KR100979271B1 - Dna chip, kit for detecting genotype of human papillomavirus and detection method using the same - Google Patents

Abstract

A probe, a test method, and a kit used to test a human Papillomavirus (HPV) genotype using a polymerase chain reaction (PCR) method and a DNA chip are disclosed. In addition, it can test 36 types of HPV genotypes with high specificity, and it is also possible to quickly detect a case of overlapping infection of HPV which cannot be confirmed by the existing method of testing HPV.

Human papillomavirus, PCR, genotype, probe, fluorescence, primer, HPV gene

Description

DNA chip for human papilloma virus genotyping, kit and test method using same {DNA CHIP, KIT FOR DETECTING GENOTYPE OF HUMAN PAPILLOMAVIRUS AND DETECTION METHOD USING THE SAME}

The present invention relates to a gene chip, kit for testing human papillomavirus (HPV) genotype, and a test method thereof. More specifically, the present invention relates to a method of testing HPV genotype using a polymerase chain reaction (PCR) method and a DNA chip.

Conventional cervical cancer screening is mainly examined for cervical cell dysplasia by using a plasma smear and a microscope. Cell smears depend on human vision under a microscope, often resulting in both positive and false negatives, and require highly trained personnel to distinguish cell dysplasia. We need a way to test it. However, as the US Food and Drug Administration (FDA) approved a test for HPV infection in March 2003, the HPV test centered on which test method to choose.

Recently, molecular molecular methods have revealed that the cause of cervical cancer is human papillomavirus (HPV), which has been reported to be more effective than cell smear for screening purposes. HPV is a cyclic double-stranded DNA virus of about 8,000 base pairs. To date, about 100 different genotypes of HPV are known and low-risk groups (HPV-6, -11, -42, -43, -44, -55, etc.) and high risk groups (HPV -16, -18. -31. -33. -35, -39, -45, -51, -52, -56, -58, -59, etc.) genotype (see Schiffiman MM; J Natl). Cancer Inst 1992; 84: 394-398, IARC working Group, IRAC, Lyon; 1995, Bosch FX et al., J Natl Cancer Inst 1995; 87; 796-802, Ferenczy A, Int J Gynecol Cancer 1994; 4; 73-78, Kataja V, et al., 1992; Sex Transm Dis; 19; 154). Therefore, HPV genotyping is a very important test for the diagnosis and prevention of cervical cancer.

Molecular biological methods for testing HPV genotypes have been based on Southern blotting hybridization, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism, and DNA-RNA hybridization. Hybrid capture method (Hybride Capture, Digene, USA) is used (Reference: Smith HL, et al., J Gen Virol , 1992; 73; 3236-3268, Lungnu OX, et al., JAMA 1989; 267; 2493-2496, Manos MM, et al., Cancer Cell 1989; 7; 209-214, Lorincz AT, J Obstet Genaecol Res , 1996; 22; 629-639). However, these methods are difficult to use for routine testing because of the risks associated with the use of radioisotopes and cancer-causing gene staining reagents, difficulties in sample processing, and time-consuming and laborious efforts.

Recently used in many laboratories, Digene's Hybrid Capture HPV test kit can classify HPV genotypes into high and low risk groups, cannot test each genotype, and is expensive. Therefore, there is a need for HPV genotyping that can be conveniently used by anyone in the daily routine without the above problems from a large number of specimens.

An object of one embodiment of the present invention is to solve the above problems.

Another object of one embodiment of the present invention is to provide a method capable of simultaneously testing dozens of HPV genotypes.

Another object of one embodiment of the present invention, to provide a kit containing the HPV gene chip used in the method.

HPV genotyping gene chip according to an embodiment of the present invention is characterized in that it includes all the probes having a sequence of SEQ ID NO: 29 to 40.

HPV genotyping kit according to an embodiment of the present invention, DNA extraction reagent; Reagents for PCR of the HPV gene; And it characterized in that it comprises a gene chip for HPV genotyping including all probes having a sequence of SEQ ID NO: 29 to 40.

