JP2006345800A - Primer set for detecting human papilloma virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a primer set capable of specifically detecting a plurality of types of human papilloma virus (HPV) genes with high sensitivity. <P>SOLUTION: The kit for detecting HPV contains six or more primer sets comprising specific sequence, and a method specifically detecting HPV in a sample comprises performing, by using the kit, polymerase chain reaction using cDNA as a template, which cDNA is prepared by reverse transcription from DNA or mRNA obtained from the sample. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a specific primer set for detecting an HPV gene and a method for detecting an HPV gene using the primer set.

  Human papillomavirus (HPV) is a DNA virus belonging to the Papovaviridae family, and is deeply involved in skin diseases such as hemorrhoids and cervical carcinogenesis. In fact, HPV is detected from most cervical cancers. HPV has genomic DNA of about 8000 base pairs, and more than 100 subtypes have been reported due to differences in the base sequences. These subtypes are roughly classified into skin types that are mainly involved in skin lesions and mucosal types that are involved in cervical lesions. Furthermore, HPV subtypes called mucosal types are classified into three types: high risk type, intermediate type, and low risk type because of the risk of transition from precancerous lesions to cancer in the cervix.

  In the cytology and histology of precancerous lesions, HPV-infected cells are multinucleated, nuclear atypical, complexed, enlarged, etc. It has long been known to be an atypical cell called tosis. However, HPV DNA is detected by genetic testing in recent years regardless of the presence or absence of corocytosis, and it is said that a precancerous lesion of cervical cancer called dysplasia itself is caused by HPV infection. Therefore, HPV genetic testing can surely capture cervical lesions at an earlier stage than search for colocytosis and dysplasia by cytological examination.

  There are mainly HPV genetic testing methods involving PCR amplification and methods for direct detection without amplification.

  A typical method that does not involve gene amplification is Daigene's hybrid capture method. This method is a method for detecting a double strand obtained by hybridizing an RNA probe to HPV DNA using an antibody that recognizes a DNA-RNA double strand. It is necessary and the sensitivity is not high. Furthermore, since a non-specific reaction is likely to occur, a better method is demanded. This kit is used for the purpose of high-risk / low-risk classification, and each type cannot be identified.

  As a method involving amplification by PCR, after amplification, a labeled probe is hybridized using a PCR-RFLP method in which a band is detected by electrophoresis after digestion with a restriction enzyme and a DNA array (chip) equipped with the probe. It is roughly divided into methods for detecting signals.

  Although the PCR-RFLP method for electrophoretic detection is not so high in detection sensitivity, it is technically simple and does not require a special instrument, so that the cost can be reduced. The PCR-RFLP method is a method in which PCR products are dispensed into a plurality of tubes, and electrophoresis is performed using a plurality of restriction enzymes. Therefore, the operation is complicated because it is necessary to operate 5 to 8 types of tubes per specimen, and when a plurality of HPVs exist in the sample, a pattern different from an expected cutting pattern is obtained. In addition, the type of HPV cannot be identified. In recent years, it has been shown that multiple infections due to multiple types of HPV have occurred on a daily basis, and there is an increasing need in the clinical field to analyze multiple infections. The purpose of identifying the type cannot be fully achieved.

  The latter method using hybridization has high detection sensitivity and high identification specificity, but takes time in all steps of target adjustment, hybridization, washing, and detection. In addition, the production of a DNA array (chip) on which a probe is mounted is expensive and requires special equipment, so that versatility is low. There is currently no equipment that can be installed at the laboratory level of a typical city hospital.

  Therefore, in the field of inspection, there is a demand for a method that can perform HPV detection and typing quickly, simply, and at low cost.

Prior art document information related to the present invention includes the following.
JP-A-2004-121240

  An object of the present invention is to provide a primer set capable of detecting a plurality of types of HPV genes specifically and with high sensitivity.

  The present invention provides a kit including a PCR primer set that can specifically detect 17 types of HPV simultaneously. The kit includes 6 or more primer sets selected from the group consisting of primer sets 1-17 shown in Table 1 below.

