtagagaa actgcactgt gacgtgtaaa 30 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> HPV-35 probe <400> 7 tgtattacat gtcaaaaacc gctgtgtcca gtt 33 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> HPV-39 probe <400> 8 aagagaaacc caagtataac atcagatatg cg 32 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> HPV-45 probe <400> 9 tgcatttgga acctcagaat gaattagatc ct 32 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> HPV-51 probe <400> 10 atgtaccaca attaaaagat gtagtattgc at 32 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> HPV-52 probe <400> 11 accccgacct gtgacccaag tgtaac 26 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> HPV-53 probe <400> 12 cacttccaca atatattata gaacttatac ca 32 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HPV-56 probe <400> 13 ctgcaagacg ttgtattaga actaacacct 30 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> HPV-58 probe <400> 14 ccatgagagg aaacaaccca acgctaag 28 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HPV-59 probe <400> 15 aacaatgcat ggaccaaaag caacactttg 30 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> HPV-66 probe <400> 16 taccaacgtt gcaagaggtt atattagaac ttg 33 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> HPV-67 probe <400> 17 agtgtgttgg agacctcaac gaacg 25 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> HPV-68 probe <400> 18 tgcaatgaaa tagagccggt cgaccttg 28 <210> 19 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> HPV-70 probe <400> 19 cggtcgacct tgtatgtcac gagcaattag a 31 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HPV-73 probe <400> 20 aggcgatata gacaatcagt atatggcact 30 <210> 21 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HPV-6 probe <400> 21 aggtaaaaca tatactaacc aaggcgcgg 29 <210> 22 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HPV-11 probe <400> 22 ttacctgtgt cacaagccgt tgtgtgaaa 29 <210> 23 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> HPV-34 probe <400> 23 tagaagatat aaccaatcag tgtatggacg g 31 <210> 24 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> HPV-40 probe <400> 24 ctgcaccctg aacctgtatg tctaaact 28 <210> 25 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> HPV-43 probe <400> 25 catggaaaaa agccgacgat cagagactat g 31 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HPV-44 probe <400> 26 gaaacaaata agtcaattct ggacgtgctg 30 <210> 27 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HPV-54 probe <400> 27 acacaggcat aagggtactg caggaactg 29 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HPV-55 probe <400> 28 cgttcggctg gttgtgcagt gcacaggaac 30 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HPV-61 probe <400> 29 aaccataaag gacatagtcc ttgaagagcg 30 <210> 30 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> GAPDH probe <400> 30 ctattaaagg ttcctttgtt ccctaagtcc aact 34 <210> 31 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> TSP-16 forward primer <400> 31 gagcgaccca gaaagttacc acagttatgc acagag 36 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TSP-16 reverse primer <400> 32 gcacacaatt cctagtgtgc ccattaacag gtc 33 <210> 33 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> TSP-18 forward primer <400> 33 gaggatccaa cacggcgacc ctacaagcta c 31 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TSP-18 reverse primer <400> 34 tgctgagctt tctactacta gctcaattct ggc 33 <210> 35 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> TSP-26 forward primer <400> 35 gagctatgtg aaagcttgaa tactactttg c 31 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TSP-26 reverse primer <400> 36 gtgcagcaca ctgatggcac acc 23 <210> 37 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> TSP-30 forward primer <400> 37 caccatcttt gtgaggtaca agaaacatcg ttgc 34 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> TSP-30 reverse primer <400> 38 gcacacaggg gacacactag ctccag 26 <210> 39 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> TSP-31 forward primer <400> 39 cctgcagaaa gacctcggaa attgcatgaa c 31 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TSP-31 reverse primer <400> 40 ggcacacgat tccaaatgag ccca 24 <210> 41 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> TSP-33 forward primer <400> 41 gcagtaaggt actgcacgac tatgtttcaa gacactg 37 <210> 42 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TSP-33 reverse primer <400> 42 taggtcactt gctgtactgt tgacacataa acgaactg 38 <210> 43 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> TSP-35 forward primer <400> 43 ttacaaactg catgatttgt gcaacgaggt agaag 35 <210> 44 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> TSP-35 reverse primer <400> 44 gaacagccgg ggcacactat tccaaatg 28 <210> 45 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> TSP-39 forward primer <400> 45 tagcctgtgt ctattgcaga cgaccac 27 <210> 46 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> TSP-39 reverse primer <400> 46 gttgcacacc acggacacac aaatcctagt g 31 <210> 47 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> TSP-45 forward primer <400> 47 tacaagacgt atctattgcc tgtgtatatt gcaaagc 37 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> TSP-45 reverse primer <400> 48 acggacacac aaaggacaag gtgctc 26 <210> 49 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> TSP-51 forward primer <400> 49 caagagggaa agaccacgaa cgctgcatg 29 <210> 50 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> TSP-51 reverse primer <400> 50 taacatctgc tgtacaacgc gaagggtgtc 30 <210> 51 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> TSP-52 forward primer <400> 51 