HPV genotyping method according to an embodiment of the present invention, the step of extracting DNA from the sample cells; Amplifying the DNA using PCR; Hybridizing the amplified gene to the HPV gene chip; And identifying the HPV genotype specifically bound in the hybridization reaction.

By using the gene chip, kit and test method according to an embodiment of the present invention, 36 types of HPV genotypes present in cervical cells can be tested with high specificity and cannot be identified by conventional test methods. Multiple infections with HPV can also be tested. In addition, by using the gene chip, kit and test method according to an embodiment of the present invention, DNA can be quickly extracted within a total of 8 hours to determine the HPV genotype by oligobase chip by extracting DNA from the cervical cell sample Therefore, it is very useful industrially.

Hereinafter, the present invention will be described in more detail at each step.

1) from sample cells DNA  extraction

This step extracts DNA for use in the PCR process of HPV genes from cells obtained from the cervix.

The sample cell can be any cell of the organism. Especially, it is preferable that they are cervical cells. DNA extraction from cells should be free of contamination and easy to manipulate.

Cervical cells are centrifuged, for example, from exfoliated cervical cells to collect only cells, and then the cells are lysed with a cell lysis solution to extract DNA to obtain DNA. Reagents necessary for extracting DNA from cells are protease, extraction solution and washing solution. For example, a combination of the following reagents can be used:

① 30 ml tube containing proteinase K (Sigma, USA) and extract solution (50 mM KCl, 10 mM Tris-HCl (pH8.4), 1.5 mM MgCl ₂ , 0.1% TritonX-100); And

② 10X washing solution (Phosphate buffered saline, PBS).

The extraction process of the cells can be carried out as follows, for example.

Cervical cells are isolated and centrifuged. The supernatant was removed and resuspended in PBS and then centrifuged again. After removing the supernatant, the extraction solution was added and resuspended and warmed in a thermostat. Through this step, dozens of samples can be quickly and easily processed for about 2 hours and 30 minutes, and there is no merit of false positives by preventing contamination between samples. In addition, such extraction method, because it does not use the organic solvent used in the conventional extraction method can eliminate the risk and inconvenience of environmental pollution.

2) PCR Gene amplification by

A part of the DNA obtained in step 1) is amplified by PCR using a primer specific for the HPV gene. The primer is preferably used in sequence of two sets of primers. For example, the first PCR performed by the first PCR primer set and the second PCR performed by the second primer set can be performed sequentially. It is particularly desirable to bind the fluorescent material to the five ends of the upstream primers of the second primer set, particularly the second primer set. Cy5 may be used as the fluorescent material.

Reagents required to perform PCR of the HPV gene include two sets of primers, buffers, DNA polymerase and dNTP. Examples of specific combinations are as follows:

① 20 mmol / μl of each set of HPV PCR 1st and 2nd primers in Table 1 below;

② 10X PCR buffer solution;

③ Taq DNA polymerae 250 units (Amplitaq, PE, USA); And

④ 2.5mM dNTP.

Primer Name Sequence structure (5 3) seq.1 1st PCR MY09 5'- CGTCCMARRGGAWACTGATC -3 ' seq.2 MY11 5'- GCMCAGGGWCATAAYAATGG -3 ' seq.3 2nd PCR GP + 5 5'-TTTGTTACTGTGGTAGATACTAC -3 ' seq.4 Cy5-GP + 6 5'Cy3-GAAAAATAAACTGTAAATCATATTC -3

3) the amplified gene HPV On a gene chip Hybridization  step

Oligobase chip fabrication to analyze the genotype of the amplified HPV gene, for example, 36 HPV probe oligo bases that specifically bind to 36 different HPV genes, 5 5 It can be prepared by combining to amine groups (-NH ₂₎ is attached to the terminal. The prepared probes are attached to the probes on a glass plate for chip fabrication at regular intervals and in an array using a microarray device, and then washed and fixed. The fixed probes and the HPV gene PCR product obtained in step 2) are hybridized at a constant temperature and then washed to remove non-specifically bound ones.

The probes preferably include all probes having a sequence of SEQ ID NOs: 29 to 40.

