KR101401940B1 - Kit for analyzing high-risk HPV gene and method for analyzing the same - Google Patents

Abstract

The present invention relates to a kit for analyzing a high-risk HPV genome gene and a method of analyzing the same, and more particularly, to a method for simply and effectively analyzing a high-risk HPV type virus. Especially, the genotype of papillomavirus is highly related to cervical cancer, And a method for detecting the genotype only.

Description

[0001] The present invention relates to a kit for analyzing a high-risk HPV genome and a method for analyzing the HPV gene,

The present invention relates to a kit for analyzing a high-risk HPV genome gene and a method for analyzing the same, and more particularly, to a method for simply and effectively analyzing a high-risk HPV type virus. Especially, the genotype of HPV is highly related to cervical cancer, And a method for detecting the genotype only.

Cervical cancer is the second most common female cancer worldwide, following breast cancer. Epidemiologic studies have shown that cervical cancer is caused by infection with human papillomavirus (HPV), and HPV DNA was detected in 99.7% of patients with cervical dysplasia and cervical cancer.

HPV is proliferating in the nucleus and is found mainly in the cytoplasm in the form of an episome. The HPV genome consists of about 8 kb of double-stranded circular DNA, an open reading frame (ORF) that encodes the E1, E2, E4, E5, E6, and E7 genes that act on their DNA replication and cell transformation, It consists of L1 and L2 genes, which are expressed only in epithelial cells and make capsid proteins. The classification of HPV genotypes is based on the similarity of the DNA sequences. If the sequences of E6, E7, and L1 ORFs differ by more than 10% from the existing sequences, they are defined as new genotypes and 90-98% (Subtype), and more than 98% variants.

To date, about 100 HPV subtypes have been known, of which about 40 subtypes are known to be associated with cervical cancer. The HPV genotypes are classified into high risk group, probable high risk group, and low risk group based on patient / control comparative epidemiological studies. Genotypes belonging to the high risk group associated with invasive cervical cancer were 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, There were 26, 53, and 66 genotypes belonging to the high risk group that were not associated with the development of cervical cancer but 6, 11, 40, 42, 43, 44, 54 , 61, 70, 72, 81 and so on.

In a study of 426 women in Korea, HPV positive cases were distributed in the order of 16, 52, 53, 62, 58, 81, 56, 66, 18 and 44 in order of invasive squamous cervical cancer And HPV was found in 86% of patients with high grade squamous intraepithelial lesion (HSIL). High-risk HPV infection occurs in the 10th to 20th years with the onset of sexual life. Periodic and repeated examination is necessary because epithelial dysplasia occurs 10 years later and cervical cancer occurs in the 40s and 50s. In particular, the risk of cervical cancer incidence is higher by 12-350 times in these persistently high-risk HPV-infected women, especially if they are tested every 6 months and the likelihood that certain high-risk HPVs are continuously detected increases the likelihood of developing cervical cancer

The odds ratio of high-risk HPV group and cervical cancer is 260.63 and the likelihood ratio of HPV group with high risk and high risk HPV group is 464.1, which supports the reason why high-risk group should be included in screening for cervical cancer . However, current high-risk screening products sold commercially do not include potential risk groups.

Screening test for cervical cancer is very important because of the close relationship between HPV infection and cervical cancer and high incidence of cervical cancer. In general, screening test can be divided into cytological examination and high risk HPV DNA test.

Liquid-based Pap smears were obtained by observing changes in cell morphology, and histopathologically confirmed the lesion stage as atypical squamous cells of undetermined significance (ASCUS), mild cervical dysplasia (low grade squamous intraepithelial lesion, LSIL), high grade squamous intraepithelial lesion HSIL, and invasive squamous cervical cancer (SCC). This method has advantages of economical efficiency and easiness of examination method, but it is diagnosed according to the shape of the cell. Therefore, not only the objectivity of the diagnosis is low but also the false negative rate is high (15 ~ 45%) and the reproducibility and accuracy of the result is poor .

Hybrid capture (Qiagen) method and PCR method are known as HPV DNA screening method.

Hybrid capture (Qiagen) method is a method of detecting HPV using a hybridization reaction of a probe. It is convenient because there is no separate DNA extraction process. However, when a large amount of DNA is required, Because RNA probes are used, the stability is low and the equipment is expensive considering that it is a screening test. In addition, it is pointed out that the false positives caused by the cross activity which is reported consistently since the launch are a big problem. The probes detecting the high risk group may show false positives in response to the HPV DNA rather than the high risk group.

The PCR method is a rapid, simple, and large-scale screening method for amplifying a small amount of DNA by preparing oligomers that specifically react with HPV nucleic acids and has a higher specificity than the cytogenetic method or the hybrid capture method. However, it has been reported that the PCR detection method shows a difference in the sensitivity of the genotypes depending on the composition and conditions of the primer used and the reaction, and amplification products are obtained using the PGMY11 / 09 and GP5 + / 6 + primer sets which are generally used In the absence of a separate genotyping process, it is not possible to identify high-risk HPV associated with cervical cancer. In order to improve the sensitivity, a second PCR (nested PCR method) is used but it takes more time than one PCR because of the two PCR steps, and the false positive rate may increase.

Therefore, the specificity of detection of only high risk groups, which are highly related to cervical cancer, including potential high risk groups, increases the practicality and efficacy of cervical cancer screening, and at the same time requires sensitive, accurate and reproducible test methods.

Korean Patent Publication No. 1020060059063 discloses a novel oligonucleotide probe for detecting human papilloma virus, a DNA chip for analysis of human papilloma virus genotype, an assay kit, and a method for producing the same, and more particularly, A probe in which guanine is bonded to the 5 'end of a novel 30 kinds of oligonucleotides capable of binding to each of the probes, and DNA for analysis of HPV genotype using microarray technology so that each type of HPV can be detected and identified using the probe Chip and an assay kit and a method of manufacturing the same.

The present invention solves the above problems and is accomplished by the above-mentioned need. It is an object of the present invention to provide a method and apparatus for detecting high-risk HPV, which is susceptible to infection or susceptible to infection, And to provide a method for detecting high-risk HPV.

Another object of the present invention is to provide a primer which specifically detects only the genotypes of 18 high-risk HPV types highly associated with cervical cancer, and a kit containing the same.