cggccatgtt tgaggatcca gcaacac 27 <210> 52 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> TSP-52 reverse primer <400> 52 caacagcatt tgctgtagag tacgaaggtc cgtc 34 <210> 53 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> TSP-53 forward primer <400> 53 gcactgtgta caacacccag gacatcc 27 <210> 54 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> TSP-53 reverse primer <400> 54 ctgcaccaac gactcacacc tacaacactg 30 <210> 55 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> TSP-56 forward primer <400> 55 ccacaattca acaatccaca ggaacgtcca cg 32 <210> 56 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TSP-56 reverse primer <400> 56 caggtcctct ttggtactct gaatgtccaa ctg 33 <210> 57 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> TSP-58 forward primer <400> 57 gatttgtgtc aggcgttgga gacatctgtg catg 34 <210> 58 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> TSP-58 reverse primer <400> 58 gtgcacagct agggcacaca atggtacatg 30 <210> 59 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> TSP-59 forward primer <400> 59 atttgagcac aacattgaat attcctctgc atgatattcg c 41 <210> 60 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> TSP-59 reverse primer <400> 60 cagctgctgt aaggctcgca atccgtc 27 <210> 61 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> TSP-66 forward primer <400> 61 ccatattcag caatacacag gaacgtccac gaagc 35 <210> 62 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> TSP-66 reverse primer <400> 62 cgtagctcct ctttggtact ctgaatgtcc aactgc 36 <210> 63 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> TSP-67 forward primer <400> 63 cagacgaaaa accacgcaac ctgcacgaat tgtg 34 <210> 64 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> TSP-67 reverse primer <400> 64 ctattcctag tgtgttcata agcatctgct ggattgttcg g 41 <210> 65 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> TSP-68 forward primer <400> 65 cattggacac cacattgcat gacgttacaa tagactg 37 <210> 66 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> TSP-68 reverse primer <400> 66 gtccataaac agcagttcta cgttccgcag g 31 <210> 67 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> TSP-70 forward primer <400> 67 tagactgtgt ctattgtaaa acacagctac agcaaac 37 <210> 68 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> TSP-70 reverse primer <400> 68 gatgcacacc agggacacac aaatgacagt g 31 <210> 69 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> TSP-73 forward primer <400> 69 gtttcccaat tcagaagaac gaccatacaa gctac 35 <210> 70 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> TSP-73 reverse primer <400> 70 ggcacacaat acctagtgta cccataagca actc 34 <210> 71 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TSP-6 forward primer <400> 71 ggcattatgg aaagtgcaaa tgcctccacg tctgcaac 38 <210> 72 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> TSP-6 reverse primer <400> 72 ggtcttcggt gcgcagatgg gacacactat gtttag 36 <210> 73 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TSP-11 forward primer <400> 73 gcagacgagg cattatggaa agtaaagatg cctccacg 38 <210> 74 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> TSP-11 reverse primer <400> 74 gtgtgcccag caaaaggtct tgtagttgtc tgatgtctc 39 <210> 75 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TSP-34 forward primer <400> 75 caatcctgag gaacggccat acaagctacc agc 33 <210> 76 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TSP-34 reverse primer <400> 76 cacccataag caggtcttct aacactaata ggtcagcg 38 <210> 77 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> TSP-40 forward primer <400> 77 ccaggaccct gtatgaactg tgtgaccagt gc 32 <210> 78 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> TSP-40 reverse primer <400> 78 cactctgtag ctgcacagtt ggggcacact atatgtaatg tg 42 <210> 79 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> TSP-43 forward primer <400> 79 gactgcacgt agctgctccc aaaacg 26 <210> 80 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> TSP-43 reverse primer <400> 80 cccaacagca ggtcttctag cttcttgatg 30 <210> 81 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> TSP-44 forward primer <400> 81 gcaagcgggg cataatggaa agtgcaaatg c 31 <210> 82 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> TSP-44 reverse primer <400> 82 cacacaggac acaatatatc cagtgaaccc agcag 35 <210> 83 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TSP-54 forward primer <400> 83 ccgaaaccgg tacatataaa agcggttgta gaaaacag 38 <210> 84 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> TSP-54 reverse primer <400> 84 gtcgtgaagc acaggtggga cacactattt gcagtgc 37 <210> 85 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> TSP-55 forward primer <400> 85 gcaagcgggg cataatggaa agtgcaaatg c 31 <210> 86 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> TSP-55 reverse primer <400> 86 caggacacag tatttccagt gaacccagca gaagcgtatg 40 <210> 87 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> TSP-61 forward primer <400> 87 cgtagggtca gcaaagcaca ctcatctatg ggaccg 36 <210> 88 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> TSP-61 reverse primer <400> 88 cagccaggac acactatgga caagtcgccc agcag 35 <210> 89 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 89 cccacaagta tcactaagct cgctttcttg ctgtcc 36 <210> 90 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 90 gtccataaac agcagttcta cgttccgcag g 31

Claims (18)

A DNA chip for human papillomavirus (HPV) genotyping, comprising at least one oligonucleotide selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 29 or their complementary nucleotide sequences.