Configurations necessary for the preparation of oligo base chips (DNA chips) for testing HPV genotypes include probes of HPV genes, glass slides, microarray devices, hybridization reaction chambers and solutions, and the like. Specifically, they can be combined as follows:

(1) 36 types of HPV probes (concentration: 40 mmol / μl) shown in Table 2 below having an amine group attached to 5 terminals;

A glass slide coated with an aldehyde group on the surface;

③ microarray device (Bio-Robotics, UK);

④ hybridization chamber for dividing the glass slide into 8 zones (Multiwell hybridization chamber, Grace, USA); And

0.2% SDS solution, NaBH4 (Sigma, USA), PBS solution, 100% ethanol.

No. Probe Sequence (5'-3 ') seq.5 HPV6 ATC CGT AAC TAC ATC TTC CAC ATA CAC CAA seq.6 HPV11 ATC TGT GTC TAA ATC TGC TAC ATA CAC TAA seq.7 HPV16 GTC ATT ATG TGC TGC CAT ATC TAC TTC AGA seq.8 HPV18 TGC TTC TAC ACA GTC TCC TGT ACC TGG GCA seq.9 HPV31 TGT TTG TGC TGC AAT TGC AAA CAG TGA TAC seq.10 HPV33 TTT ATG CAC ACA AGT AAC TAG TGA CAG TAC seq.11 HPV34 TAC ACA ATC CAC AAG TAC AAA TGC ACC ATA seq.12 HPV35 GTC TGT GTG TTC TGC TGT GTC TTC TAG TGA seq.13 HPV39 TCT ACC TCT ATA GAG TCT TCC ATA CCT TCT seq.14 HPV40 GCT GCC ACA CAG TCC CCC ACA CCA ACC CCA seq.15 HPV42 CTG GAA CAT CTG GTG ATA CAT ATA CAG CTG seq.16 HPV43 TCT ACT GAC CCT ACT GTG CCC AGT ACA TAT seq.17 HPV44 GCC ACT ACA CAG TCC CCT CCG TCT ACA TAT seq.18 HPV45 ACA CAA AAT CCT GTG CCA AGT ACA TAT GAC seq.19 HPV51 AGC ACT GCC ACT GCT GCG GTT TCC CCA ACA seq.20 HPV52 TGC TGA GGT TAA AAA GGA AAG CAC ATA TAA seq.21 HPV54 TAC AGC ATC CAC GCA GGA TAG CTT TAA TAA seq.22 HPV56 GTA CTG CTA CAG AAC AGT TAA GTA AAT ATG seq.23 HPV58 ATT ATG CAC TGA AGT AAC TAA GGA AGG TAC seq.24 HPV53 GCA AAT TAA ACA GTA TGT TAG ACA TGCAGA seq.25 HPV59 TGT ATA CAC ACC TAC CAG TTT TAA AGA ATG seq.26 HPV66 ACT AAA TAT GAT GCC CGT GAA ATC AAT CAA seq.27 HPV68 ACC AAA TAT TTA TGA TCC TAA TAA ATT TAA seq.28 HPV70 CTG CTG TAT ATA GCC CTA CAA AGT TTA AGG seq.29 HPV32 TAC TGT AAC AAC TGA AGA CAC ATA CAA GTC seq.30 HPV61 TAC TGC TAC ATC CCC CCC TGT ATC TGA ATA seq.31 HPV62 CCA CTG CTG CAG CAG AAT ACA AGG CTA CCA seq.32 HPV67 CAA TCT GCA TCT GCC ACT TTT AAA CCA TCA seq.33 HPV69 TGT ATC TGC ACA ATC TGC ATC TGC CAC TTT seq.34 HPV71 CCA AAA CTG TTG AGT CTA CAT ATA AAG CCT seq.35 HPV72 TGC CAC AGC GTC CTC TGT ATC AGA ATA TAC seq.36 HPV81 GCA CAG CTA CAT CTG CTG CTG CAG AAT ACA seq.37 HPV82 TTT ACT CCA GCA AAC TTT AAG CAG TAC ATT seq.38 HPV83 CAG CTG CTG CTACAC AGG CTA ATG AAT ACA seq.39 HPV84 GTG CTG CTA CCA ACA CCG AAT CAG AAT ATA seq.40 HPV90 CAC ACA AAC ACC CTC TGA CAC ATA CAA GGC HPV 36 types

In order to hybridize the PCR product obtained in step 2) to the DNA chip prepared as described above, a buffer solution, Tris-HCl, SDS solution, etc. are required. Examples of specific combinations are as follows:

① HPV genotyping oligonucleotide chip divided into 8 zones;

② hybridization buffer solution (6X SSC);

③ 1M Tris-HCl (pH 7.2); And

④ 10% SDS.

