KR100452103B1 - Primers and Method for Detecting Oncogenic Human Papillomavirus by PCR - Google Patents

Abstract

The present invention relates to a primer capable of amplifying the DNA of a human papillomavirus (HPV) and a method for detecting the presence of HPV using the primer, which is classified as a genotype of oncogenic HPV. Providing a synthesized oligonucleotide sequence capable of amplifying or detecting a nucleic acid and amplifying various oncogenic HPV DNAs through a single polymerase chain reaction (PCR) using the oligonucleotide synthesized as such as a primer By using a combination of the above primers and a PCR technique for amplifying a variety of tumorigenic HPV type nucleic acid by one PCR.

Description

Primer and Method for Detecting Oncogenic Human Papillomavirus by PCR for primers for detecting oncogenic human papillomavirus by polymerase chain reaction

The present invention relates to a primer that can be used for polymerase chain reaction (hereinafter, referred to as "PCR") and an amplification method using the primer, and more specifically to a tumor papillomavirus (Human Papillomavirus, hereinafter). A nucleotide capable of amplifying a nucleic acid of "HPV" and a method capable of detecting the presence of HPV using the nucleotide as a primer.

Cervical cancer is the second highest incidence of cancer among women in the world, behind breast cancer, with an annual incidence of 50 to 5 million. In Korea, the number of deaths from cervical cancer is 24 per 100,000 population, compared to 7, 9 and 4 per 100,000 population in the United States, Japan and France, respectively.

HPV is a circular double-stranded DNA virus of about 8 kb in length. The HPV genome contains the E1 to E7 genes involved in the initial process and the L1 and L2 genes involved in the late process after infecting the host cell. In particular, tumorigenic proteins E6 and E7 are known to be involved in tumor formation by transforming infected cells by binding to p53 and pRb, which are tumor suppressor proteins of host cells, thereby inhibiting the activity of these proteins. HPV has not only been associated with genital cancer, laryngeal cancer and tongue cancer, but has also been identified as an essential maintenance factor for onset of carcinogenesis and carcinogenesis, particularly in cervical cancer.

To date, more than 120 different types of HPV are known depending on the differences in open reading frame (ORF) sequences of the E6, E7, and L1 genes. If the nucleotide sequence of the ORF is more than 10% different, it is defined as a new type, if it is more than 2% and less than 10%, it is defined as a subtype, and less than 2% is defined as a variant. Specifically HPV 2, 3, 6, 7, 10, 11, 13, 16, 26, 30, 31, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 66, 68, 70, 73 and the like are classified as tumorigenic HPVs that infect tumors of the genital mucosa and cause tumors. Due to the close relationship between HPV infection and cervical cancer and high mortality due to cervical cancer, a method for early diagnosis of cervical cancer has been sought.

HPV detection methods and cervical cancer diagnostic methods that have been recognized to date can be largely divided into cytological and molecular biological methods, the latter is further divided into hybridization and PCR methods.

Cytoscopic methods for morphological changes of HPV-infected cells include vaginal magnification, in addition to the cell smear, which diagnoses cervical cancer based on perinuclear halo (koliocytosis). (colposcopy), cervicography, biopsy, etc. are used, but these methods are all based on the clinical findings of the cytological or histopathological findings of HPV infection. It has a fundamental limitation that is not effective for early diagnosis. As HPV is regarded as a etiological factor of cervical cancer, HPV DNA detection method has been sought as a molecular biological method for early diagnosis of cervical cancer.

First, diagnostic methods using hybridization methods between target HPV DNA and specific probes include dot blot hybridization, filter in situ hybridization, and Southern blot hybridization. blot hybridization has been developed, and recently, the `` Hybrid Capture II '' method, a soluble hybridization method using a specific hybridization reaction between HPV DNA and RNA oligomer probes in solution (Digene), has been developed. However, hybridization method requires a large amount of HPV DNA or genome (basic), so if there is a small amount of genome of HPV, it cannot be confirmed and has a disadvantage of low sensitivity.

The PCR method adds a complementary nucleic acid (oligomeric primer) that specifically binds to the nucleic acid sequence of the target pathogen, and repeats the process of denaturation, annealing, and polymerization according to the temperature to remove the trace pathogen nucleic acid. As a technique capable of amplifying and detecting the presence (US Pat. Nos. 4683195 and 4683202), it has a very high relative superiority in accuracy and sensitivity compared to the above-described cytological and hybridization methods. However, to date, primers used in the HPV detection method by PCR have the following limitations.

First, a general primer was chosen that was designed to amplify all types of HPV. Such primers have oligonucleotide sequences capable of complementary binding to nucleotide sequences present in specific genes of various HPV types. To date, as a target gene, primers such as WD72, WD73, WD76, WD67, WD67, WD154, WD155, which can complementarily complement E6 or E7 genes, and TYP01, TYP02, TYN01, TYN02, etc., which can bind to E1 genes. Primers, MY11, MY09, PEG01 primers and the like that can bind to the L1 gene are known. However, these inclusive primers cannot distinguish between HPV type and tumorigenic HPV types, and thus, even if amplification occurs by PCR reaction, the HPV type present in the sample is tumorigenic or non-tumorogenic. Can't. Therefore, amplification may occur due to non-tumorogenic HPV, and it is difficult to diagnose whether cervical cancer is infected only by amplification.

To overcome this problem, a method of using a primer capable of complementarily binding with type specific HPV DNA has been recently proposed. However, if type-specific primers are used, amplification may not occur, depending on the type of oncogenic HPV. Therefore, the diagnosis of cervical cancer has a practical limitation because it has to use both type-specific primers.

As a result, amplification of HPV DNA by PCR selectively amplifies only oncogenic HPV types as a primer capable of detecting HPV, thereby raising the need for a primer for early diagnosis of cervical cancer caused by oncogenic HPV. It is becoming.

The present invention has been made to solve the above problems in the method of detecting HPV by PCR method, an object of the present invention is to provide a nucleotide that can amplify a variety of tumorigenic HPV type DNA by PCR process. .

Another object of the present invention is to provide a method for amplifying tumorigenic HPV type DNA by PCR by selecting one or more trinucleotide primers as described above.

It is another object of the present invention to provide a method for amplifying target DNA using the above-described primers and analyzing the presence of oncogenic HPV types according to whether they are amplified.

Still another object of the present invention is to provide a kit including the primers described above and capable of detecting a variety of tumorigenic HPV by amplifying a nucleic acid of a tumorigenic HPV type by a PCR procedure.

Figures 1a and 1b are photographs electrophoresed after PCR using the primers synthesized in the present invention using the target DNA as CaSki and HeLa, the infected cell lines of HPV-16, respectively.

Figures 2a to 2l is a picture of the electrophoresis after the PCR for the four sets of primers and various HPV DNA synthesized in the present invention.

Figures 3a to 3o is a picture of the electrophoresis after performing a PCR on the DNA obtained in the human body diagnosed as being associated with cervical cancer using the four sets of primers synthesized in the present invention and the conventional HPV DNA amplification primers.

4A to 4O are electrophoresis photographs after PCR of DNA obtained from a human body diagnosed as not related to cervical cancer using four sets of primers synthesized in the present invention and conventional HPV DNA amplification primers.