In order to achieve the above object, the present invention provides a primer pair for detection of HPV type 16 of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, or SEQ ID NOs: 7 and 8;

A pair of primers for detection of HPV 18 type of SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, or SEQ ID NOs: 13 and 14;

A pair of primers for detection of HPV type 31 of SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, or SEQ ID NOs: 19 and 20;

A pair of primers for detection of HPV type 33 of SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, or SEQ ID NOs: 25 and 26;

A pair of primers for detection of HPV type 35 of SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30, or SEQ ID NOs: 31 and 32;

A pair of primers for detection of HPV 39 type of SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, or SEQ ID NOs: 37 and 38;

A pair of primers for detection of HPV type 45 of SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, or SEQ ID NOs: 43 and 44;

A pair of primers for detection of HPV type 51 of SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, or SEQ ID NOs: 49 and 50;

A pair of primers for detection of HPV type 52 of SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54, or SEQ ID NOs: 55 and 56;

A pair of primers for detection of HPV type 56 of SEQ ID NOs: 57 and 58, SEQ ID NOs: 59 and 60, or SEQ ID NOs: 61 and 62;

A pair of primers for detection of HPV type 58 of SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 66, or SEQ ID NOs: 67 and 68;

A pair of primers for detection of HPV type 59 of SEQ ID NOs: 69 and 70, SEQ ID NOs: 71 and 72, or SEQ ID NOs: 73 and 74;

A pair of primers for detection of HPV type 68 of SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 78, or SEQ ID NOs: 79 and 80;

A pair of primers for detection of HPV type 73 of SEQ ID NOs: 81 and 82, SEQ ID NOs: 83 and 84, or SEQ ID NOs: 85 and 86;

A pair of primers for detection of HPV type 82 of SEQ ID NOs: 87 and 88, or SEQ ID NOs: 89 and 90;

A pair of primers for detection of HPV type 26 of SEQ ID NOS: 91 and 92, SEQ ID NOS: 93 and 94, or SEQ ID NOS: 95 and 96;

A pair of primers for detection of HPV type 53 of SEQ ID NOs: 97 and 98, SEQ ID NOs: 99 and 100, or SEQ ID NOs: 101 and 102;

A pair of primers for analyzing one or more high-risk HPV genotypes selected from the group consisting of SEQ ID NOS: 103 and 104, SEQ ID NOS: 105 and 106, or SEQ ID NOS: 107 and 108,

The present invention also provides a composition for detecting a high-risk HPV gene comprising the primer pair of the present invention.

HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 selected by HPV subtypes of the present invention, , 68, 73, 82, 26, 53, and 66, but is not limited thereto.

In another embodiment of the present invention, the concentrations of the primers of the HPV 16, 51, and 26 types are higher than those of the HPV 18, 33, 35, 39, 45, 52, 56, The HPV 59 and HPV type primers of the 31 types are the HPV 18, 33, 35, 39, 45, and 52 types, respectively, But is not limited to, three times the concentration of the primers of type 56, 58, 68, 73, 82, 53, and 66.

The present invention also provides a kit for detecting a high-risk HPV genome gene comprising the primer pair of the present invention.

In one embodiment of the present invention, the kit preferably further comprises a buffer solution, a DNA polymerase, and dNTP, but is not limited thereto.

The present invention also relates to a method for the preparation of a pharmaceutical composition comprising the steps of: obtaining a sample from an individual; And a method for detecting a high-risk HPV genome comprising the steps of: (a) detecting a high-risk HPV genome; and (b) detecting a high-risk HPV genome.

In one embodiment of the present invention, the annealing temperature in the PCR is preferably 65 to 70 ° C, but is not limited thereto.

Hereinafter, the present invention will be described.

The present invention relates to genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 of the HPV types classified as high risk and low risk groups and belonging to high risk group highly associated with invasive cervical cancer. 68, 73, 82, 26, 53, 66 of the present invention can be specifically detected.

The high-risk group used in the present invention was 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, including genotypes of high- And 66, respectively.

The HRP (high risk primer) used in the present invention refers to primers that specifically amplify the high-risk HPV group.

The high-risk HPV detection method of the present invention can be summarized as follows. The high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 66 genotypes are analyzed in a fast, simple, sensitive, accurate, and efficient way, and other genotypes, other types of viruses or other microorganisms, except HPV high risk, are not detected, and HRPs that specifically react with high risk HPV Mixture (HRP Mix) is used. The reaction time is about 1 hour and the reaction is carried out at 65 ℃ or higher.

In order to accomplish the object described in the subject to be solved, the present invention provides a method of amplifying a specific HPV nucleic acid, comprising the steps of: a) selecting a primer capable of selecting each genotype in the primers represented by SEQ ID NOS: 1 to 108, b) confirming the detection of HPV DNA of a high risk group from the amplified reactant (PCR product).

The HRP used in the present invention has a base sequence that specifically amplifies only HPV high-risk genes, and is characterized by a primer composition that specifically hybridizes under stringent hybridization conditions.

In the present invention, the HRP is characterized by having multiple sequences of the HPV genotype, and not only in DNA viruses or microorganisms of humans or other species, but only in 18 HPV high risk groups.

In the present invention, the HRP is composed of 108 forward or reverse nucleotides, and specifically reacts at 65-70 ° C. It is characterized by sequencing, RFMP, real- time PCR) and HPV DNA chips.

Generally, the primer used is generally designed to operate at 65 DEG C or less. However, HRP used in the present invention is designed such that the temperature of the guanine and cytosine arrangement of the primer sequence and the temperature of Tm (Melting Temperature) is 65 Lt; 0 > C or more.

The high-risk HPV detection kit of the present invention comprises HRP Mix comprising a total of 18 sets of primers, one DNA primer set for each genotype among the primers of SEQ ID NOS: 1 to 108, DNA polymerase, dNTPs and buffer solution .

According to the HPV high-risk detection method of the present invention, the positive detection rate is more effective than the conventional high-risk HPV detection method. It is also a powerful detection method that is more sensitive, specific, and has high throughput processing than conventional HPV high risk assays when considering all HPV diagnoses detected in infected patients.