The method of claim 1, wherein the DNA chip is a high risk HPV 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-58, HPV-59, HPV-66, HPV-67, HPV-68, HPV-70, and HPV-73 and HPV- as low risk HPV 6, HPV-11, HPV-34, HPV-40, HPV-43, HPV-44, HPV-DNA chip, characterized in that for the analysis of HPV genotypes, including HPV-61.
The DNA chip according to claim 1 or 2, wherein the oligonucleotide binds complementarily with a type-specific E6 / E7 gene region of HPV.
The DNA chip according to claim 1 or 2, wherein the DNA chip comprises an oligonucleotide having a nucleotide sequence of SEQ ID NO: 30 or a complementary nucleotide sequence thereof that binds complementarily with a human GAPDH gene as a positive control. chip.
The DNA chip according to claim 1 or 2, wherein the DNA chip is divided into eight wells capable of accumulating oligonucleotides.
The DNA chip according to claim 1 or 2, wherein the concentration of the oligonucleotide is 50 pmol or more.
The DNA chip according to claim 1 or 2, wherein the oligonucleotide is bonded onto a support coated with a superaldehyde group by combining C6 amine-modified dideoxythymidine as a linker.
8. The DNA chip of claim 7, wherein the support is selected from the group consisting of glass slides, paper, nitrocellulose membranes, microplate wells, plastics, silicon, DVDs and beads.
The cervical or vaginal swab according to claim 1 or 2, wherein the sample comprises: a cervical or vaginal swab; Tissue of the cervix; Tissue of male penis; Pee; DNA chip, characterized in that selected from the group consisting of a swab of the anus, rectum, pharynx, oral cavity or head and neck.
The DNA chip according to claim 1 or 2, wherein the specimen is selected from the group consisting of cancer of male genital organs, cancer of male urinary tract, anal cancer, head and neck cancer, and precancerous cells thereof.
Primer for HPV E6 / E7 gene amplification, characterized in that selected from the group consisting of the nucleotide sequence of SEQ ID NO: 31 to 88.
A kit for genotyping of human papillomavirus (HPV), comprising a DNA chip according to claim 1 or claim 2, a primer of claim 11 and a labeling means for detecting amplified DNA.
The kit of claim 12, wherein the primer further comprises a primer for amplifying a human GAPDH gene having the nucleotide sequences of SEQ ID NOs: 89 and 90.
The method of claim 12, wherein the label means Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM, FITC, Fluor X, ROX, Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor Orange 546, Calfluor red 610, Quasar 670, Biotin Kit selected from the group consisting of Au, Ag and polystyrene.
Genotyping of human papillomavirus (HPV) comprising the following steps:
(a) amplifying a target gene of a sample by a multiplex PCR method using a primer selected from the group consisting of SEQ ID NOs: 31 to 88;
(b) hybridizing the amplified PCR product to a DNA chip according to claim 1 or 2; And
(c) detecting the hybridization product obtained by the hybridization.
The method of claim 15, wherein the DNA chip is oligonucleotide Cy3, Cy5, Cy5.5, Bodipy, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 660, Rhodamine, TAMRA, FAM , FITC, Fluor X, ROX, Texas Red, Orange green 488X, Orange green 514X, HEX, TET, JOE, Oyster 556, Oyster 645, Bodipy 630/650, Bodipy 650/665, Calfluor Orange 546, Calfluor red 610, Quasar 670 and biotin, characterized in that the labeling with a substance selected from the group consisting of.
The analysis method according to claim 15, further comprising analyzing the plasmid vectors into which the E6 / E7 gene of the HPV type according to claim 2 is inserted, as positive control clones.
The method of claim 15, wherein the sample comprises: a cervical or vaginal swab; Tissue of the cervix; Tissue of male penis; Pee; Analytical method characterized in that it is selected from the group consisting of a swab of the rectum, rectum, pharynx, oral cavity or head and neck.

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