In order to achieve the above object, the present invention provides a primer consisting of nucleotides capable of amplifying DNA of various types of tumorigenic human papillomavirus (HPV).

In the present invention, the nucleotide consisting of SEQ ID NO: 1 and SEQ ID NO: 2;

Nucleotides consisting of SEQ ID NO: 3 and SEQ ID NO: 4;

Nucleotides consisting of SEQ ID NO: 5 and SEQ ID NO: 6;

Nucleotides consisting of SEQ ID NO: 7 and SEQ ID NO: 8;

Nucleotides consisting of SEQ ID NO: 9 and SEQ ID NO: 10;

Nucleotides consisting of SEQ ID NO: 11 and SEQ ID NO: 12;

Nucleotides consisting of SEQ ID NO: 13 and SEQ ID NO: 14;

Nucleotides consisting of SEQ ID NO: 15 and SEQ ID NO: 16;

Nucleotides consisting of SEQ ID NO: 17 and SEQ ID NO: 18;

Nucleotides consisting of SEQ ID NO: 19 and SEQ ID NO: 20;

Nucleotides consisting of SEQ ID NO: 21 and SEQ ID NO: 22;

Nucleotides consisting of SEQ ID NO: 23 and SEQ ID NO: 24;

Nucleotides consisting of SEQ ID NO: 25 and SEQ ID NO: 26;

Nucleotides consisting of SEQ ID NO: 27 and SEQ ID NO: 28; And

Provided are synthetic nucleotides that amplify the DNA of oncogenic HPV selected from the group consisting of nucleotides consisting of SEQ ID NOs: 29 and 30.

In another aspect, the present invention provides a method for amplifying the oncogenic HPV type DNA in a sample by selecting one or more of the above nucleotides as a primer.

The present invention also provides a method of selecting one or more sets of the above primers to amplify target DNA in a sample and to analyze the presence of oncogenic HPV type in the sample depending on whether the amplification is performed.

The present invention also provides a kit including the primers described above and capable of detecting a variety of tumorigenic HPV by amplifying a tumorigenic HPV type nucleic acid by PCR.

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

In the conventional method of searching for HPV by PCR, oligonucleotides that can hybridize with nucleotide sequences specific to each type are used as primers, or common nucleotides that can hybridize with all types of HPV are used as primers. However, in the present invention, a primer capable of hybridizing with various tumorigenic HPV types was designed, and one-time PCR was performed by the primers to detect various tumorigenic HPV types.

As mentioned above, there are more than 120 types of HPV depending on the sequence of a particular gene. In particular, the E1, E7, and L1 genes of HPV genes are used as criteria for distinguishing HPV types, and thus include not only specific sequences but also conserved sequences. Therefore, selecting a nucleotide capable of complementarily binding to a tumorigenic HPV type DNA in the sequence of the gene as a primer can amplify the DNA of various HPV types.

In the present invention, after classifying the HPV into three groups according to the internationally accepted criteria based on the sequence of the genes (E1, E7, L1 gene), oncogenic HPV is divided into various types by using a computer simulation program After identifying oligonucleotides capable of amplifying the oncogenic HPV DNA by complementary binding to DNA, a DNA synthesizer was used to synthesize primers identified through the virtual amplification test, and to target DNA obtained from HPV infected cell lines and samples. PCR was performed to confirm the presence or absence of HPV type by comparing the amplification and the size of the obtained product.

To this end, in the present invention, the appropriate oligonucleotide sequence was designed as a primer through a simulation amplification test process. First, based on the nucleotide sequence of a total of 65 HPV type full-length DNA is published in three groups according to the similarity of the sequence, using the computer program primer premier 5, PREMIER Biosoft International Co. PCR primers were designed. At this time, the length of the primer consists of 33 to 35 nucleotides and the size of the resulting PCR product was limited to 300 ~ 500bp, it was adjusted so that the heterologous base is not synthesized by the wobble (wobble).

Using a computer program, Amplify 1.2, University of Wisconsin, for a total of 180 trillion primers designed through the virtual simulation, each primer was subjected to virtual amplification experiments on 65 different HPV types. The amplification degree of was confirmed. In the present invention, the amplification degree of a total of 35 oncogenic HPV types closely related to cervical cancer is expressed as sensitivity, and the amplification degree of 35 oncogenic HPV types with respect to the amplified HPV type is expressed as a specificity ( specificity). That is, in relation to the present invention, the sensitivity and specificity are defined as follows.

% Sensitivity = (number of amplified oncogenic HPV types ÷ total number of oncogenic HPV types) × 100

% Specificity = (number of amplified oncogenic HPV types ÷ total number of amplified HPV types) × 100

A total of 15 trillion primers were selected based on the degree of amplification confirmed through the virtual amplification test, and the same nucleotides were synthesized using a DNA synthesizer. In order to synthesize primers capable of complementarily binding to various oncogenic HPV DNA in connection with the object of the present invention, it is preferable to select primers having high amplification degree through the virtual amplification test. In particular, it is preferable to select a primer capable of complementarily binding to various oncogenic HPV DNAs, that is, high specificity primers, without binding to non-tumorogenic HPV.

In the present invention, 15 sets of primers are selected from a total of 103 sets of primers having a specificity of 90% or more in the virtual amplification test, and an amplification type, amplification position, and a virtual amplification test of the selected primers are performed on a total of 65 sets of HPV types. Checking the size of the amplification product confirmed that it is distinguished from the existing HPV DNA amplification primers.

Primers selected above were synthesized using a nucleic acid synthesizer (Expedite ™ 8909 synthesizer, ABI). PCR was performed using the primers synthesized as described above and DNA in the HPV infected cell line as target DNA to amplify tumorigenic HPV.

In addition, by using two or more trillion of the above-selected primers capable of amplifying DNA of various HPVs, more various oncogenic HPVs can be detected. In this case, it is particularly preferable to use primers that amplify different HPV types in the virtual amplification of the selected primers. PCR amplification tests confirmed that the combined primers amplify a wider range of HPV types.

In addition, by using the primers identified in the present invention, PCR amplification may be performed on samples obtained from the human body, thereby allowing early diagnosis of cervical cancer depending on whether amplification is performed. In the present invention, 15 abnormal patients and 15 normal patients were identified through clinical pathology examination such as colposcopy. For some patients, hybrid capture II (Digiene) and DNA chip test (Biomedlab) were performed. Finally, the PCR test was performed using four sets of primers identified in the present invention and a primer known as MY09 / 11. By performing the amplification by the primers identified in the present invention, not only the same results as the cytology, hybrid capture II, and DNA chip diagnostics can be obtained, but also more sensitive and accurate than those of the existing primers. Early diagnosis of infection and cervical cancer was confirmed.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1

In this example, 15 sets of primers identified through computer simulations were synthesized and PCR amplification tests were performed on CaSki (ATCC CRL-1550), an HPV-16 infected cell line, and HeLa (ATCC CCL-2), an HPV-18 infected cell line. It was.