  That is, the kit of the present invention includes primer set 1 including oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. A primer set 2 comprising: a primer set 3 comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6 respectively; and an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8, respectively Primer set 4, primer set 5 including oligonucleotides having base sequences represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively, primer set including oligonucleotides having base sequences represented by SEQ ID NO: 11 and SEQ ID NO: 12, respectively 6, each Primer set 7 including an oligonucleotide having the base sequence represented by column number 13 and SEQ ID NO: 14, primer set 8 including an oligonucleotide having the base sequence represented by SEQ ID NO: 15 and SEQ ID NO: 16, respectively, SEQ ID NO: Primer set 9 comprising an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 17 and SEQ ID NO: 18, primer set 10 comprising an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 19 and SEQ ID NO: 20, respectively, SEQ ID NO: 21 and Primer set 11 including an oligonucleotide having a base sequence represented by SEQ ID NO: 22, primer set 12 including an oligonucleotide having a base sequence represented by SEQ ID NO: 23 and SEQ ID NO: 24, respectively, SEQ ID NO: 25 And a primer set 13 including an oligonucleotide having a base sequence represented by SEQ ID NO: 26, a primer set 14 including an oligonucleotide having a base sequence represented by SEQ ID NO: 27 and SEQ ID NO: 28, respectively, SEQ ID NO: 29 and a sequence Primer set 15 including the oligonucleotide having the base sequence represented by No. 30 and Primer set 16 including the oligonucleotide having the base sequence represented by SEQ ID No. 31 and SEQ ID No. 32, respectively, SEQ ID No. 33 and SEQ ID No. 34 6 or more primer sets selected from the group consisting of a primer set 17 including an oligonucleotide having a base sequence represented by:

  Each primer set included in the kit of the present invention can specifically amplify each HPV gene even when these primer sets are mixed together and have different lengths depending on the HPV type. Selected to give amplification product. Therefore, the presence or absence of a large number of HPVs can be examined simultaneously by performing multiplex PCR in one test tube, performing electrophoresis in one lane, and examining the length of the amplified product.

  As shown in the examples below, when the 17 types of primer sets shown in Table 1 and the internal control and external control primers were all mixed, PCR was performed using a mixture of 16 types of HPV genome as a template. Specific amplification products were obtained for all types of HPV and were confirmed by electrophoresis as bands of different lengths (FIG. 1). This means that, out of the 17 types of primer sets shown in Table 1, any 2 sets, 3 sets, 4 sets, or more primer sets can be selected according to the number of HPV types to be detected. Thus, it is shown that the HPV gene present in the sample can be identified by performing multiplex PCR and electrophoresis of amplification products.

  Preferably, the kit of the present invention comprises 7 or more primer sets selected from the group consisting of primer sets 1-17 shown in Table 1 above, more preferably 8 or more, and even more preferably 9 or more. Most preferably, it contains 10 or more primer sets.

  More preferably, the kit of the present invention comprises a primer set capable of detecting one or more of HPV 16, 18, 31, 51, 52, 56 and 58 that have been suggested to be associated with cervical cancer. That is, the kit of the present invention preferably includes at least one of the following primer sets: primer set 3 including two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6, respectively Primer set 4 comprising two oligonucleotides consisting of the base sequences represented by No. 7 and SEQ ID No. 8, respectively, Primer set 5 comprising oligonucleotides having the base sequences represented by SEQ ID No. 9 and SEQ ID No. 10, respectively Primer set 7 comprising two oligonucleotides consisting of base sequences represented by No. 13 and SEQ ID No. 14, primer set 12 comprising two oligonucleotides comprising base sequences represented by SEQ ID No. 23 and SEQ ID No. 24, respectively SEQ ID NO: 25 Primer set 13 including two oligonucleotides each having a base sequence represented by SEQ ID NO: 26, primer set 14 including oligonucleotides having the base sequences represented by SEQ ID NO: 27 and SEQ ID NO: 28, respectively, SEQ ID NO: 29 And a primer set 15 comprising two oligonucleotides consisting of the base sequence represented by SEQ ID NO: 30.

  Even more preferably, the kit of the present invention is a primer set 4 comprising two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and a base represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively. Primer set 5 including oligonucleotides having sequences, primer set 12 including two oligonucleotides each having a base sequence represented by SEQ ID NO: 23 and SEQ ID NO: 24, bases represented by SEQ ID NO: 25 and SEQ ID NO: 26, respectively Primer set 13 including two oligonucleotides composed of sequences, primer set 14 including oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 27 and SEQ ID NO: 28, respectively, bases represented by SEQ ID NO: 29 and SEQ ID NO: 30, respectively 2 consisting of arrays Including the primer set 15 comprising an oligonucleotide.

  Most preferably, the kit of the present invention comprises 34 types of oligonucleotides each having the base sequence set forth in SEQ ID NOs: 1-34.