After the hybridization reaction is complete, it is desirable to wash the DNA chip to remove nonspecifically bound fragments. It can be washed with a washing solution. Examples of cleaning solution combinations include:

① Wash solution I solution (2X SSC, 0.1% SDS);

② wash solution II (0.2X SSC); And

③ Wash solution III solution (0.1X SSC).

4) specifically Combined HPV  Genotyping

This step is to identify specifically bound HPV genotypes. For example, a chip scanner can identify HPV genotypes specifically bound to HPV probes.

The HPV DNA chip read may use a microarray scanner or the like. Specifically, a microarray scanner (GSI Lumonics, USA) etc. are mentioned.

HPV genotyping kit according to an embodiment of the present invention may include a DNA extraction reagent, a reagent for PCR of the HPV gene and a gene chip for HVP genotyping. An example is as follows:

1) DNA Extraction Reagent:

① 30 ml tube containing proteinase K (Sigma, USA) and extract solution (50 mM KCl, 10 mM Tris-HCl (pH8.4), 1.5 mM MgCl ₂ , 0.1% TritonX-100);

② 10X washing solution (Phosphate buffered saline, PBS).

2) Reagents for HPV Gene PCR:

① 1.5 ml tubes with GP + 5 and Cy5-GP + 6 primers at 20 mmol / μl concentrations, respectively;

② 1.5 ml tube of 10XPCR buffer solution; And

③ 1.5 ml tube of Taq DNA polymerase 250 unit (Ampli Tag., PE, USA).

3) Reagents for HPV Genotyping of HPV Gene PCR Products:

① HPV genotyping oligobase chip; And

② 8 well hybridization chamber (Multiwell hybridization chamber, sigma, USA).

Using the kit above, DNA extraction from cells obtained from the cervix is easily performed, and amplification of the HPV gene by PCR takes about 8 hours to test the HPV genotype with an HPV DNA chip. In addition, eight samples can be tested simultaneously on one HPV oligobase chip. Thus, the kit can be used as a routine test method for HPV genotyping and is an industrially useful invention that can replace the HPV test kit (Hybride Capture, Digene, USA) currently imported for HPV gene testing.

Hereinafter, the present invention will be described in more detail with reference to a preferred embodiment of the present invention, which is intended to illustrate the present invention but does not limit the scope of the present invention.

Example

Example 1 DNA Extraction Method from Cells

Cytobrush preserved in 5 ml of phosphate buffered saline (PBS) was strongly vortexed to separate cervical cells from the cytobrush, centrifuged at 1,300 g for 10 minutes, and the supernatant was removed. Resuspended in ㎖ PBS, transferred to 1.5ml microtube and centrifuged for 5 minutes at 1,300g. The supernatant was removed and resuspended in 200 μl of extract solution (50 mM KCl, 10 mM Tris-HCl (pH 8.4), 1.5 mM MgCl ₂ , 0.1% TritonX-100). After warming in a 55 ° C. thermostat for 2 hours, it was further warmed at 95 ° C. for 10 minutes to remove the activity of proteinase K.

Example 2 Amplification Method of HPV Gene by PCR.

Nested PCR method was used. Primers used for the diagnosis of HPV are as shown in Table 1 above. Two types of first (1st) PCR (5'-CGTCCMARRGGAWACTGATC-3 '/ 5'-GCMCAGGGWCATAAYAATGG-3') and second (2nd) PCR (5 '-TTTGTTACTGTGGTAGATACTAC-3 / 5'-Cy5-GAAAAATAAACTGTAAA TCATATTC-3') Β-globin primer (5′-Cy5-CAACTTCAT CCACGTTCACC-3 ′ / 5′-GAAGAGCCAAGGACAGGTAC-3 ′) was used as a control.