(1) PCR primer design

A nucleotide was designed as a PCR primer capable of amplifying the HPV DNA by binding complementarily with oncogenic HPV DNA, which is the object of the present invention. First, HPV-1a, -2a, -3, -4, -5, -6b, -7, -8, -9, -10 from the National Center for Biotechnology Information (NCBI) and the US National Los Alamos HPV database. , -11, -12, -13, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27,- 28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -41, -42, -44, -45, -47, -48, -49, -50, -51, -52, -53, -54, -55, -56, -57, -58, -59, -60, -61, -63, -65 A total of 65 HPV types, including -66, -68, -70, -72, and -73, were obtained. Group A containing HPV-16, -31, -33, -35, -52, Group B of HPV-18, -45, -59, -70 and HPV-11, based on the similarity of the obtained DNA sequences C-groups of -32, -44, and -55, their entire DNA sequences were sorted by group, and then PCR primers were designed using the computer program primer premier 5 (Premier Biosoft International Co.). It was. At this time, the length of the primer was set to 35 ± 2 oligonucleotides and the size of the PCR product was limited to 300 ~ 500bp. 35, 45, and 100 sets of PCR primers were designed for the A, B, and C groups, respectively.

(2) Virtual amplification of the designed PCR primers and screening of primers

Amplification degree was confirmed using another computer program (Amplify 1.2, University of Wisconsin) for a total of 180 trillion primers designed in the above process. A virtual amplification experiment was conducted on a total of 65 different HPV types already acquired during the design process. In the present embodiment, HPV-2a, -3, -6b, -7, -10, -11, -13, -16, -18, -26, -30, -31, 32, -33, -34, -35, -39, -40, -42, -44, -45, -51, -52, -53, -54, -55, -56, -57, -58, Amplification Degree of Sensitivity for a total of 35 Tumorogenic HPV Types of -59, -61, -66, -68, -70, -73 and Sensitivity of Tumorogenic HPV Type to Total Number of Amplified HPV Types Was confirmed by specificity.

Based on the selective amplification confirmed through the virtual amplification test, 15 of the total 103 trillion primers having a specificity of more than 90% was randomly selected and named by aibio number order, which is shown in Table 1 below.

Table 1. Primers Selected for Amplification of Tumorogenic HPV DNA

Screened primers SEQ ID NO: Specificity (%) ^* Sensitivity (%) ^** albio-1 SEQ ID NO: 1 SEQ ID NO: 2 100 17.1 albio-2 SEQ ID NO: 3 SEQ ID NO: 4 91.7 31.4 albio-3 SEQ ID NO: 5 SEQ ID NO: 6 100 20 albio-4 SEQ ID NO: 7 SEQ ID NO: 8 100 28 albio-5 SEQ ID NO: 9 SEQ ID NO: 10 100 20 albio-6 SEQ ID NO: 11 SEQ ID NO: 12 100 22.9 albio-7 SEQ ID NO: 13 SEQ ID NO: 14 100 17.1 albio-8 SEQ ID NO: 15 SEQ ID NO: 16 100 22.9 albio-9 SEQ ID NO: 17 SEQ ID NO: 18 100 17.1 albio-10 SEQ ID NO: 19 SEQ ID NO: 20 100 42.9 albio-11 SEQ ID NO: 21 SEQ ID NO: 22 100 34.3 albio-12 SEQ ID NO: 23 SEQ ID NO: 24 100 28.6 albio-13 SEQ ID NO: 25 SEQ ID NO: 26 100 22.9 albio-14 SEQ ID NO: 27 SEQ ID NO: 28 100 45.7 albio-15 SEQ ID NO: 29 SEQ ID NO: 30 100 25.7

* Specificity (%) = (number of amplified oncogenic HPV types ÷ number of amplified HPV types) × 100

**% Sensitivity = (number of amplified oncogenic HPV types ÷ number of oncogenic HPV types) × 100

Odd sequence numbers of the sequence numbers in Table 1 are used as forward primers and even sequence numbers are used as reverse primers.

(3) Synthesis of Selected Primers

In the process, 15 sets of primers selected were synthesized. The primers were synthesized using an Expedite ^™ 8909 nucleic acid synthesizer (ABI Co., Ltd.) to which a solid-phase synthesis technique based on oligonucleotide phosphoramidite synthesis chemistry was applied. First, the synthesis reaction was performed on a CPG column immobilized with nucleosides located at the 3 'end of the oligonucleotide, and basically detritylation, coupling, capping, and oxidation reactions. The oligonucleotide polymerization reaction of the selected PCR primers was carried out in a repeat cycle. After completion of the synthesis, aqueous ammonia was added to the CPG column to isolate the oligomer, followed by deprotection at 55 ° C. for at least 12 hours, concentrated to dryness, and further purified by reverse phase liquid chromatography and anion exchange chromatography. Finally, the purified oligomer was quantified by measuring absorbance at 260 nm.

(4) virtual amplification of primers

Virtual amplification test was performed for each of the 15 sets of primers selected in the above process. Using the same computer program (Amplify 1.2, University of Wisconsin), we predicted the amplification type, amplification product size, and amplification position according to 15 sets of primers selected from a total of 65 HPV types. The results are shown in Table 2 below.

Table 2. Virtual Amplification of Selected Primers

primer Primer base characteristics Amplification Type Amplification product size (bp) (HPV type) Amplification position albio-1 Unwobble 16,31,33,35,44,52,58 319-340 (16) 630-918 albio-2 Unwobble 2a, 6b, 18,29,39,45,51,54,59,68,70 273-321 (18) 645 to 949 albio-3 Unwobble 11,16,31,33,35,52,58 311-323 (16) 627-918 albio-4 Unwobble 11,16,26,31,33,35,44,45,51,52,53,58 187-315 (16) 635-949 albio-5 Unwobble 11,16,31,33,35,52,58 313-325 (16) 625-918 albio-6 Unwobble 11,16,26,31,33,35,52,58 305 ~ 317 (16) 633-918 albio-7 Unwobble 16,31,33,35,52,58 342 ~ 345 (16) 2372-2702 albio-8 Unwobble 16,31,33,34,35,52,56,58 344-347 (16) 2366-2704 albio-9 Unwobble 16,31,33,35,52,58 342 ~ 345 (16) 2364-2700 albio-10 Unwobble 6b, 11,13,16,26,31,32,33,35,42,44,52,55,58,61 400-406 (16) 6615-7228 albio-11 Unwobble 6b, 11,13,26,32,35,42,44,52,53,55,56 404-410 (11) 6604-7232 albio-12 Unwobble 6b, 11,13,26,32,42,44,52,53,55,56 406-412 (11) 6615-7139 albio-13 Unwobble 6b, 11,13,32,42,44,53,55 411-417 (11) 6676 ~ 7239 albio-14 Unwobble 6b, 11,13,16,34,35,39,40,44,45,51,52,55,61,66,70 411-426 (16) 6676 ~ 7239 albio-15 Unwobble 6b, 11,13,16,44,52,55 373-382 (16) 6416-6860

(5) PCR amplification test by the synthesized primer

PCR amplification tests were performed on CaSki (ATCC CRL-1550) and HeLa (ATCC CCL-2), HPV 16 and HPV 18 infected cell lines, using 15 trillion primers synthesized in the synthesis process.