  In another aspect, the present invention provides a method for detecting the presence of HPV in a sample. The method includes performing a polymerase chain reaction using the above-described kit of the present invention using DNA obtained from the sample as a template, and detecting an amplified HPV gene. In order to detect HPV expression in a sample, mRNA is prepared from the sample, cDNA is synthesized, and then the HPV gene is amplified and detected in the same manner as described above.

  The human papilloma detection kit of the present invention includes six or more kinds of primer sets shown in Table 1 above. According to the present invention, even when each primer set is used as a mixture, a specific type of HPV can be specifically detected. As shown in Examples described later, since the primer set 1-17 of the present invention has high specificity for each target HPV, when these are mixed and subjected to multiplex PCR reaction in one test tube, It is possible to specifically amplify specific types of HPV genes present in the sample. In addition, since each primer set is designed so that amplification products of different lengths can be obtained, the PCR reaction products are separated by electrophoresis in one lane, so that a specific type of HPV present in the sample can be obtained. It is possible to detect a gene specifically. The present invention is a useful method that satisfies the three elements of simplicity, rapidity, and low cost necessary for testing a large number of specimens.

  It has recently been reported that gynecological cytodiagnosis performed on a daily basis is more effective when used in combination with HPV genetic testing. In other words, cytodiagnosis is a technique for determining the state of cells by morphological characteristics, but the result may be subjective because it is determined with the naked eye. In contrast to the subjective quantitative test of cytology, the HPV genetic test according to the present invention can perform not only the presence of HPV but also the identification of the type of HPV, so that an objective qualitative test can be performed. Therefore, the HPV genetic test is very effective both as auxiliary data for cytological examination and as independent data.

  The multiplex PCR method is a method of performing PCR using a primer mix. Each primer set according to the present invention is designed so that the amplification products have different sizes even when amplification of a plurality of PCR products is present. Therefore, a specific amplification product can be detected by fractionating the size by electrophoresis. Furthermore, even when multiple types of HPV are present, amplification products can be detected for each type. Since the target gene contained in the sample can be identified by performing PCR with one tube for one sample and performing electrophoresis with one lane, the method of the present invention is suitable for multi-sample processing. . Moreover, since no operation such as hybridization is required, the cost can be reduced and the inspection time is short. In addition, it can be carried out without requiring special training at the inspection site.

  Since the HPV genetic test is a test using clinical samples, experimental accuracy control is very important. Therefore, it is preferable to set an internal standard and an external standard as amplification targets for multiplex PCR.

  As an internal standard, a gene that is necessarily contained in human genomic DNA present in a clinical specimen is selected. Even if HPV is not present, a band should be detected whenever human genomic DNA is present, and can therefore be used as a positive control for DNA extraction. As an external standard, arbitrary DNA is added to the reaction solution together with a primer set for amplifying the internal region. As a result, amplification of the external standard must occur, so that it can be used as a positive control for the amplification reaction.

  Each oligonucleotide of the primer set of the present invention can be chemically synthesized using, for example, a general-purpose DNA synthesizer (for example, Model 394 manufactured by Applied Biosystems). Oligonucleotides may be synthesized using any of the other methods well known in the art.

  In order to detect the presence of an HPV gene in a sample using the primer set of the present invention, DNA is extracted from the sample, and this is used as a template to perform polymerase chain reaction using the primer set of the present invention to amplify the HPV gene. Let As a sample, a tissue sample obtained from a subject suspected of being infected with HPV, a biopsy, a smear, or the like can be used. Furthermore, cervical squamous epithelial cells that are usually infected with HPV can be concentrated and used from the collected cells by density gradient centrifugation. Alternatively, cultured cells such as HeLa cells may be used. Target diseases considered useful for detecting the presence of the HPV gene according to the present invention include warts, intraepithelial neoplasia, skin cancer, oral and oropharyngeal cancer, genital neoplasia, cervical cancer, condyloma, Papilloma is included.

  Methods for extracting DNA from a sample are well known in the art, and can be performed, for example, by extraction with phenol / chloroform or extraction using a commercially available DNA extraction reagent. Alternatively, extraction with a column kit (GENERATION (registered trademark) Capture Column Kit Gentra) may be performed.

  Furthermore, since the primer set of the present invention is selected so that the sequence in the coding region of the HPV gene is amplified, it can be used to detect the expression of the HPV gene in the sample. In order to detect the expression of the HPV gene, RNA is extracted from the sample, a template cDNA is prepared, and this is used as a template to perform polymerase chain reaction using the primer set of the present invention. Amplify the gene.