(1) first PCR

5.0 μl of the sample was added to a 0.2 ml PCR tube and pre-denaturated at 95 ° C. for 10 minutes before PCR. 2.5 μl 10 × buffer (100 mM-KCl, 20 mM Tris-HCl (pH8.0), 2.0 mM MgCl ₂ ) _{, 2} μl _of 2.5 mM dNTP, 0.3 μl of Taq polymerase (5 units), 1.0 μl of 20 pmol MY09 / MY11 primer, 5 μl of template DNA was added to 25 μl with distilled water. PCR was performed 40 times, 30 seconds at 95 ° C, 30 seconds at 55 ° C, and 30 seconds at 72 ° C (9700, Applied Biosystems).

(2) second PCR

2 μl of the first PCR DNA template, 2.5 μl of 10 × buffer, 1.0 μl of each 10 pmol GP5 + / GP6 + primer, 2.0 μl of 2.5 mM dNTP, and 0.3 μl of Taq polymerase (5 units) were added to 25 μl in distilled water. . DNA amplification was performed 30 times at 95 ° C., 30 seconds at 55 ° C., and 30 seconds at 72 ° C. (9700, Applied Biosystems). After PCR amplification, each PCR product was electrophoresed with 3.0 µl in 2.5% agarose gel and subjected to image analysis (Multimage, BioRad). The time required at this time was about 2 hours 30 minutes.

<Example 3: HPV genotyping oligobase chip production>

A) Spotting 36 kinds of HPV genotype probes (Table 2 above) equal to 40 mmol / μl concentration by dividing the microscope glass slider into 8 zones in the microarray apparatus. ) And spotted β-globin probes as fluorescent markers in the middle.

B) The glass slider attached with the probe of a) is washed twice in 500 ml 0.2% SDS solution for 2 minutes and washed twice with distilled water and then mixed with 1.3 g NaBH ₄ , 375 ml PBS and 125 ml ethanol. The mixture was stirred vigorously for 5 minutes, and then washed again three times with 0.2% SDS solution and three times with distilled water for one minute and then dried with water to store in a sealed box.

Example 4 HPV Genotyping Test Genotyping of HPV Gene PCR Products Using Oligobase Chips

A) HPV genotyping oligobase chip preparation

The 8 well hybridization chamber was firmly attached to the HPV oligo base chip prepared in Example 4, thereby preparing oligo base chips divided into 8 zones (FIG. 1).

B) Hybridization of HPV gene PCR product and oligobase chip.

The PCR product was denatured at 95 ° C. for 5 minutes to hybridize the oligobases spotted on the HPV DNA chip by adding 8 µl of PCR product and 32 µl of 2X hybridization buffer to a 1.5 ml tube. It was made to react at 43 degreeC for 10 minutes immediately before use. The hybridization reaction solution and the PCR product were mixed well and carefully dispensed into each well of each gene chip in the well of each chip in the well, and fixed with a tape to prevent evaporation. The hybridization reaction was performed at 43 ° C. for 1 hour.

C) Method of washing oligobase chip after hybridization reaction.

After completion of the hybridization, the gene chip was washed 5 min / 2 times with washing solution I (2X SSC, 0.1% SDS), 5 min / 2 times with washing solution II (0.2X SSC) and 5 min / 1 times with washing solution III (0.1X SSC). . After washing, dried at room temperature and stored in a light-blocked place.

D) HPV genotyping of oligobase chips after washing.

After setting the chip scanner to detect the excitation wavelength of 550ml and the emission wavelength of 570nm, insert the oligobase chip washed in a) and read the position of the developed probe by binding the HPV gene product to the HPV genotype probe. HPV genotype was confirmed. The results are shown in FIGS. 2 and 3.