First, CaSki and HeLa were incubated according to the manufacturer's instructions, and then removed from the culture flask by adding 0.25% trypsin solution and centrifuged. The cells were washed once with Dulbecco's phosphate-buffered saline (Gibco) and the cell number was measured under a microscope. Then, 2 × 10 ⁴ cells were suspended in 230 μl of distilled water and heated at 100 ^° C. for 15 minutes to extract cellular DNA. Finally, they were completely cooled at room temperature, centrifuged at 12000 rpm for 5 minutes, and the supernatant was taken as a template DNA solution.

The composition of the reactants used in the PCR experiment (TakaRa) is as follows;

10 x buffer 2.5 μl, 10 mM MgCl ₂ 3.75 μl, 10 mM dNTP 0.5 μl, Taq polymerase 0.5 μl (5 units), 1 μl primer (50 pmoles), distilled water 5.25 μl, template DNA 11.5 μl.

The mixture was allowed to react for 5 minutes at 94 ^o C and sufficiently denatured, followed by 49 times in the order of 94 ^o C 1 minute-49 ^o C 1 minute-72 ^o C 1 minute, and finally at 72 ^o C for 5 minutes. Reacted. 5 μl of the final reaction solution was electrophoresed on 2% agarose gel with DNA size standard. At this time, the electrophoretic gel was stained with 0.00005% ethidium bromide solution. The validity of the band appearing on each path of the electrophoretic gel was determined by comparing the expected size of the PCR product obtained in the computer virtual amplification experiment (see Table 2 above), and the results of PCR were compared with those obtained in the virtual amplification experiment. Matched.

Figure 1a and Figure 1b is a picture of the electrophoresis after PCR for HeLa, HPV-16 infected cell line CaSki, HPV-18 infected cell line, respectively using 15 sets of primers synthesized in the present invention. Where lanes 1 to 15 are used to represent albio primers 1 to 15, respectively, and numbers on the left indicate the amplification products. As can be seen in Figure 1a for the HPV-16 infected cell line CaSki amplification occurs except for albio primers 2, 11, 12, 13. As can be seen in Figure 1b for the HPV-18 infected cell line HeLa can be seen that amplification occurs only when using the albio-2 primer.

Example 2 Use and Amplification Test of Primer

In this example, a PCR procedure was performed according to the combination of some of the 15 sets of primers selected in Example 1 for the purpose of developing a kit capable of comprehensively and selectively detecting tumorigenic HPV types.

(1) Prediction of amplification according to the combination of primers

The amplification degree when the primer was used in combination was confirmed with reference to the amplification degree of the 15 sets of primers identified in Example 1 above. First, we calculated the specificity and sensitivity of all 15 sets of primers and predicted a combination that could have the same amplification degree as when all albio primers were used simultaneously using as few primers as possible. As shown in Table 3 below, even when using four sets of primers in combination, it was confirmed that the amplification was significantly increased compared to each primer.

Table 3. HPV Amplification Degree of Selected Total and Combination Primers

Primer combination Detectable HPV Types Specificity (%) ^* Sensitivity (%) ^** albio-1 ~ 15 2, 6, 11, 13, 16, 18, 26, 29, 31, 32, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 70 96.8 85.7 albio-1 + 2 + 4 + 14 2, 6, 11, 13, 16, 18, 26, 29, 31, 33, 34, 35, 39, 40, 44, 45, 51, 52, 53, 54, 55, 58, 59, 61, 66, 68, 70 96.2 74.2 albio-2 + 4 + 11 + 14 2, 6, 11, 13, 16, 18, 26, 29, 31, 32, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 70 96.6 82.8 albio-2 + 4 + 12 + 14 2, 6, 11, 13, 16, 18, 26, 29, 31, 32, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 70 96.6 82.8 albio-2 + 6 + 11 + 14 2, 6, 11, 13, 16, 18, 26, 29, 31, 32, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 70 96.6 82.8 albio-2 + 6 + 12 + 14 2, 6, 11, 13, 16, 18, 26, 29, 31, 32, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 70 96.6 82.8 albio-2 + 10 + 11 + 14 2, 6, 11, 13, 16, 18, 26, 29, 31, 32, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 70 96.6 82.8 albio-2 + 10 + 12 + 14 2, 6, 11, 13, 16, 18, 26, 29, 31, 32, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 70 96.6 82.8

* Specificity (%) = (number of amplified oncogenic HPV types ÷ number of amplified HPV types) × 100

**% Sensitivity = (number of amplified oncogenic HPV types ÷ number of oncogenic HPV types) × 100

(2) Amplification test of combined primer

Among the combination primers identified through the amplification test, PCR was performed on various tumorigenic HPV DNA, HPV infected cell lines CaSki and HeLa, and HPV uninfected cell lines K-562 by combining albio primers 1, 2, 4, and 14 It was. The strains used in this example are as follows:

HPV-16 (ATCC 45113);

HPV-18 (ATCC 45152);

HPV-31 (ATCC 65446);

HPV-2 (ATCC 45022);

HPV-6 (ATCC 45150);

HPV-1 (ATCC 45021);

HPV-11 (ATCC 45151);

PV-rabbit (ATCC 45072);

PV-bovine (ATCC 45029);

CaSki (ATCC CRL-1550);

HeLa (ATCC CCL-2);

K-562 (KCLB-10243, Korean Cell Line Bank).

First, each E. coli strain containing plasmid DNA of each HPV type was shaken incubated for 16 hours in a 37 ° C LB liquid medium, and then separated and purified by 'Qiafilter Plasmid Maxi Kit (Cat. No. 12263, QIAGEN)' to 10pg. The concentration was adjusted to / μl, and the whole DNA of HPV infected cell line CaSki and HeLa and non HPV infected cell line K-562 was prepared in the same manner as in Example 1.

PCR amplification was performed independently for each of the constituent primers for each template DNA according to the same reactions and procedures as in Example 1. The validity of the bands appearing on each pathway of the electrophoretic gel was determined by comparing the expected size of the PCR product obtained in the computer virtual amplification experiment of HPV type DNA for each primer (see Table 4 below). As a result of PCR, the amplification by each primer was found to be in agreement with that predicted by computer virtual experiments.

Figures 2a to 2l is a picture of the electrophoresis after PCR for a variety of HPV strains and HPV non-infected cell lines using four sets of primers, such as albio primer 1, 2, 4, 14. Figure 2a is HPV-16, Figure 2b is HPV-18, Figure 2c is HPV-31, Figure 2d is HPV-2, Figure 2e is HPV-6, Figure 2f is HPV-1, Figure 2g is HPV-11, Figure 2h is PV-rabbit, FIG. 2I is PV-bovine, FIG. 2J is CaSki, FIG. 2K is HeLa, and FIG. 2L is K-562. In each figure, lane 1 represents an albio-1 primer, lane 2 represents an albio-2 primer, lane 3 represents an albio-4 primer, and lane 4 represents an albio-14 primer, and the number on the side indicates the size of the amplification product. will be. albio-1 primers for HPV-16 (FIGS. 2A and 2J) and HPV-31 (FIG. 2C), albio-2 primers for HPV-18 (FIGS. 2B and 2K), HPV-2 (FIG. 2D) and For HPV-6 (FIG. 2E), albio-4 primers are for HPV-16 (FIGS. 2A and 2J), HPV-31 (FIG. 2C) and HPV-11 (FIG. 2G), and albio-14 primers are for HPV. It can be seen that amplification occurred for −16 (FIGS. 2A and 2K), HPV-6 (FIG. 2E), and HPV-11 (FIG. 2G). In addition, the four sets of primers were amplified against HPV-1 (FIG. 2F), PV-rabbit (FIG. 2H), PV-bovine (FIG. 2I) and non-HPV infected cell line K-562 (FIG. 2L). This did not happen. It can be seen that this is consistent with the prediction results of Table 4 below.