1 to 1-18 in FIG. 1, 18 HPV plasmid DNAs were diluted with TE (Tris-ETDA) buffer (containing 1 ng / ml Human Genomic DNA) to find an optimal annealing temperature in HRP, Were subjected to polymerase reaction under the conditions of 50 ° C, 55 ° C, 60 ° C, 65 ° C and 70 ° C, and the amplified product was verified using an automatic electrophoresis apparatus. Lane 1 is 50 ° C, lane 2 is 55 ° C, lane 3 is 60 ° C, lane 4 is 65 ° C, lane 5 is 70 ° C, lane 6 is negative control, and L is a size marker. 1-1 is 16 type, 1-2 is 18 type, 1-3 is type 31, 1-4 is 33 type, 1-5 is 35 type, 1-6 is 39 type, 1-7 is 45 Type 1-8 is 51, 1-9 is 52, 1-10 is 56, 1-11 is 58, 1-12 is 59, 1-13 is 68, 1-14 is 73, , 1-15 indicates 82 type, 1-16 indicates 26 type, 1-17 indicates 53 type, and 1-18 indicates 66 type.
FIG. 2 (a) is a photograph of the amplification product of the HRP mix prepared using HRP mix at a concentration of 0.05 μM, which is lower than the primer concentration of 0.2 μM to 0.5 μM used in the conventional polymerase chain reaction, . Where L is the size marker, lane 1 is 16, lane 2 is 18, lane 3 is 31, lane 4 is 33, lane 5 is 35, lane 6 is 39, lane 7 is 45, Lane 9 is 52 type, lane 10 is 56 type, lane 11 is 58 type, lane 12 is 59 type, lane 13 is 68 type, lane 14 is 73 type, lane 15 is 82 type, lane 16 is 26 type Lane 17 shows 53 type and lane 18 shows 66 type. Fig. 2B shows a sample diluted with PBS buffer (containing 1 ng / ml Human Genomic DNA) of 18 types of HPV plasmid DNA and 0.025 mu M HRP PCR amplification products were verified using automated electrophoresis equipment. Where L is the size marker, lane 1 is 16, lane 2 is 18, lane 3 is 31, lane 4 is 33, lane 5 is 35, lane 6 is 39, lane 7 is 45, Lane 9 is 52 type, lane 10 is 56 type, lane 11 is 58 type, lane 12 is 59 type, lane 13 is 68 type, lane 14 is 73 type, lane 15 is 82 type, lane 16 is 26 type Lane 17 represents 53 type, lane 18 represents 66 type, and FIGS. 2c to 2g show 18 kinds of HPV plasmid DNAs in 10 consecutive dilutions with PBS buffer (containing 1 ng / ml Human Genomic DNA) The PCR product was amplified with 0.025 μM HRP mix at the same concentration, and amplification products were verified using automatic electrophoresis equipment. Among the 18 types of HPV genotypes, five kinds of sensitivities are significantly low. 2c represents 16 type, 2d represents 31 type, 2e represents 51 type, 2f represents 59 type, and 2g represents 26 type. Lane 1 was 5x10 ³ copies / PCR, lane 2 was 2.5x10 ³ copies / PCR, lane 3 was 1x10 ³ copies / PCR, lane 4 was 5x10 ² copies / PCR, lane 5 was 2.5x10 ² copies / 6, 1x10 ² copies / PCR for lane 7, 5x10 ¹ copies / PCR for lane 7, 2.5x10 ¹ copies / PCR for lane 8, 1x10 ¹ copies / copies / PCR.
3, 3a-3e, HRP mixes were prepared by varying the magnification according to the genotypes of five kinds of primers having significantly low sensitivity among 18 types of HPV genotypes. Then, 18 kinds of HPV plasmid DNA were dissolved in PBS buffer (1ng / ml PCR amplification was carried out using an automated electrophoresis system. The amplification products were verified by PCR amplification using sixteen-type and 31-type amplified products, , 51 type, 59 type, and 26 type. 3a represents 16 type, 3b represents 31 type, 3c represents 51 type, 3d represents 59 type, and 3e represents 26 type. Lane 1 was 5x10 ³ copies / PCR, lane 2 was 2.5x10 ³ copies / PCR, lane 3 was 1x10 ³ copies / PCR, lane 4 was 5x10 ² copies / PCR, lane 5 was 2.5x10 ² copies / 6, 1x10 ² copies / PCR for lane 7, 5x10 ¹ copies / PCR for lane 7, 2.5x10 ¹ copies / PCR for lane 8, 1x10 ¹ copies / copies / PCR.
4a in FIG. 4 is a high-risk HPV determination method. L is a size marker, lane 1 is HPV high-risk group, lane 2 is high-risk HPV group, and lane 3 is a sample that has failed to extract DNA due to lack of an internal control band. FIG. 4B is a spectrum of lane 1 shown in FIG. 4A, FIG. 4C is a spectrum of lane 2 shown in FIG. 4A, and HPV high-risk group is positive or negative, respectively. FIG. 4D is a spectrum of lane 3 shown in FIG. 4A. Since the DNA extraction has failed or the sample is insufficient, sampling should be repeated.
FIG. 5 shows the results of the detection of HPV 6, 11, 40, 42, 44, 54, 55, 61, 62, 67, 68, 70, 71, The PCR products were analyzed by HRP mix using 72, 74, 81, 83, 84, 86, 87, 89, Are size markers and each lane represents a genotype determined to be a low-risk HPV group by the sequencing method. PC represents a positive control and NC represents a negative control.
FIG. 6 is a photograph of 564 women in the Department of Obstetrics and Gynecology, Konkuk University Hospital after performing the test according to the method of the present invention and verifying the amplification product using an automatic electrophoresis apparatus. Only 21 out of 564 patients showed results. In all 21 patients, the band was observed at 520 bp, which is an amplification product of human-derived β-golbin, which is an internal control, and HPV The corresponding band appeared around 300 bp, a high-risk amplification product. L is a size marker and lanes 1, 2, 3, 5, 6, 9, 10, 11, 15, 16, 17, 20 and 21 are considered HPV high risk groups and lanes 4, 7, 8, 12, , 18 and 19 were judged to be high-risk HPV negative.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not limited by the following examples.

Example  One: primer  Design and selection

The HPV-associated HPV group was found to be closely associated with human cervical cancer induction, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26 To design 18 pairs of primers capable of amplifying 53,66 types, HPV genomic sequences were amplified using NCBI GeneBank (Aceession No. HE798687, AJ388068, X66269, A25077, GQ161734, K02718, L22674, AF115831, M83860, M83862, S67066, L22664, M83882, EU918764, EF140814, AF404703, JF728187, FJ528600, X05015, AY262282, GQ180787, AY863152, FJ610150, GQ180791, U45889, HM596525, EF202153, U45893, M12732, DQ059013, AF548837, EF140827, EF140828, JN041163, HQ537681, HQ537680, HQ537679, HM596539, EU911366, DQ241373, JN041158, HQ537677, HM596536, X05015, J04353, GU296036, EU911364, HQ537694, HQ537693, HQ537691, GU797245, GQ479014, EU779744, DQ486473, M12732, HQ537707, FJ202006, U45896, HE798575, M74117, M62849, JN104070, U45902, M62849, EU911263, EF202167, EU911266, JN104069, U45903, U45916, M96309, M62877, HM596533, HV509953, EU779728, GQ472848, EF177181, EU779717, DQ003074, EF202166, EF202167, X74479, Y13218, GQ487711, AB438955, M62877, U45917, D90400, GU296060, GU296065, JN874436, HQ537743, X77858, DQ057319, JN874423, DQ080079, FJ797780, X94165, EU911637, HQ537745, AB027021, U45920, GQ472849, EF546481, EF546480, DQ486475, JN393897, X74482, DQ007180, EF177176, HM585462, X74483, EF177179, X74472, X74482, X77858, U45930, U45932, EU911290, AB437933, FJ797805, EF177191, DQ486474, DQ007162, EF177189, U31794, AY147908, U12498, JN661514, EU918769, AJ831568, FR751040, U45934, GQ472851, DQ080079).