Example 5 Examination of Cervical Cell Specimens Using HPV Genotyping Oligobase Chips

The HPV genotype test oligobase chip prepared in Example 3 was diagnosed according to the test method of Example 4, and HPV 36 type was developed by randomly selecting 300 samples from the already analyzed swab samples. The results were compared to the HPV chip. The sample was extracted from the uterine cervix and extracted from the gene and stored in DNA. Comparative analysis revealed no differences in HPV type, infection type from single infection to double infection, double infection to single infection, or negative HPV and vice versa in each sample. The results are shown in Table 3 below.

HPV type  Existing Results 36 type HPV chip % One HPV6 3 3 1.0 2 HPV11 5 5 1.7 3 HPV16 52 52 17.3 4 HPV18 12 12 4.0 5 HPV31 2 2 0.6 6 HPV33 9 9 3.0 7 HPV34 0 8 HPV35 One One 0.3 9 HPV39 0 10 HPV40 0 11 HPV42 One One 0.3 12 HPV43 0 13 HPV44 0 14 HPV45 0 15 HPV51 2 2 0.6 16 HPV52 6 6 1.8 17 HPV54 2 2 0.6 18 HPV56 5 5 1.7 19 HPV58 20 20 6.7 20 HPV53 22 22 7.3 21 HPV59 0 22 HPV66 0 23 HPV68 0 24 HPV70 18 18 6.0 25 HPV32 0 26 HPV61 One One 0.3 27 HPV62 12 12 4.0 28 HPV67 0 29 HPV69 0 30 HPV71 4 4 1.3 31 HPV72 8 8 2.6 32 HPV81 6 6 2.0 33 HPV82 0 34 HPV83 One One 0.3 35 HPV84 2 2 0.6 36 HPV90 0 Double infection 21 21 7.0 voice 87 87 29.0 00 Sum 302 302 100

<Example 6: Test method for HPV genotyping and test kit method of oligobase chip>

The test kit of the gene chip for HPV genotyping of the present invention includes a kit kit for retrieving cells from the cervix and a DNA extraction kit, a kit kit for a reagent for PCR of the HPV gene, and It consists of three parts of HPV gene oligo chip to identify genotype of HPV gene PCR product.

A) Kitization of DNA Extraction Method

① 10X washing solution in a 30ml tube containing 5ml PBS solution,

② Protienase K (Sigma, USA) It consists of 30ml tube containing 20mg / ml concentration and 5% chelex. Store all at room temperature. The kit is capable of processing 80 cervical cell specimens and the processing operations and methods of use are the same as in Examples 1 and 2.

B) kit the reagents configured for HPV polymerase chain reaction (PCR);

Reagents used for PCR of the HPV gene of the HPV gene were kitted as described in Example 2. The composition is ①. 1 ml PCR primer, HPV gene PCR primer, 1.5 ml tube containing 20 pmol / l, 50ul volume, respectively, ②. 1.5 ml tube containing 1 ml of 10 × PCR buffer, ③. 1.5 ml tube containing 100ul of 10mM dNTP (mixed solution of dATP, dGTP, dCTP, dTTP) solution, ④. Taq. It consists of 1.5ml tube containing 200 units of DNA polymerase (Amplitaq, PE, USA), ⑤ 1.5ml tube containing hybridization reaction solution, ⑥ beta globin primer 20 pmol/l, 1.5ml tube containing 50ul volume. All of these are stored in a -20 ° C. freezer and the method of use is the same as in Example 2.

C) HPV gene oligobase chip kit

The HPV oligobase chip prepared according to the fabrication method of Example 4 was composed of 10 oligobase chips capable of processing eight specimens in one sheet, and the method of use was the same as in Example 4.

The above HPV genotyping kit was configured to process 80 cervical cell samples.

Example 7 Comparison of HPV DNA Chip and Sequence Analysis Method

When the HPV subtype genotypes were determined by the chip results, sequencing was performed to confirm whether the genotypes accurately show the results. According to the results of each HPV chip, according to each sample, 5 μl of PCR product was added to 8 μl of Bigdye and 1 μl of 5 ′ primer of each gene, so that the total was 20 μl. The sequencing reaction was performed at 95 ° C. for 2 minutes. The cells were denatured and subjected to 25 cycles of 95 ° C., 20 seconds, 50 ° C., 5 seconds, and 60 ° C. for 2 minutes (ABI3100, Applied Biosystems). The sequencing results confirmed that for each HPV subtype genotype, that is, all of the results match the results of the HPV DNA chip.