Table 4. Estimated Sizes of Selected Albio Primers 1, 2, 4, and 14 Amplified Products

primer Size of amplified product (bp) HPV-16 HPV-18 HPV-31 HPV-2a HPV-6 HPV-1 HPV-11 PV-rabbit PV-bovine albio-1 335 - ^* 335 - - - - - - albio-2 - 309 - 293 314 - - - - albio-4 310 - 306 - - - 303 - - albio-14 420 - - - 417 - 414 - -

^* Non-amplified

Example 3 Amplification of Selected Primers for Clinical Samples

In this example, it was confirmed whether cervical cancer can be diagnosed early according to whether amplification of HPV type DNA was clinically performed using the four sets of primers identified in Example 2.

(1) Diagnosis of cervical cancer by conventional methods

First of all, a randomized 30 patients who visited the outpatient clinic of the Women's Cancer Center of the Pocheon College of Medicine, Korea were diagnosed with colposcopy, cervicography, biopsy, and cytoplasmic smear under the responsibility of the clinician. Specialized checkups, such as a pap-smear test, were performed. Examination results As shown in Table 5 below, subject 1 is an ovarian cancer having cervical atypical cells, and subjects 2, 5, 6, 7, 8, 9, 10, 11, 12, and 15 are uterine. Cervical cancer patients, subjects 3 and 4, were patients with dysplasia (CIS), a precursor of cervical cancer, and subjects 13 and 14 were patients with squamous cell tumors (15 in total), an early stage of cervical cancer. . The remaining subjects had normal cells (subjects 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25) or only insignificant modified cells (subjects 26 and 27) in the Pap smear smear or ovarian teratoma. (ovary teratoma) (subjects 16 and 30), myoma (subjects 19, 20, 22, 23, 24, 25, 26, 27 and 28), uterine gland adenomyosis (subjects 18, 21 and 29) And cervical cancer unrelated patients (15 patients in total).

In addition, HPV DNA testing of some patients (2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 19, and 24) revealed that the hybrid capture II method (hybrid) 2, 3, 4, 5, 6, 12 were positive, and 19, 24 were negative in capture II, Digene), and 7, 7, 8, 9, 10, 13, 14, in DNA chip (Biomedlab) method. 15 proved to be a proton. Table 5 shows the results of the diagnosis.

Table 5. Clinical Trial Results

Subject No. disease HPV DNA Diagnostic Test Hybrid Capture II (high / low) DNA chip One Ovarian Cancer - ^* - 2 Cervical cancer Positive / negative - 3 Dysplasia Positive / negative - 4 Dysplasia Positive / negative - 5 Cervical cancer Positive / negative - 6 Cervical cancer recurrence Positive / negative - 7 Cervical cancer - 16 8 Cervical cancer - 16 9 Cervical cancer - 58 10 Cervical cancer - 16 11 Cervical cancer - - 12 Cervical cancer Positive / negative - 13 Squamous cell tumor - 16 14 Squamous cell tumor - 16 15 Cervical cancer - 16 16 Ovarian teratoma - - 17 - - - 18 Uterine gland myoma - - 19 Myoma Voice / voice - 20 Myoma - - 21 Uterine gland myoma - - 22 Myoma - - 23 Myoma - - 24 Myoma Voice / voice - 25 Myoma - - 26 Myoma - - 27 Myoma - - 28 Myoma - - 29 Adenoma - - 30 Ovarian teratoma - -

^* Non-irradiation

(2) Diagnosis of cervical cancer with 4 sets of selected primers

PCR was performed on DNA samples obtained from the subjects using four sets of primers identified in Example 2 and MY09 / MY11 primer pairs, which are already disclosed HPV detection primers, and electrophoresis. DNA from the subjects was obtained according to a conventional method, and PCR reaction conditions were carried out in the same procedure as in Example 1 or 2 above. The primer of the present invention amplified only for DNA samples obtained from abnormal patients (subjects 2 to 15) and no amplification to DNA samples obtained from normal patients (subjects 16 to 30). This is consistent with the etiological reasoning of 14 patients with cervical cancer and 15 patients without cervical cancer, except for ovarian cancer patients with only cervical atypical cells (No. 1).

Figures 3a to 3o is a picture of the electrophoresis and PCR performed DNA samples obtained from the abnormal patient using a pair of primers and MY09 / MY11 primer pair according to the present invention. FIG. 3A shows a subject 1, FIG. 3B shows a subject 2, FIG. 3C shows a subject 3, FIG. 3D shows a subject 4, FIG. 3E shows a subject 5, FIG. 3F shows a subject 6, FIG. 3G shows a subject 7, FIG. 3H shows a subject 8, and FIG. 3I. Figure 3j is a subject 10, Figure 3k is a subject 11, Figure 3l is a subject 12, Figure 3m is a subject 13, Figure 3n is a subject 14, Figure 3o is a DNA obtained from the subject 15 . Herein, lane 1 represents aibio-1 primer, lane 2 represents albio-2 primer, lane 3 represents albio-3 primer, lane 4 represents albio-14 primer, and lane 5 represents MY09 / MY11 primer as a comparison. In addition, the numbers on the left side of each picture indicate the size of the amplification product. It can be seen that amplification has occurred with respect to at least one of the primers of the present invention, and it can be seen that amplification has also occurred with the comparative group MY09 / MY11 pair. In particular, DNA samples obtained from patients with recurrent cervical cancer (FIG. 3F) amplified from albio-1 primers and albio-3 primers of the present invention, but it was found that amplification did not occur from MY09 / MY11. have.

Figures 4a to 4b are photographs subjected to PCR and electrophoresis of DNA samples obtained from normal patients using a set of four primers and MY09 / MY11 primer pair according to the present invention. 4A is a subject 6, FIG. 4B is a subject 17, FIG. 4C is a subject 18, FIG. 4D is a subject 19, FIG. 4E is a subject 20, FIG. 4F is a subject 21, FIG. 4G is a subject 22, FIG. 4H is a subject 23, FIG. 4I 4A is a subject 25, FIG. 4K is a subject 26, FIG. 4L is a subject 27, FIG. 4M is a subject 28, FIG. 4N is a subject 29, and FIG. 4O is a DNA obtained from a patient 30. . Herein, lane 1 represents aibio-1 primer, lane 2 represents albio-2 primer, lane 3 represents albio-4 primer, lane 4 represents albio-14 primer, and lane 5 represents MY09 / MY11 primer as a control. In addition, the numbers on the left side of each picture indicate the size of the amplification product. It can be seen that no effective band (300 to 500 bp) was found for both the primer of the present invention and the MY09 / MY11 primer.