The sequence of the primer (HRP) for amplifying the high-risk HPV gene to be analyzed is as follows.

HPV genotype Primer set number Sequences (5 '-> 3') Estimated size of amplification product (bp) SEQ ID NO: 16 type Primer set 1 AAGGGGTCGGTGGACCGGTC 293 One TGTGTGCTTTGTACGCACAACCG 2 Primer set 2 CCCTGTTTTCCTGACCTGCACTGCT 302 3 TCGGTTTGCACACACCCATGTGCA 4 Primer set 3 TGCAAGCAACAGTTACTGCGACGT 307 5 GACCGGTCCACCGACCCCTT 6 Primer set 4 TAACAGCAGCCTCTGCGTTTAGGT 290 7 AAAGTTGGGTAGCCGATGCACGT 8 18 type Primer set 5 GGTGGTGCCAGCCTATAACATTTCA 299 9 CCCTGTGACTTACACAGGTAGCGG 10 Primer set 6 CCGCTACCTGTGTAAGTCACAGGGG 300 11 CACAGTGTCCAGGTCGTGTAGCAGC 12 Primer set 7 AAGTTGCGTGCAGGACAAAATCA 280 13 TAGTTCCTCGCATGTGTCTTGCAGT 14 Type 31 Primer set 8 CGACACACCACACGGAGTGTGTACA 310 15 GCAACGTAGGTGTTTCTCCACGCAT 16 Primer set 9 CAACCTGAGGCAACTGACCTCCACT 301 17 TGCATCCCGTCCCCTCCCCA 18 Primer set 10 ACTTGTTCCTACTTGTTCCTGCTCC 288 19 GGCAAGGTGTGTTAGGCAGGAAACT 20 33 type Primer set 11 ACAGGTTAAGGTTGTTGACCCTGC 318 21 GGCACGGTGTGGTCTAAAGGCA 22 Primer set 12 GGCAATTTGGTTTAACACCTCCTCC 281 23 GCGTTTTGCAGACGATGTGCG 24 Primer set 13 GGCTGGGATGGGGTGTACTGGTTGG 305 25 ACGTTCTACACGGGTTTGCAGCACG 26 35 type Primer set 14 ACAGGTAGAGGGGCATGATACAGT 280 27 ATGTTTAAAGTTCGCCACACTTGGG 28 Primer set 15 TACCAGACAGCGGTTATGGCAA 310 29 GTTCGCCACACTTGGGGCTATT 30 Primer set 16 CTGTGGCGGTCTAACGAAGCCACT 300 31 ACAGGCCCAAACCAAACGCTGGAG 32 39 type Primer set 17 ACGCAGTCACAGTTTCTGTCCA 291 33 AGTTGCATACACCGAGTCCGAG 34 Primer set 18 TGGGGTAAGGGAAAGGCATGCAAGC 305 35 GCGTCACCCACCATACCACCACGAT 36 Primer set 19 GCCAATCGTGAAGGTACAGACGGG 292 37 ACGGTCTAGTGTCGCCACTGCT 38 45 type Primer set 20 CACGTCGACCCCCAAAACCGC 304 39 ACCTGTCCAATGCCAGGTGGAGG 40 Primer set 21 TGCTGCAATGTCCCGCTTGTGCA 300 41 TGCTGCACGGTGGGATACCATGGT 42 Primer set 22 GACGGGGAGGGAACGGGGTG 301 43 CCGTCTCCACACTTAGCTGCTCCC 44 51 type Primer set 23 TCTTTGTCTAGGCAGTTGCCCTCCT 300 45 ACCTTGCTGTCATTAGTGCGCCA 46 Primer set 24 AGGGGGCGGGGTGTAATGGG 315 47 CGTTTGCCTGACTATGGCTGTGTGT 48 Primer set 25 TGCTGGCAACGTACACGACAACGT 302 49 GCTGTACAACGCGAAGGGTGTCTCC 50 52 type Primer set 26 CATGTGGGTGGTCGGGTAATTGT 300 51 ACAAGTTGTATGTGCAACCCGTCC 52 Primer set 27 GCGACGGACCTTCGTACTCTACAGC 297 53 ACAATGCCCGGGCTGCCTCA 54 Primer set 28 AGCACTGCGACGGACCTTCGTACT 305 55 AAACAATGCCCGGGCTGCCTCA 56 56 type Primer set 29 CAGGTCCTGACATGGTGTTG 299 57 GCAAGCAATCGTGAACTGCC 58 Primer set 30 GGCGCCGTAAACGTATTCCCT 322 59 ACCGTTCCTGGTCCGGATT 60 Primer set 31 ATGGTGTTGCCTACAGGCCCCAGT 301 61 GGGATGTCCTACGGCAAGCAATCGT 62 58 type Primer set 32 TGAAGGTACAAACGGGGTTGGGGCG 301 63 AGCCCGGTCCACACAGTCCTCTACA 64 Primer set 33 GGAGGTACATGTGGGTAGTCGGGT 280 65 TTTAGAACTACACACGTTCCGCCC 66 Primer set 34 GCTGGCAGTTCCAGACTTTTGGC 320 67 TCAGACCCTGGCTGTGCGGG 68 59 type Primer set 35 TGGACACCCTTTCGCAGCGT 300 69 GCCAAGAACTATGGCAAACAGCACC 70 Primer set 36 CGCAAGACGGATTGCGAGCCTTACA 300 71 TAAACAAGGCCTGTGCTGTCTCGCG 72 Primer set 37 GGACACCCTTTCGCAGCGTT 318 73 TGGACAATGCAAGAAACATGCCA 74 68 type Primer set 38 AGGCCTGCCTTAACATCCCGAAGA 316 75 TGGATGCAGCAGAAGCCAATGAAGG 76 Primer set 39 CCAATTGGCTGCACAAGGCACAAGG 304 77 GGCGGAGGGGCAACACCAAAATTCC 78 Primer set 40 ACCAGGTGGTGCCGCCTGTAAACAT 305 79 ACCCCAGTAATCCACACGCCCTTGG 80 73 type Primer set 41 ACTGGTGATTGTCCCCCACTGGAAT 314 81 GGTGTTGCAGTATTGCCTGTGCCTT 82 Primer set 42 GCGCCTAGTATGGGCCTGTTCTG 281 83 ATTCCAGTGGGGGACAATCACCA 84 Primer set 43 GGGGGTGGGCAAAGGTAGGTAGCA 304 85 TCCGAGTCACACTGTCTTTGCCGT 86 82 type Primer set 44 AGACATTACACCTTCCGCTGGTACA 288 87 ACCTTAACCTGTGAAAATGCCCTGC 88 Primer set 45 CACCTGCACCAGTGTCACCCATTGT 305 89 GCCAACACCTAACGGCTGACCCCTA 90 26 type Primer set 46 GGCGGGGGTGTACAGGGTGGTT 301 91 TGCTGACTGTCGCTTTGCTGTCTGT 92 Primer set 47 AGTACTGTAGCACCTGACCCCGAT 301 93 TGCGCGGCGTATGTATGCTAGG 94 Primer set 48 CGTGGACTTTAAACAAGAGGCGGAA 307 95 TGACTGTCCGGGCTGTGTGG 96 53 type Primer set 49 GTTGCCCACCGGGCACGCAG 303 97 TGAAGGGTCAGTGGGGCCTACCGA 98 Primer set 50 CCTCCATTTTGCCTTGCAACCG 299 99 AGGTGCACACCTGGTTTACTAGACA 100 Primer set 51 TTGCAACCGGTTTCGGTTGTGC 308 101 ACGGTTGCGGTGCATGAGTAATAGG 102 66 type Primer set 52 TGCAACGTGCACAGGGCCA 293 103 TGCAACTGGTGGGGATAAGCCA 104 Primer set 53 AGGCCGAGGTCAACCTTTAGGTGCT 301 105 TGCACCAAACCCGGTGTCCACCA 106 Primer set 54 TGCAACGTGCACAGGGCCATA 295 107 GTTGCAACTGGTGGGGATAAGCCAA 108