1 shows a gene chip for HPV genotyping according to an embodiment of the present invention.

Figure 2 shows the results of the hybridization reaction using the HPV genotype testing gene chip according to an embodiment of the present invention.

Figure 3 shows the results of the hybridization reaction using the HPV genotype testing gene chip according to an embodiment of the present invention.

<110> MYGENE CO., LTD.          JUNG, WOON WON          KIM, HYUN SOOK          HAN, IN KWON <120> DNA CHIP, KIT FOR DETECTING GENOTYPE OF HUMAN PAPILLOMAVIRUS AND          DETECTION METHOD USING THE SAME <130> P07-10-24 <160> 40 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cgtccmarrg gawactgatc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gcmcagggwc ataayaatgg 20 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tttgttactg tggtagatac tac 23 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gaaaaataaa ctgtaaatca tattc 25 <210> 5 <211> 30 <212> DNA <213> Human papillomavirus type 6 <400> 5 atccgtaact acatcttcca catacaccaa 30 <210> 6 <211> 30 <212> DNA <213> Human papillomavirus type 11 <400> 6 atctgtgtct aaatctgcta catacactaa 30 <210> 7 <211> 30 <212> DNA <213> Human papillomavirus type 16 <400> 7 gtcattatgt gctgccatat ctacttcaga 30 <210> 8 <211> 30 <212> DNA <213> Human papillomavirus type 18 <400> 8 tgcttctaca cagtctcctg tacctgggca 30 <210> 9 <211> 30 <212> DNA <213> Human papillomavirus type 31 <400> 9 tgtttgtgct gcaattgcaa acagtgatac 30 <210> 10 <211> 30 <212> DNA <213> Human papillomavirus type 33 <400> 10 tttatgcaca caagtaacta gtgacagtac 30 <210> 11 <211> 30 <212> DNA <213> Human papillomavirus type 34 <400> 11 tacacaatcc acaagtacaa atgcaccata 30 <210> 12 <211> 30 <212> DNA <213> Human papillomavirus type 35 <400> 12 gtctgtgtgt tctgctgtgt cttctagtga 30 <210> 13 <211> 30 <212> DNA <213> Human papillomavirus type 39 <400> 13 tctacctcta tagagtcttc cataccttct 30 <210> 14 <211> 30 <212> DNA <213> Human papillomavirus type 40 <400> 14 gctgccacac agtcccccac accaacccca 30 <210> 15 <211> 30 <212> DNA <213> Human papillomavirus type 42 <400> 15 ctggaacatc tggtgataca tatacagctg 30 <210> 16 <211> 30 <212> DNA <213> Human papillomavirus type 43 <400> 16 tctactgacc ctactgtgcc cagtacatat 30 <210> 17 <211> 30 <212> DNA <213> Human papillomavirus type 44 <400> 17 gccactacac agtcccctcc gtctacatat 30 <210> 18 <211> 30 <212> DNA <213> Human papillomavirus type 45 <400> 18 acacaaaatc ctgtgccaag tacatatgac 30 <210> 19 <211> 30 <212> DNA <213> Human papillomavirus type 51 <400> 19 agcactgcca ctgctgcggt ttccccaaca 30 <210> 20 <211> 30 <212> DNA <213> Human papillomavirus type 52 <400> 20 tgctgaggtt aaaaaggaaa gcacatataa 30 <210> 21 <211> 30 <212> DNA <213> Human papillomavirus type 54 <400> 21 tacagcatcc acgcaggata gctttaataa 30 <210> 22 <211> 30 <212> DNA <213> Human papillomavirus type 56 <400> 22 gtactgctac agaacagtta agtaaatatg 30 <210> 23 <211> 30 <212> DNA <213> Human papillomavirus type 58 <400> 23 attatgcact gaagtaacta aggaaggtac 30 <210> 24 <211> 30 <212> DNA <213> Human papillomavirus type 53 <400> 24 gcaaattaaa cagtatgtta gacatgcaga 30 <210> 25 <211> 30 <212> DNA <213> Human papillomavirus type 59 <400> 25 tgtatacaca cctaccagtt ttaaagaatg 30 <210> 26 <211> 30 <212> DNA <213> Human papillomavirus type 66 <400> 26 actaaatatg atgcccgtga aatcaatcaa 30 <210> 27 <211> 30 <212> DNA <213> Human papillomavirus type 68 <400> 27 accaaatatt tatgatccta ataaatttaa 30 <210> 28 <211> 30 <212> DNA <213> Human papillomavirus type 70 <400> 28 ctgctgtata tagccctaca aagtttaagg 30 <210> 29 <211> 30 <212> DNA <213> Human papillomavirus type 32 <400> 29 tactgtaaca actgaagaca catacaagtc 30 <210> 30 <211> 30 <212> DNA <213> Human papillomavirus type 61 <400> 30 tactgctaca tccccccctg tatctgaata 30 <210> 31 <211> 30 <212> DNA <213> Human papillomavirus type 62 <400> 31 ccactgctgc agcagaatac aaggctacca 30 <210> 32 <211> 30 <212> DNA <213> Human papillomavirus type 67 <400> 32 caatctgcat ctgccacttt taaaccatca 30 <210> 33 <211> 30 <212> DNA <213> Human papillomavirus type 69 <400> 33 tgtatctgca caatctgcat ctgccacttt 30 <210> 34 <211> 30 <212> DNA <213> Human papillomavirus type 71 <400> 34 ccaaaactgt tgagtctaca tataaagcct 30 <210> 35 <211> 30 <212> DNA <213> Human papillomavirus type 72 <400> 35 tgccacagcg tcctctgtat cagaatatac 30 <210> 36 <211> 30 <212> DNA <213> Human papillomavirus type 81 <400> 36 gcacagctac atctgctgct gcagaataca 30 <210> 37 <211> 30 <212> DNA <213> Human papillomavirus type 82 <400> 37 tttactccag caaactttaa gcagtacatt 30 <210> 38 <211> 30 <212> DNA <213> Human papillomavirus type 83 <400> 38 cagctgctgc tacacaggct aatgaataca 30 <210> 39 <211> 30 <212> DNA <213> Human papillomavirus type 84 <400> 39 gtgctgctac caacaccgaa tcagaatata 30 <210> 40 <211> 30 <212> DNA <213> Human papillomavirus type 90 <400> 40 cacacaaaca ccctctgaca catacaaggc 30  