The above embodiments are only presented to explain the present invention in detail, but the present invention is not limited thereto.

As described above, the oligonucleotides synthesized in the present invention are PCR primers capable of amplifying the HPV type by binding complementarily with various oncogenic HPV types, each primer being oncogenic HPV individually or in combination with other primers. It can be used to selectively amplify types of DNA. For example, the present invention can be applied to a multiplex PCR method using two or more sets of 15 primers identified in the present invention. In addition, the primer can be used as a probe that can directly detect tumorigenic HPV, HPV can be used to detect the presence of HPV type by PCR and amplification of the primer and the target DNA. It can be used for the early diagnosis of cervical cancer caused by.

<110> ALBIOMED Co., LTD <120> Primers and Method for Detecting Oncogenic Human Papillomavirus by PCR <160> 30 <170> Kopatent In 1.71 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 1 tgcaacctga gacaactgac ctatactgtt atgag 35 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 2 ttactgcttc tacataaaac catccattac atccc 35 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 3 ccttctatgt cacgagcaat taagcgactc aga 33 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 4 aaaccatcca ttacaccccg tcccctcccc gtc 33 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <2 23> Primer for amplifying HPV <400> 5 gacctatact gttatgagca attaagtgac agctc 35 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifying HPV <400> 6 ttactgcttc tacataaaac catccattac atccc 35 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplifyin HPV <400> 7 ctgttatgag caattaagtg acagctcaga tgagg 35 <210> 8 <211> 35 <212> DNA < 213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 8 ttactgcttc tacataaaac catccattac atccc 35 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 9 ctgacctata ctgttatgag caattaagtg acagc 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 10 ttactgcttc tacataaaac catccattac atccc 35 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 11 tactgttatg agcaattaag tgacagctca gatga 35 <210> 12 < 211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 12 ttactgcttc tacataaaac catccattac atccc 35 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220 > <223> Primer for amplyfing HPV <400> 13 caaccattag cagatgccaa aataggtatg ttaga 35 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 14 cctcgtgcaa atttaatctg caccacgtcc ttgag 35 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 15 caaccattag ca gatgccaa aataggtatg ttaga 35 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 16 ttcctcgtgc aaatttaatc tgcaccacgt ccttg 35 <210> 17 <211> 35 <212 > DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 17 tacaaccatt agcagatgcc aaaataggta tgtta 35 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 18 tcgtgcaaat ttaatctgca ccacgtcctt gagaa 35 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 19 actgttgtag atactacccg tagtacaaac atgac 35 <210 > 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 20 ccaaggggaa actgatctaa ctcactagaa aactt 35 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 21 actgttgtag taactacccg tagtacaaac atgac 35 <210> 22 <211> 35 <212> DNA < 213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 22 cctaccaagg ggaaactgat ctaactcact agaaa 35 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyginf HPV <400> 23 actgttgtag taactacccg tagtacaaac atgac 35 <210> 24 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 24 ttcctaccaa ggggaaactg atctaactca ctaga 35 <210> 25 < 211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 25 actgttgtag taactacccg tagtacaaac atgac 35 <210> 26 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 26 aaaacttcct accaagggga aactgatcta actca 35 <210> 27 <211> 35 <212> DNA < 213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 27 ttttatttac gaaaggaaca aatgtttgct agaca 35 <210> 28 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 28 gagcagttaa ggtaatacta cataattgaa acata 35 <210> 29 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 29 tatttacgaa aggaacaaat gtttgctaga cactt 35 <210> 30 < 211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer for amplyfing HPV <400> 30 aactgtaaat caaactcctc aacatggcgc atgta 35

Claims (13)