Table 1 is a table showing primer sequences and designs.

Example  2: HPV  High-risk detection method

(1) Screening of primers according to HPV genotype

The primers shown in Table 1 are primers that can selectively amplify the type-specific HPV DNA by complementary binding with a specific type of HPV DNA through computer simulation. As a result, The same results can be obtained by using any of the HPV genotype-specific primers listed in Table 1.

For example, in the HRP Mix used in the present invention, the HPV type 16 and the HPV type 18, 23 and 4, the HPV 18 type 13 and 14, the HPV 31 type 17 and 18, the HPV 33 type 25, HPV type 45 was 41 and 42, HPV 51 was 47 and 48, HPV 52 was 53 and 54, and HPV 56 was HPV 35 The HPV type 58 and the HPV type 59 were 57 and 58, the HPV 58 type was 67 and 68, the HPV type 59 was 69 and 70, the HPV type 68 was 77 and 78, the HPV type 73 was 83 and 84, The HPV type 26, the HPV type 26, the HPV type 26, the HPV type 26, the HPV type 26, and the HPV type 26, respectively.

(2) Polymerase reaction temperature setting

To find the optimal annealing temperature, 18 kinds of HPV genomic plasmid DNAs were diluted with PBS buffer (1 ng / ml of Human Genomic DNA) in the primer set used in the HRP Mix, and the polymerase reaction Respectively.

100 ng of each of the 18 HPV plasmid DNAs to be analyzed were added to each of the primers of 1x buffer, 1 mM MgSO ₄ , 200 mM dNTP, 0.5 U of DNA polymerase, and 18 kinds of HPV genotypes, Lt; / RTI >

94 DEG C for 5 minutes,

94 ° C 15 sec 50 ° C to 70 ° C 30 sec 72 ° C 30 sec (45 cycles)

72 ℃ 5 minutes

The final reaction solution used was MultiNA (MCE-202, Shimadzu) automatic electrophoresis equipment and DNA-1000Kit capable of analyzing the size of 100bp-1,000bp. Prepare the separation buffer, which is a component of the kit, by mixing 62 μl of each sample and 0.63 μl of the fluorescent dye solution. 6.2 μl of the marker solution and 5 μl of the PCR product are prepared and the PCR product is determined according to the MultiNA user protocol . As shown in FIGS. 1-1 to 1-18, the validity of the band appeared in each lane of the photograph was judged to be in agreement with the expected size of the amplification product of Table 1 above. All the 18 HPV genotypes were amplified in all lanes except for the negative control, and the size of the amplified product was the same as the expected size in Table 1. As shown in Table 2, At 65 ℃ and 70 ℃, no nonspecific bands were observed in all genotypes.

HPV genotype Temperature 50 ℃ 55 ° C 60 ° C 65 ℃ 70 ℃ 16 O X X X X 18 O O O X X 31 O O O X X 33 O O O X X 35 O O X X X 39 O O O X X 45 O O O X X 51 O O O X X 52 O O O X X 56 O O O X X 58 O O O X X 59 O O O X X 68 O O X X X 73 O X X X X 82 O O X X X 26 O O O X X 53 O O O X X 66 O O X X X

 Table 2 shows the presence or absence of nonspecific bands according to temperature.

Therefore, it is preferable to use it at an annealing temperature of 65 캜 to 70 캜. The subsequent PCR was carried out at 94 ° C for 5 minutes, at 94 ° C for 15 seconds, at 65 ° C for 30 seconds, at 72 ° C for 30 seconds (45 cycles), and at 72 ° C for 5 minutes.

 (2) Polymerase reaction Reaction condition setting

To determine the optimal concentration of the HRP mix for detecting all 18 genotypes, an HRP mix was prepared by using 0.05 μM of each primer, which is lower than the concentration of 0.2 μM to 0.5 μM used in the conventional polymerase reaction, The template was prepared by diluting 18 HPV plasmid DNAs with PBS buffer (containing 1 ng / ml Human Genomic DNA).

Buffer solution 1x, MgSO ₄ 2 mM, dNTP 200 mM, DNA polymerase 0.5 U, HRP Mix 1 μM, and 100 ng of the HPV DNA to be analyzed, and the total reaction volume was adjusted to 20 μl. The polymerase reaction was carried out under the following conditions.

94 DEG C for 5 minutes,

94 占 폚 15 sec 65 占 폚 30 sec 72 占 폚 30 sec (45 cycles)

72 ℃ 5 minutes

The final reaction solution was a PCR product using MultiNA (MCE-202, Shimadzu) (Fig. 2a). As shown in Figure 2a, amplification products were formed in all lanes of 18 HPV genotypes and the size of the amplification product was approximately 300 bp, which was consistent with the expected size of the amplification products in Table 1 above, but non-specific products Respectively.