  Methods for extracting RNA from a sample are well known in the art, and for example, guanidine-isothiocyanate and phenol / chloroform extraction can be used. Alternatively, a commercially available RNA extraction reagent may be used.

  A method for synthesizing cDNA using RNA as a template is also well known in the art. A commercially available random primer can be used as a primer, and cDNA can be synthesized using reverse transcriptase in the presence of dNTPs. As the reverse transcriptase, for example, SuperScript II (Invitrogen) can be used.

  Next, a polymerase chain reaction (PCR) is performed using the DNA or cDNA thus prepared as a template and a mixture of the primer set of the present invention as a primer. As the polymerase, for example, Ex Taq (registered trademark) (Takara), Z-Taq, AccuPrime Taq DNA Polymerase, a polymerase contained in an M-PCR kit (QIAGEN), or the like can be used. Methods for selecting appropriate PCR reaction conditions based on the Tm of the primer are well known in the art, and those skilled in the art will know the primer length, GC content, desired specificity and sensitivity, the characteristics of the polymerase used, etc. Based on the above, the optimum condition can be selected. For example, in the examples below, PCR was performed at 95 ° C for 15 minutes, then 94 ° C for 30 seconds, 70 ° C for 1.5 minutes, 72 ° C for 1 minute, 35 cycles, then 72 ° C for 2 minutes. Carried out. In addition, the amount of primer included in the PCR solution can be appropriately adjusted so as to achieve a predetermined detection sensitivity and reduce variations in detection sensitivity depending on the virus type.

  Next, the presence or absence of each HPV in the sample can be detected by separating the amplification products by electrophoresis and determining the length of the band.

  The kit of the present invention is characterized in that each HPV gene can be specifically amplified using a primer set corresponding to 17 types of HPVs mixed together. Furthermore, the primer of the present invention is designed to give amplification products having different lengths depending on the HPV type. That is, the presence or absence of 17 types of HPV can be examined simultaneously by performing PCR in one test tube, performing electrophoresis in one lane, and examining the length of the amplified product. This has the advantage that the assay is simple and requires less time, money and samples for the assay compared to prior art methods that perform PCR in separate tubes using type specific primers. Moreover, even in the case of multiple infections, that is, when multiple types of HPV are present in a sample, these can be detected simultaneously.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Primer design Genetic testing in clinical tests, including HPV, requires detecting the presence of DNA of the target virus from the presence of large amounts of human genomic DNA and other DNA such as viruses and microorganisms. It is necessary to design the sequence with high specificity.

  In addition, since the primers are mixed and used, it is necessary to design a primer having high specificity between the primers so that primer dimers are not easily generated. Furthermore, it is very difficult to design primers that can specifically identify multiple subtypes of HPV with high homology. In the case of HPV, it is almost the case that the sequence of one of the other types of HPV is matched at a ratio of approximately 70% or more by simply designing primers normally. Therefore, it is necessary to design primers carefully so that non-specific amplification does not occur.

  In genetic testing by HPV multiplex PCR according to the present invention, subtype identification is performed according to the size of the band. Therefore, the PCR product size must be designed in advance. Separation of PCR products by electrophoresis requires sufficient design as the number of targets increases because sufficient resolution cannot be obtained unless at least 10 to 20 bases are separated. In other words, it is necessary to adjust the size of the band by several base units at the same time as raising the primer specificity to the limit, and the difficulty increases relatively as the number of targets to be identified increases. Become.

  Even if primers are designed randomly without considering the above factors, HPV present in the sample can be detected in a band without any non-specific cross-reaction in the mixed state of primers for 16 types of HPV. It is almost impossible to do. In order to obtain a primer having high specificity as described above, it is essential to optimize the primer set while simultaneously checking specific amplification both in silico and in vitro. In the identification of genes having completely different sequences by the multiplex PCR method, there have been reported examples in which several targets are amplified, but few systems are known that can specifically amplify and confirm 6 or more targets. In particular, for viruses that are highly homologous to each other, such as HPV, there is no known multiplex PCR system that can distinguish the types according to the length of the amplification product. The biggest reason why it is difficult to increase the number of targets in the multiplex PCR method is that it takes a lot of time and cost to check all non-specific amplifications both in silico and in vitro.