Nucleotides (first primers) consisting of SEQ ID NO: 1 and SEQ ID NO: 2; Nucleotides (second primer) consisting of SEQ ID NO: 3 and SEQ ID NO: 4; Nucleotides consisting of SEQ ID NO: 5 and SEQ ID NO: 6 (third primer); Nucleotides (fourth primer) consisting of SEQ ID NO: 7 and SEQ ID NO: 8; Nucleotides (fifth primer) consisting of SEQ ID NO: 9 and SEQ ID NO: 10; Nucleotide (sixth primer) consisting of SEQ ID NO: 11 and SEQ ID NO: 12; Nucleotide (seventh primer) consisting of SEQ ID NO: 13 and SEQ ID NO: 14; Nucleotides (eighth primer) consisting of SEQ ID NO: 15 and SEQ ID NO: 16; Nucleotides consisting of SEQ ID NO: 17 and SEQ ID NO: 18 (ninth primer); Nucleotides (the tenth primer) consisting of SEQ ID NO: 19 and SEQ ID NO: 20; Nucleotides (11th primer) consisting of SEQ ID NO: 21 and SEQ ID NO: 22; Nucleotides (the 12th primer) consisting of SEQ ID NO: 23 and SEQ ID NO: 24; Nucleotides consisting of SEQ ID NO: 25 and SEQ ID NO: 26 (Thirteenth Primer); A nucleotide consisting of SEQ ID NO: 27 and SEQ ID NO: 28 (fourteenth primer); and A synthesized primer for amplifying DNA of tumorigenic HPV, wherein the DNA is selected from the group consisting of nucleotides consisting of SEQ ID NO: 29 and SEQ ID NO: 30 (15 primer). The method of claim 1, Said first primer is a primer for amplifying HPV types 16, 31, 33, 35, 44, 52 and 58 DNA, The second primer is a primer for amplifying HPV types 2a, 6b, 18, 29, 39, 45, 51, 54, 59, 68 and 70 DNA, The third primer is a primer for amplifying HPV types 11, 16, 31, 33, 35, 52 and 58 DNA, The fourth primer is a primer for amplifying HPV types 11, 16, 26, 31, 33, 35, 44, 45, 51, 52, 53 and 58 DNA, Said fifth primer is a primer for amplifying HPV types 11, 16, 31, 33, 35, 52 and 58 DNA, The sixth primer is a primer for amplifying HPV types 11, 16, 26, 31, 33, 35, 52 and 58 DNA, The seventh primer is a primer for amplifying HPV types 16, 31, 33, 35, 52 and 58 DNA, The eighth primer is a primer for amplifying HPV types 16, 31, 33, 34, 35, 52, 56 and 58 DNA, Said ninth primer is a primer for amplifying HPV types 16, 31, 33, 35, 52 and 58 DNA, The tenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 26, 31, 32, 33, 35, 42, 44, 52, 55, 58 and 61 DNA, The eleventh primer is a primer for amplifying HPV types 6b, 11, 13, 26, 32, 35, 42, 44, 52, 53, 55 and 56 DNA, The twelfth primer is a primer for amplifying HPV types 6b, 11, 13, 26, 32, 42, 44, 52, 53, 55, and 56 DNA; The thirteenth primer is a primer for amplifying HPV types 6b, 11, 13, 32, 42, 44, 53 and 55 DNA, The fourteenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 34, 35, 39, 40, 44, 45, 51, 52, 55, 61, 66 and 70 DNA, Wherein said fifteenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 44, 52 and 55 DNA. The method of claim 2, The first primer is a primer for amplifying HPV types 16 and 31 DNA, The second primer is a primer for amplifying HPV type 18 DNA, The third primer is a primer for amplifying HPV type 16 DNA, Said fourth primer is a primer for amplifying HPV types 11, 16 and 31 DNA, The fifth primer is a primer for amplifying HPV type 16 DNA, The sixth primer is a primer for amplifying HPV type 16 DNA, The seventh primer is a primer for amplifying HPV type 16 DNA, Said eighth primer is a primer for amplifying HPV type 16 DNA, The ninth primer is a primer for amplifying HPV type 16 DNA, The tenth primer is a primer for amplifying HPV type 16 DNA, Said fourteenth primer is a primer for amplifying HPV types 6, 11 and 16 DNA, Wherein said fifteenth primer is a primer for amplifying HPV type 16 DNA. Nucleotides (first primers) consisting of SEQ ID NO: 1 and SEQ ID NO: 2; Nucleotides (second primer) consisting of SEQ ID NO: 3 and SEQ ID NO: 4; Nucleotides consisting of SEQ ID NO: 5 and SEQ ID NO: 6 (third primer); Nucleotides (fourth primer) consisting of SEQ ID NO: 7 and SEQ ID NO: 8; Nucleotides (fifth primer) consisting of SEQ ID NO: 9 and SEQ ID NO: 10; Nucleotide (sixth primer) consisting of SEQ ID NO: 11 and SEQ ID NO: 12; Nucleotide (seventh primer) consisting of SEQ ID NO: 13 and SEQ ID NO: 14; Nucleotides (eighth primer) consisting of SEQ ID NO: 15 and SEQ ID NO: 16; Nucleotides consisting of SEQ ID NO: 17 and SEQ ID NO: 18 (ninth primer); Nucleotides (the tenth primer) consisting of SEQ ID NO: 19 and SEQ ID NO: 20; Nucleotides (11th primer) consisting of SEQ ID NO: 21 and SEQ ID NO: 22; Nucleotides (the 12th primer) consisting of SEQ ID NO: 23 and SEQ ID NO: 24; Nucleotides consisting of SEQ ID NO: 25 and SEQ ID NO: 26 (Thirteenth Primer); A nucleotide consisting of SEQ ID NO: 27 and SEQ ID NO: 28 (fourteenth primer); and Nucleotides selected from the group consisting of nucleotides consisting of SEQ ID NO. 29 and SEQ ID NO. A method for amplifying DNA of tumorigenic HPV by performing a polymerase chain reaction with a nucleic acid obtained from a biological sample as a target DNA. The method of claim 4, wherein Said first primer is a primer for amplifying HPV types 16, 31, 33, 35, 44, 52 and 58 DNA, The second primer is a primer for amplifying HPV types 2a, 6b, 18, 29, 39, 45, 51, 54, 59, 68 and 70 DNA, The third primer is a primer for amplifying HPV types 11, 16, 31, 33, 35, 52 and 58 DNA, The fourth primer is a primer for amplifying HPV types 11, 16, 26, 31, 33, 35, 44, 45, 51, 52, 53 and 58 DNA, Said fifth primer is a primer for amplifying HPV types 11, 16, 31, 33, 35, 52 and 58 DNA, The sixth primer is a primer for amplifying HPV types 11, 16, 26, 31, 33, 35, 52 and 58 DNA, The seventh primer is a primer for amplifying HPV types 16, 31, 33, 35, 52 and 58 DNA, The eighth primer is a primer for amplifying HPV types 16, 31, 33, 34, 35, 52, 56 and 58 DNA, Said ninth primer is a primer for amplifying HPV types 16, 31, 33, 35, 52 and 58 DNA, The tenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 26, 31, 32, 33, 35, 42, 44, 52, 55, 58 and 61 DNA, The eleventh primer is a primer for amplifying HPV types 6b, 11, 13, 26, 32, 35, 42, 44, 52, 53, 55 and 56 DNA, The twelfth primer is a primer for amplifying HPV types 6b, 11, 13, 26, 32, 42, 44, 52, 53, 55, and 56 DNA; The thirteenth primer is a primer for amplifying HPV types 6b, 11, 13, 32, 42, 44, 53 and 55 DNA, The fourteenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 34, 35, 39, 40, 44, 45, 51, 52, 55, 61, 66 and 70 DNA, Said fifteenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 44, 52 and 55 DNA. The method of claim 5, The first primer is a primer for amplifying HPV types 16 and 31 DNA, The second primer is a primer for amplifying HPV type 18 DNA, The third primer is a primer for amplifying HPV type 16 DNA, Said fourth primer is a primer for amplifying HPV types 11, 16 and 31 DNA, The fifth primer is a primer for amplifying HPV type 16 DNA, The sixth primer is a primer for amplifying HPV type 16 DNA, The seventh primer is a primer for amplifying HPV type 16 DNA, Said eighth primer is a primer for amplifying HPV type 16 DNA, The ninth primer is a primer for amplifying HPV type 16 DNA, The tenth primer is a primer for amplifying HPV type 16 DNA, Said fourteenth primer is a primer for amplifying HPV types 6, 11 and 16 DNA, The fifteenth primer is a primer for amplifying HPV type 16 DNA. Nucleotides (first primers) consisting of SEQ ID NO: 1 and SEQ ID NO: 2; Nucleotides (second primer) consisting of SEQ ID NO: 3 and SEQ ID NO: 4; Nucleotides consisting of SEQ ID NO: 