Therefore, in order to improve the specificity of the detection method of the present invention, the HRP Mix was reduced from 0.05 μM to 1/2, 1/4, 1/5, and 1/10, Confirmed that nonspecific products disappeared without reducing DNA amplification of 18 high-risk HPV groups at 0.025 μM. (Fig. 2b) It was confirmed that the amplification amount of DNA of 18 high-risk HPV types was reduced even though the concentration of HPV DNA was lower than that of 0.025 μM. Therefore, it is preferable to use HRP Mix at a concentration lower than 0.025 μM . In addition, since HPV samples were obtained from human vaginal part, an internal control was added to the HRP Mix to confirm that the DNA extraction failed or the amount of the sample was insufficient.

The internal control primer consists of a forward 5'-AGGGAGGGCAGGAGCCAGGG-3 '(SEQ ID NO: 109) nucleotide and a reverse 5'-CGTGCAGCTTGTCACAGTGCAGC -3' (SEQ ID NO: 110) nucleotide.

The internal control is a primer capable of detecting human-derived β-golbin, and the amplification product is 520 bp. If the band is not present, it means that the DNA extraction has failed or the sample is deficient.

As shown in Figure 2b, amplification products were formed in all lanes of 18 HPV genotypes and the size of the amplification product was approximately 300 bp, which is consistent with the predicted results in Table 1 above. Especially, in every lane of all photographs, no bands were found which are difficult to distinguish from effective bands.

The 18 kinds of HPV plasmid DNAs were diluted with 10 steps of serial dilution with PBS buffer (1 ng / ml of Human Genomic DNA) (5000, 2500, 1000, 500, 250, 100, 50, 25, As a result, HPV genotypes of 18 types, 33 types, 35 types, 39 types, 45 types, 52 types, and 18 types were selected as shown in FIGS. 2c to 2g. Sensitivity of less than 300copies / PCR was shown for 56 type, 58 type, 68 type, 73 type, 82 type, 53 type and 66 type. In case of 16 type, 31 type, 51 type, 59 type, and 26 type And the detection ability was remarkably low. (Figures 2C-2G)

Thus, in order to maintain sensitivity of less than 300copies / PCR in all 18 HPV genotypes, primer concentrations of 16, 31, 51, 59, and 26 types in HRP Mix were doubled, tripled, and quadrupled, respectively As a result of performing the polymerase reaction in the same manner as described above, the primers set 2, 24 and 47 were amplified twice (0.05 μM), the primer sets 9 and 35 were amplified three times (0.075 μM) When HRP mix was constructed, it was confirmed that all 18 HPV genotypes showed sensitivity of less than 300copies / PCR. As shown in FIGS. 3A to 3E, in the HPV genotypes of 18 kinds including 5 species with low sensitivity in FIGS. 2C to 2G, amplification products were formed at less than 300copies / PCR, and the size of amplification products was about 300 bp, 1 of the amplified product. In particular, no bands were found which were difficult to distinguish from the effective bands in each lane of all photographs, and this fact indicates that the method of the present invention does not deteriorate at all compared to the conventional hybrid capture method in which the sensitivity and specificity according to the HPV genotype I confirmed the fact.

(3) Result judgment method

The results of the method of the present invention were determined using a MultiNA (MCE-202, Shimadzu) automatic electrophoresis equipment and DNA-1000Kit capable of analyzing the size of 100bp-1,000bp. Prepare the separation buffer, which is a component of the kit, by mixing 62 μl of each sample and 0.63 μl of the fluorescent dye solution. 6.2 μl of the marker solution and 5 μl of the PCR product are prepared and the PCR product is determined according to the MultiNA user protocol do. Whether the bands or spectra appearing in each lane of the photograph are valid is determined by the HPV high risk group (lane 1 or 4b in FIG. 4a) when the corresponding band or spectrum of about 300 bp, which is the expected size of the PCR product of Table 1, If the band or spectrum is not present, HPV is not high risk or HPV negative (lane 2 or 4c in Figure 4a). In addition, if the corresponding band or spectrum is not observed at 520 bp, which is an amplification product of human-derived β-golbin, which is an internal control, it means that the DNA extraction has failed or the sample is insufficient. (Lane 3 or 4d in Fig. 4A)

Experimental Example  1: Minimum detection limit

18 HPV plasmid DNAs were serially diluted 10 times with PBS buffer (containing 1 ng / ml Human Genomic DNA) in the same manner as in Examples 1 and 2, and then repeated 20 times.

Table 3 is a representative representation of the minimum detection limit for 16 types of 18 HPV genotypes.

Table 3 shows HPV type 16 minimum detection limits.

When 18 HPV genotypes were repeatedly tested in the same manner as above, the concentration that showed a positive result of 95% or more in each genotype was set as a minimum detection limit and analyzed by probit analysis. As a result, The minimum detection limit was less than 300copies / PCR in all 18 high-risk HPV types, as shown in Table 4 below.

HPV genotype The minimum detection limit
(Number of copies / PCR) 16 26.91 (95% CI: 14.93-70.33) 18 78.48 (95% CI: 46.23-183.02) 31 42.78 (95% CI: 28.78-93.90) 33 49.63 (95% CI: 27.87-129.93) 35 58.75 (95% CI: 43.10-104.86) 39 57.35 (95% CI: 40.43-113.18) 45 72.84 (95% CI: 48.74-157.48) 51 91.46 (95% Cl: 56.81-207.41) 52 34.24 (95% CI: 18.58-92.57) 56 134.10 (95% CI: 91.38-265.27) 58 50.97 (95% CI: 28.96-125.43) 59 129.74 (95% CI: 93.70-238.61) 68 40.20 (95% CI: 22.51-101.79) 73 57.48 (95% CI: 33.66-134.86) 82 83.67 (95% CI: 54.40-186.06) 26 259.80 (95% CI: 178.36-496-21) 53 173.56 (95% CI: 121.97-327.87) 66 36.77 (95% CI: 20.54-93.75)

Table 4 shows the minimum detection limits for HPV genotypes.

Therefore, it was confirmed that the minimum detection limit for the HPV high risk group was 10 times that of the HPV high risk group detection method according to the present invention, compared with the conventional method, Hybrid Capture 2. (Lorincz, AT Hybrid Capture ^TM method for detection of human papillomavirus DNA in clinical specimens: a tool for clinical management of equivocal pap smears and for population screening, J. Obstet. Gynecol Res. 1996a; 22 (6): 629-636.).

Experimental Example  2: Specificity of the method of the present invention

HPV 6, 11, 40, 42, 44, 54, 55, 61, 62, 67, 69, 70, 71, 72, and 74 were identified as HPV low risk group , 81, 83, 84, 86, 87, 89, and 102 were subjected to polymerase reaction in the same manner as in Examples 1 and 2.