  In silico checks check that there are no sequences similar to the designed primers in other subtype sequences. At this time, if an attempt is made to design by narrowing down the region, the region that can be used as a primer is limited, and primer candidates are often not found. Therefore, a primer sequence designed for a certain HPV was searched for the sequences of all other subtypes of HPV, and primer candidates were selected with a mismatch rate (%). The above operation was repeated several tens of times to select all candidate primers. When a candidate primer could not be obtained, the condition setting was changed, and finally a candidate primer was selected.

  Next, using these candidate primer sets, PCR is performed using a sample containing each of the genomes of 16 types of HPV as a template and a sample containing a template mix in which these genomes are mixed. It was confirmed that a band was specifically observed. When non-specific amplification was observed, the primer set was excluded, in-silico verification was performed again, the primer was redesigned, and the specificity was confirmed. This in silico and in vitro verification cycle was repeated dozens of times, and as a result of optimization, a set of primer sets shown in Table 1 was selected.

Since the HPV genetic test is a test using clinical samples, experimental accuracy control is very important. Therefore, an internal standard and an external standard were set as amplification targets for multiplex PCR. As an internal standard, the genomic region of ALAS1 (aminolevulinate, delta-, synthase 1, NM_000688) was used. As an external standard, a plasmid obtained by cloning genomic DNA of Brevundimonas diminuta (syn. Pseudomonas diminuta) was used in an amount of 10 ⁴ copies per reaction solution together with a primer set for amplifying the internal region.

  Primers for multiplex PCR capable of amplifying a total of 19 kinds of targets including these internal and external standards could be designed. Using a mixture of these 19 primer sets, it was possible to identify 16 HPV types by band size (FIG. 1). The PCR amplification product was cloned and sequenced to confirm that it was the nucleotide sequence of the target type of HPV gene.

Next, using various commercially available polymerases, the temperature conditions of PCR were changed in various ways to optimize the conditions. As a result, using QIAGEN's M-PCR kit, 95 cycles at 95 ° C for 15 minutes, followed by 35 cycles of 94 ° C for 30 seconds, 70 ° C for 1.5 minutes, 72 ° C for 1 minute, 72 ° C for 2 minutes When PCR was performed under the conditions described above, the presence of all types of HPV was confirmed as a band with a sensitivity of 10 ² copies per reaction solution (FIG. 2).

Detection of HPV in a clinical sample As a clinical sample, a part of a specimen to be subjected to cytological examination was used as a material, and epithelial cells were concentrated by density gradient centrifugation. When the number of cells was large, a column kit (GENERATION (registered trademark) Capture Column Kit Gentra) was used. When the number of cells was small, DNA extraction was performed by phenol chloroform extraction and ethanol precipitation.

When DNA extraction was performed using phenol chloroform extraction, a PCR solution having the following composition was prepared.
PCR master mix 12.5
Primer mix 4
Template 1
External standard plasmid 1
DDW 6.5
25 μl total

When DNA extraction was performed using a column extraction kit (GENERATION (registered trademark) Capture Column Kit Gentra), a PCR solution having the following composition was prepared.
PCR master mix 12.5
Primer mix 4
Template 5
External standard plasmid 1
DDW 2.5
25 μl total

  PCR was performed using the QIAGEN M-PCR kit. PCR conditions were 95 ° C for 15 minutes, then 94 ° C for 30 seconds, 70 ° C for 1.5 minutes, 72 ° C for 1 minute for 35 cycles, 72 ° C for 2 minutes, and finally 4 ° C. The obtained amplification product was analyzed using a bioanalyzer (Agilent Technology) as a capillary electrophoresis apparatus.

  When the relationship between the diagnosis result by the cytological day classification and the HPV positive rate was examined, a very high correlation was found (Table 2).

  In class I with negative cytology, the HPV positive rate was 4.2%. Class III was 20.6%, whereas Class III was 88.2%, showing a significant increase. Furthermore, there was a clear relationship between cytological results and infection rate, with 85.7% for IIIb and 100% for IV and V.

  Furthermore, according to the classification by the Bethesda system, which is a US classification method, the positive rate is 96.9% (31/31) among HSIL (class IIIa class nuclear atypia and classes IV and V combined). 32) and a positive rate close to 100% (Table 3).

  Class III is classified when morphologically cells with nuclear dysplasia are included. Dysplasia refers to abnormalities in the size of the nucleus / the size of the cytoplasm (N / C) and the amount of chromatin when viewed morphologically. In the first place, since cytodiagnosis itself is a study that has been focused on morphology, the judgment may differ depending on the cytologist. The HPV test has been shown to be very useful because it can qualitatively check the presence or absence of infected cells regardless of human subjectivity.