5 and SEQ ID NO: 6 (third primer); Nucleotides (fourth primer) consisting of SEQ ID NO: 7 and SEQ ID NO: 8; Nucleotides (fifth primer) consisting of SEQ ID NO: 9 and SEQ ID NO: 10; Nucleotide (sixth primer) consisting of SEQ ID NO: 11 and SEQ ID NO: 12; Nucleotide (seventh primer) consisting of SEQ ID NO: 13 and SEQ ID NO: 14; Nucleotides (eighth primer) consisting of SEQ ID NO: 15 and SEQ ID NO: 16; Nucleotides consisting of SEQ ID NO: 17 and SEQ ID NO: 18 (ninth primer); Nucleotides (the tenth primer) consisting of SEQ ID NO: 19 and SEQ ID NO: 20; Nucleotides (11th primer) consisting of SEQ ID NO: 21 and SEQ ID NO: 22; Nucleotides (the 12th primer) consisting of SEQ ID NO: 23 and SEQ ID NO: 24; Nucleotides consisting of SEQ ID NO: 25 and SEQ ID NO: 26 (Thirteenth Primer); A nucleotide consisting of SEQ ID NO: 27 and SEQ ID NO: 28 (fourteenth primer); and Amplifying the target DNA using a nucleotide selected from the group consisting of nucleotides consisting of SEQ ID NO: 29 and SEQ ID NO: 30 (15 primer) as a primer and using nucleic acid obtained from a biological sample as a target DNA; And A method for analyzing the presence of oncogenic HPV type comprising the step of determining the presence of HPV type in the sample when the amplification product is present in the amplification process. The method of claim 7, wherein Said first primer is a primer for amplifying HPV types 16, 31, 33, 35, 44, 52 and 58 DNA, The second primer is a primer for amplifying HPV types 2a, 6b, 18, 29, 39, 45, 51, 54, 59, 68 and 70 DNA, The third primer is a primer for amplifying HPV types 11, 16, 31, 33, 35, 52 and 58 DNA, The fourth primer is a primer for amplifying HPV types 11, 16, 26, 31, 33, 35, 44, 45, 51, 52, 53 and 58 DNA, Said fifth primer is a primer for amplifying HPV types 11, 16, 31, 33, 35, 52 and 58 DNA, The sixth primer is a primer for amplifying HPV types 11, 16, 26, 31, 33, 35, 52 and 58 DNA, The seventh primer is a primer for amplifying HPV types 16, 31, 33, 35, 52 and 58 DNA, The eighth primer is a primer for amplifying HPV types 16, 31, 33, 34, 35, 52, 56 and 58 DNA, Said ninth primer is a primer for amplifying HPV types 16, 31, 33, 35, 52 and 58 DNA, The tenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 26, 31, 32, 33, 35, 42, 44, 52, 55, 58 and 61 DNA, The eleventh primer is a primer for amplifying HPV types 6b, 11, 13, 26, 32, 35, 42, 44, 52, 53, 55 and 56 DNA, The twelfth primer is a primer for amplifying HPV types 6b, 11, 13, 26, 32, 42, 44, 52, 53, 55, and 56 DNA; The thirteenth primer is a primer for amplifying HPV types 6b, 11, 13, 32, 42, 44, 53 and 55 DNA, The fourteenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 34, 35, 39, 40, 44, 45, 51, 52, 55, 61, 66 and 70 DNA, Wherein said fifteenth primer is a primer that amplifies HPV types 6b, 11, 13, 16, 44, 52 and 55 DNA. The method of claim 8, The first primer is a primer for amplifying HPV types 16 and 31 DNA, The second primer is a primer for amplifying HPV type 18 DNA, The third primer is a primer for amplifying HPV type 16 DNA, Said fourth primer is a primer for amplifying HPV types 11, 16 and 31 DNA, The fifth primer is a primer for amplifying HPV type 16 DNA, The sixth primer is a primer for amplifying HPV type 16 DNA, The seventh primer is a primer for amplifying HPV type 16 DNA, Said eighth primer is a primer for amplifying HPV type 16 DNA, The ninth primer is a primer for amplifying HPV type 16 DNA, The tenth primer is a primer for amplifying HPV type 16 DNA, Said fourteenth primer is a primer for amplifying HPV types 6, 11 and 16 DNA, Wherein said fifteenth primer is a primer that amplifies HPV type 16 DNA. The method according to any one of claims 7 to 9, Wherein said biological sample is obtained from a human. Nucleotides (first primers) consisting of SEQ ID NO: 1 and SEQ ID NO: 2; Nucleotides (second primer) consisting of SEQ ID NO: 3 and SEQ ID NO: 4; Nucleotides consisting of SEQ ID NO: 5 and SEQ ID NO: 6 (third primer); Nucleotides (fourth primer) consisting of SEQ ID NO: 7 and SEQ ID NO: 8; Nucleotides (fifth primer) consisting of SEQ ID NO: 9 and SEQ ID NO: 10; Nucleotide (sixth primer) consisting of SEQ ID NO: 11 and SEQ ID NO: 12; Nucleotide (seventh primer) consisting of SEQ ID NO: 13 and SEQ ID NO: 14; Nucleotides (eighth primer) consisting of SEQ ID NO: 15 and SEQ ID NO: 16; Nucleotides consisting of SEQ ID NO: 17 and SEQ ID NO: 18 (ninth primer); Nucleotides (the tenth primer) consisting of SEQ ID NO: 19 and SEQ ID NO: 20; Nucleotides (11th primer) consisting of SEQ ID NO: 21 and SEQ ID NO: 22; Nucleotides (the 12th primer) consisting of SEQ ID NO: 23 and SEQ ID NO: 24; Nucleotides consisting of SEQ ID NO: 25 and SEQ ID NO: 26 (Thirteenth Primer); Nucleotides (SEQ ID NO: 14) consisting of SEQ ID NO: 27 and SEQ ID NO: 28; and A primer selected from the group consisting of nucleotides (fifteenth primer) consisting of SEQ ID NO: 29 and SEQ ID NO: 30; dNTP mixtures (dTP, dCTP, dGTP, dTTP); Heat resistant polymerase; And Tumorogenic HPV detection kit comprising PCR buffer. The method of claim 11, Said first primer is a primer for amplifying HPV types 16, 31, 33, 35, 44, 52 and 58 DNA, The second primer is a primer for amplifying HPV types 2a, 6b, 18, 29, 39, 45, 51, 54, 59, 68 and 70 DNA, The third primer is a primer for amplifying HPV types 11, 16, 31, 33, 35, 52 and 58 DNA, The fourth primer is a primer for amplifying HPV types 11, 16, 26, 31, 33, 35, 44, 45, 51, 52, 53 and 58 DNA, Said fifth primer is a primer for amplifying HPV types 11, 16, 31, 33, 35, 52 and 58 DNA, The sixth primer is a primer for amplifying HPV types 11, 16, 26, 31, 33, 35, 52 and 58 DNA, The seventh primer is a primer for amplifying HPV types 16, 31, 33, 35, 52 and 58 DNA, The eighth primer is a primer for amplifying HPV types 16, 31, 33, 34, 35, 52, 56 and 58 DNA, Said ninth primer is a primer for amplifying HPV types 16, 31, 33, 35, 52 and 58 DNA, The tenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 26, 31, 32, 33, 35, 42, 44, 52, 55, 58 and 61 DNA, The eleventh primer is a primer for amplifying HPV types 6b, 11, 13, 26, 32, 35, 42, 44, 52, 53, 55 and 56 DNA, The twelfth primer is a primer for amplifying HPV types 6b, 11, 13, 26, 32, 42, 44, 52, 53, 55, and 56 DNA; The thirteenth primer is a primer for amplifying HPV types 6b, 11, 13, 32, 42, 44, 53 and 55 DNA, The fourteenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 34, 35, 39, 40, 44, 45, 51, 52, 55, 61, 66 and 70 DNA, Said fifteenth primer is a primer for amplifying HPV types 6b, 11, 13, 16, 44, 52 and 55 DNA. The method of claim 12, The first primer is a primer for amplifying HPV types 16 and 31 DNA, The second primer is a primer for amplifying HPV type 18 DNA, The third primer is a primer for amplifying HPV type 16 DNA, Said fourth primer is a primer for amplifying HPV types 11, 16 and 31 DNA, The fifth primer is a primer for amplifying HPV type 16 DNA, The sixth primer is a primer for amplifying HPV type 16 DNA, The seventh primer is a primer for amplifying HPV type 16 DNA, Said eighth primer is a primer for amplifying HPV type 16 DNA, The ninth primer is a primer for amplifying HPV type 16 DNA, The tenth primer is a primer for amplifying HPV type 16 DNA, Said fourteenth primer is a primer for amplifying HPV types 6, 11 and 16 DNA, Said fifteenth primer is a primer for amplifying HPV type 16 DNA.