As shown in Table 5, HPV high-risk genotypes were not amplified in all 22 genotypes determined to be low-risk HPV by sequencing (Figure 5)

Table 5 is a table verifying the specificity of the method of the present invention.

Therefore, in the conventional method of analyzing HPV high-risk group, a probe for detecting a high-risk group in Hybrid Capture 2 may cause false positives in response to HPV DNA rather than a high-risk group, whereas the detection method according to the present invention detects only high- And that there is no false positives due to cross errors.

Experimental Example  3: Clinical efficacy comparison between the present invention and the conventional method

In this experimental example, in order to confirm the effectiveness of the method of the present invention, the method of the present invention and the conventional methods of hybrid capture 2 and the sequencing method were carried out in a human clinical sample.

First, specialist examinations such as biopsy and pap-smear were conducted for the women who visited Konkuk University Hospital Obstetrics and Gynecology Clinic under the responsibility of the clinic. In this comparative example, a total of 564 patients were enrolled. Seventy-five cervical intraepithelial neoplasia (CIN), 13 adenocarcinoma in situ (ACIS), 5 cervical cancer, and 471 cervical cancer I had normal irrelevant findings.

According to the result of tissue biopsy and cytology smear test, 93 patients diagnosed to be related to cervical cancer and 471 normal persons not related to cervical cancer were screened by the conventional method, Hybrid Capture 2, the sequencing method, and the high- .

Only 56 out of 564 women who visited Konkuk University Hospital were surveyed by the method of the present invention, only the results of 21 were shown in FIG. As shown, the band appeared at 520 bp, which is an amplification product of human-derived β-golbin, which is an internal control in all 21 patients, and the corresponding band at around 300 bp, which is the HPV high-risk amplification product, The band appeared. Lanes 1, 2, 3, 5, 6, 9, 10, 11, 15, 16, 17, 20 and 21 were considered to be high risk HPV. Lanes 4, 7, 8, 12, 13, 14, HPV negative was judged to be negative.

As a result, Hybrid Capture 2, which is a conventional method, showed sensitivity of 81.3%, specificity of 73.5%, positive predictive value of 15.6%, negative predictive value of 98.5%, sensitivity of 93.8%, specificity of 73.7%, positive predictive value of 17.7% , And negative predictive value of 99.5%. The method of the present invention showed a sensitivity of 93.8%, a specificity of 73.3%, a positive predictive value of 17.4%, and a negative predictive value of 99.5%. It was confirmed that the high-risk HPV detection method according to the present invention matches the clinical sequence with the nucleotide sequence method used as a reference method and has superior clinical performance than Hybrid Capture 2, which is widely used in clinical practice. (Table 6)

Table 6 is a table comparing the clinical efficacy of the conventional method and the method according to the present invention.

Experimental Example  4: Comparison of agreement between the present invention and the conventional method

In this experimental example, the above three high-risk HPV detection methods were performed using the clinical sample of Experimental Example 3 in order to confirm the concordance rate between the method of the present invention and the conventional methods of Hybrid Capture 2 and the sequencing method.

As a result, 131 of Hybrid Capture 2 positive samples and 41 of negative samples, which are conventional methods out of 172 positive samples of the present invention, and 86 of Hybrid Capture 2 positive samples and 356 of negative samples, among 392 method negative samples of the present invention and 356 of negative samples, % (κ = 0.68) was confirmed.

Of the 170 samples that were positive in the conventional sequencing method, 37 samples of Hybrid Capture 2 positive samples and 40 samples of negative samples, 39 samples of Hybrid Capture 2 positive samples and 394 of negative sequence samples, And the agreement of 86.3% (κ = 0.67) was confirmed.

Finally, 165 of the 172 sequenced positive samples and 7 negative samples of the conventional method among the 172 positive samples of the present invention, 97 of the sequenced positive samples and 397 negative samples among 392 method negative samples of the present invention, and 97.9 % (κ = 0.95), respectively. (Table 7)

Table 7 is a table comparing the degree of agreement between the method of the present invention and the conventional method.

Experimental Example  5: Confirmation result of inconsistency between the present invention and the conventional method

In order to confirm the cause of the inconsistency between the hybrid capture method 2 and the sequencing method, which confirmed the result of discrepancy of 13.7% in Experimental Example 4, and the present invention method and the sequencing method, which showed a mismatching result of 2.1% Analysis was performed.

Seven mismatches identified in Experimental Example 4, positive and sequenced negative samples of the present invention, five negative method of the present invention and a nucleotide sequence positive sample papilloma virus genotype were confirmed. As a result, four low-risk groups and an unknown risk group were identified among seven positive and sequenced negative samples of the present invention, and the remaining three were not identified as HPV. In addition, high risk groups were confirmed in five positive samples of the present invention and negative sequence sequencing.

In addition, 37 disparity hybrid capture 2 positive And base sequence light negative samples, 40 hybrid capture 2 negative And HPV genotypes of the base sequence positive samples were confirmed. As a result, Hybrid Capture 2 positive Eleven low risk and unknown risk groups and four high risk groups were identified among the base sequence and negative samples and the remaining 21 were not identified as HPV. Of the 40 hybrid capture 2 negative and sequencing positive samples, 39 were identified as high risk and 1 as low risk, while the remaining one was not identified as HPV.

Finally, we identified the HPV genotypes of 36 mismatched hybrid capture 2 positive and inventive method negative samples and 41 hybrid capture 2 negative and inventive method positive samples. As a result, 11 low-risk and unknown risk groups and 4 high-risk groups among 36 hybrid-capture-2 positive and inventive method negative samples were identified, and the remaining 21 were not identified as HPV. In addition, 41 of the Hybrid Capture 2-negative and 39 positive samples of the method of the present invention were identified as high-risk and one low-risk, and the remaining one was not confirmed as HPV. It has been confirmed that the high-risk HPV detection method according to the present invention can be used as a part for correcting nonspecific binding errors of Hybrid Capture 2, which is a conventional method. (Table 8)

Table 8 shows the result of discrepancy between the method of the present invention and the conventional method.