  It should be noted that HPV positive rate of 10 to 20% was also observed in class I and II, which are negative in cytology. This means that even if the results of cytodiagnosis are negative, HPV typing allows the detection of cervical cancer as soon as possible by following the progression of nuclear atypia, and the choice of treatment, etc. This suggests that it will be advantageous.

  The result of identifying the double infection is shown (FIG. 3). Thus, when there were a plurality of types of HPV, the type could be clearly identified by the band size by a one-step method.

  As a result of multi-sample analysis of cytological specimens, multiple infections were confirmed in less than 30% of all positive specimens. Among them, specimens in which four types were simultaneously infected were also confirmed. These suggest that HPV co-infection occurs on a daily basis.

  From the above results, it was shown that HPV can be specifically detected from clinical samples by multiplex PCR using the HPV detection kit of the present invention. Furthermore, it became clear that superinfection could be detected.

FIG. 1 shows the results of verification of type specificity at the time of HPV gene amplification using the HPV detection kit of the present invention. FIG. 2 shows the result of verification of detection sensitivity using the HPV detection kit of the present invention. FIG. 3 shows the results of detection of superinfection using the HPV detection kit of the present invention.

Claims (5)

Primer set 1 including two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, Primer set including two oligonucleotides each including a base sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4 2. Primer set 3 including two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and two oligonucleotides each including a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8 Primer set 4, primer set 5 including two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively, two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12 Primer set 6 containing Primer set 7 including two oligonucleotides consisting of base sequences represented by SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and two oligonucleotides consisting of base sequences represented by SEQ ID NO: 15 and SEQ ID NO: 16, respectively Primer set 8, primer set 9 comprising two oligonucleotides consisting of base sequences represented by SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and two oligonucleotides comprising base sequences represented by SEQ ID NO: 19 and SEQ ID NO: 20, respectively A primer set 10 comprising two oligonucleotides each comprising two oligonucleotides comprising a nucleotide sequence represented by SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and two primer sequences comprising a nucleotide sequence represented by SEQ ID NO: 23 and SEQ ID NO: 24, respectively Including oligonucleotides Primer set 12, which includes two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 25 and SEQ ID NO: 26, and two oligonucleotides each comprising a base sequence represented by SEQ ID NO: 27 and SEQ ID NO: 28 A primer set 14 including two oligonucleotides each including two oligonucleotides each having a nucleotide sequence represented by SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and two primer sequences each including a nucleotide sequence represented by SEQ ID NO: 31 and SEQ ID NO: 32. A primer set 16 including an oligonucleotide, and 6 or more primer sets selected from the group consisting of a primer set 17 including two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 33 and SEQ ID NO: 34, A kit for detecting HPV. Primer set 3 including two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6, respectively, Primer set including two oligonucleotides each including a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8 4. Primer set 5 including oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively, Primer set comprising two oligonucleotides each comprising the nucleotide sequences represented by SEQ ID NO: 13 and SEQ ID NO: 14 7. Primer set 12 comprising two oligonucleotides consisting of base sequences represented by SEQ ID NO: 23 and SEQ ID NO: 24, respectively, comprising two oligonucleotides comprising base sequences represented by SEQ ID NO: 25 and SEQ ID NO: 26, respectively Primer set 3. Primer set 14 containing oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 27 and SEQ ID NO: 28, respectively, Primer set comprising two oligonucleotides consisting of the nucleotide sequences represented by SEQ ID NO: 29 and SEQ ID NO: 30, respectively The kit according to claim 1, comprising one or more primer sets selected from the group consisting of 15. Primer set 4 comprising two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8, primer set 5 comprising an oligonucleotide having a base sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively Primer set 12 including two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 23 and SEQ ID NO: 24, and primer set including two oligonucleotides each including a base sequence represented by SEQ ID NO: 25 and SEQ ID NO: 26 13. Primer set 14 comprising oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 27 and SEQ ID NO: 28, respectively, Primer set comprising two oligonucleotides comprising the nucleotide sequences represented by SEQ ID NO: 29 and SEQ ID NO: 30, respectively Including 15, kit of claim 1 wherein. The kit according to claim 1, comprising 34 kinds of oligonucleotides each consisting of the base sequence set forth in SEQ ID NOs: 1-34. A method for detecting the presence or expression of HPV in a sample, wherein DNA obtained from the sample or cDNA prepared by reverse transcription from mRNA obtained from the sample is used as a template. Performing a polymerase chain reaction using the kit described and detecting the amplified HPV gene.