Claims (8)

Gene chip for testing HPV genotype comprising all of the probe having a sequence of SEQ ID NO: 29 to 40. DNA extraction reagents; Reagents for PCR of the HPV gene; And A gene chip for testing HPV genotype including all probes having a sequence of SEQ ID NOs: 29 to 40; HPV genotyping kit comprising a. The kit for testing HPV genotype according to claim 2, wherein the reagent for PCR of the HPV gene comprises primers having a sequence of SEQ ID NOs: 1-4. The method of claim 3, wherein HPV genotyping kit, characterized in that the fluorescent material is bonded to the five ends of the primer having a sequence of SEQ ID NO: 4. a) extracting DNA from sample cells; b) amplifying the DNA using PCR; c) hybridizing the amplified gene to the HPV gene chip including all the probes having the sequences of SEQ ID NOs: 29 to 40; And d) HPV genotyping method comprising the step of identifying the HPV genotype specifically bound in the hybridization reaction. According to claim 5, wherein step b), HPV genotyping method, characterized in that for performing PCR using primers having a sequence of SEQ ID NO: 1 to 4. The method of claim 6, B), The first PCR and the second PCR are performed sequentially using two sets of primers, One of the two sets of primers has a fluorescent material bound to the five ends of the upstream primer, Characterized in that to perform the second PCR with a primer set having an upstream primer to which the fluorescent material is bound, Step d), HPV genotyping method comprising measuring the fluorescent material. delete

Images