Attach an electronic file to a sequence list

Claims (8)

A pair of primers for detection of HPV type 16 selected from the group consisting of the primers set forth in SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, and SEQ ID NOs: 7 and 8;
A pair of primers for detection of HPV 18 type selected from the group consisting of primers set forth in SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, and SEQ ID NOs: 13 and 14;
A pair of primers for detection of HPV type 31 selected from the group consisting of primers set forth in SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20;
A pair of primers for detection of HPV 33 type selected from the group consisting of the primers set forth in SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, and SEQ ID NOs: 25 and 26;
A pair of primers for detection of HPV type 35 selected from the group consisting of primers set forth in SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30, and SEQ ID NOs: 31 and 32;
A pair of primers for detection of HPV 39 type selected from the group consisting of primers set forth in SEQ ID NOS: 33 and 34, SEQ ID NOS: 35 and 36, and SEQ ID NOS: 37 and 38;
A pair of primers for detection of HPV type 45 selected from the group consisting of primers set forth in SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, and SEQ ID NOs: 43 and 44;
A pair of primers for detection of HPV type 51 selected from the group consisting of primers set forth in SEQ ID NOS: 45 and 46, SEQ ID NOS: 47 and 48, and SEQ ID NOS: 49 and 50;
51 and 52, SEQ ID NOs: 53 and 54, and SEQ ID NOs: 55 and 56;
A pair of primers for detection of HPV type 56 selected from the group consisting of primers set forth in SEQ ID NOs: 57 and 58, SEQ ID NOs: 59 and 60, and SEQ ID NOs: 61 and 62;
A pair of primers for detection of HPV type 58 selected from the group consisting of primers set forth in SEQ ID NOS: 63 and 64, SEQ ID NOS: 65 and 66, and SEQ ID NOS: 67 and 68;
A pair of primers for detection of HPV type 59 selected from the group consisting of primers set forth in SEQ ID NOS: 69 and 70, SEQ ID NOS: 71 and 72, and SEQ ID NOS: 73 and 74;
A pair of primers for detection of HPV type 68 selected from the group consisting of primers set forth in SEQ ID NOS: 75 and 76, SEQ ID NOS: 77 and 78, and SEQ ID NOS: 79 and 80;
A pair of primers for detection of HPV type 73 selected from the group consisting of primers set forth in SEQ ID NOS: 81 and 82, SEQ ID NOS: 83 and 84, and SEQ ID NOS: 85 and 86;
A pair of primers for detection of HPV type 82 selected from the group consisting of primers set forth in SEQ ID NOS: 87 and 88, and SEQ ID NOS: 89 and 90;
A pair of primers for detecting HPV 26 type selected from the group consisting of primers set forth in SEQ ID NOS: 91 and 92, SEQ ID NOS: 93 and 94, and SEQ ID NOS: 95 and 96;
A pair of primers for detection of HPV type 53 selected from the group consisting of primers set forth in SEQ ID NOS: 97 and 98, SEQ ID NOS: 99 and 100, and SEQ ID NOS: 101 and 102;
Comprising a primer pair for high-risk HPV genome analysis consisting of a pair of primers for detection of HPV type 66 selected from the group consisting of primers set forth in SEQ ID NOS: 103 and 104, SEQ ID NOS: 105 and 106, and SEQ ID NOS: 107 and 108, A composition for detecting high risk HPV virus gene. delete delete The method according to claim 1, wherein the concentrations of the HPV 16, 51, and 26 primers are selected from the group consisting of HPV 18, 33, 35, 39, 45, 52, 56, 58, The HPV 59 and HPV type primers of the present invention can be used for the HPV 18, 33, 35, 39, 45, 52, 56, Wherein the concentration of the primer is 3 times the concentration of primers of type 58, 68, 73, 82, 53, and 66. A pair of primers for detection of HPV type 16 selected from the group consisting of the primers set forth in SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, and SEQ ID NOs: 7 and 8;
A pair of primers for detection of HPV 18 type selected from the group consisting of primers set forth in SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, and SEQ ID NOs: 13 and 14;
A pair of primers for detection of HPV type 31 selected from the group consisting of primers set forth in SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20;
A pair of primers for detection of HPV 33 type selected from the group consisting of the primers set forth in SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, and SEQ ID NOs: 25 and 26;
A pair of primers for detection of HPV type 35 selected from the group consisting of primers set forth in SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30, and SEQ ID NOs: 31 and 32;
A pair of primers for detection of HPV 39 type selected from the group consisting of primers set forth in SEQ ID NOS: 33 and 34, SEQ ID NOS: 35 and 36, and SEQ ID NOS: 37 and 38;
A pair of primers for detection of HPV type 45 selected from the group consisting of primers set forth in SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, and SEQ ID NOs: 43 and 44;
A pair of primers for detection of HPV type 51 selected from the group consisting of primers set forth in SEQ ID NOS: 45 and 46, SEQ ID NOS: 47 and 48, and SEQ ID NOS: 49 and 50;
51 and 52, SEQ ID NOs: 53 and 54, and SEQ ID NOs: 55 and 56;
A pair of primers for detection of HPV type 56 selected from the group consisting of primers set forth in SEQ ID NOs: 57 and 58, SEQ ID NOs: 59 and 60, and SEQ ID NOs: 61 and 62;
A pair of primers for detection of HPV type 58 selected from the group consisting of primers set forth in SEQ ID NOS: 63 and 64, SEQ ID NOS: 65 and 66, and SEQ ID NOS: 67 and 68;
A pair of primers for detection of HPV type 59 selected from the group consisting of primers set forth in SEQ ID NOS: 69 and 70, SEQ ID NOS: 71 and 72, and SEQ ID NOS: 73 and 74;
A pair of primers for detection of HPV type 68 selected from the group consisting of primers set forth in SEQ ID NOS: 75 and 76, SEQ ID NOS: 77 and 78, and SEQ ID NOS: 79 and 80;
A pair of primers for detection of HPV type 73 selected from the group consisting of primers set forth in SEQ ID NOS: 81 and 82, SEQ ID NOS: 83 and 84, and SEQ ID NOS: 85 and 86;
A pair of primers for detection of HPV type 82 selected from the group consisting of primers set forth in SEQ ID NOS: 87 and 88, and SEQ ID NOS: 89 and 90;
A pair of primers for detecting HPV 26 type selected from the group consisting of primers set forth in SEQ ID NOS: 91 and 92, SEQ ID NOS: 93 and 94, and SEQ ID NOS: 95 and 96;
A pair of primers for detection of HPV type 53 selected from the group consisting of primers set forth in SEQ ID NOS: 97 and 98, SEQ ID NOS: 99 and 100, and SEQ ID NOS: 101 and 102;
A pair of primers for detection of HPV type 66 selected from the group consisting of primers set forth in SEQ ID NOS: 103 and 104, SEQ ID NOS: 105 and 106, and SEQ ID NOS: 107 and 108
A kit for detecting high-risk human papillomavirus genes comprising a pair of primers for analyzing high-risk human papilloma virus genes. 6. The kit according to claim 5, wherein the kit further comprises a buffer solution, DNA polymerase, and dNTP. A method for detecting a high-risk HPV genome comprising the steps of: (a) performing a polymerase chain reaction using the primer pair composition of claim 1; and (h) detecting a high-risk HPV gene. [Claim 7] The method according to claim 7, wherein the annealing temperature in the polymerase chain reaction is 65 to 70 < 0 > C.

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