WO2007148762A1 - Method of detecting human papilloma virus (hpv) and, for use therein, primer set and detection kit - Google Patents

Method of detecting human papilloma virus (hpv) and, for use therein, primer set and detection kit Download PDF

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Publication number
WO2007148762A1
WO2007148762A1 PCT/JP2007/062536 JP2007062536W WO2007148762A1 WO 2007148762 A1 WO2007148762 A1 WO 2007148762A1 JP 2007062536 W JP2007062536 W JP 2007062536W WO 2007148762 A1 WO2007148762 A1 WO 2007148762A1
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gene
hpv
oligonucleotide
menstrual blood
human
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PCT/JP2007/062536
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French (fr)
Japanese (ja)
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Masahiko Kuroda
Yoshinori Kosugi
Kosuke Oikawa
Masami Tanaka
Seiko Iizuka
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Masahiko Kuroda
Yoshinori Kosugi
Kosuke Oikawa
Masami Tanaka
Seiko Iizuka
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Application filed by Masahiko Kuroda, Yoshinori Kosugi, Kosuke Oikawa, Masami Tanaka, Seiko Iizuka filed Critical Masahiko Kuroda
Priority to JP2008522519A priority Critical patent/JPWO2007148762A1/en
Publication of WO2007148762A1 publication Critical patent/WO2007148762A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma

Definitions

  • HPV Human papillomavirus
  • the present invention relates to a method for detecting human papillomavirus (HPV), a primer set for detecting HPV52, and a detection kit.
  • HPV human papillomavirus
  • Cervical cancer is the most common gynecological malignant tumor in Japan.
  • an internal examination is conducted to examine the shape and size of the uterus, and then cells or tissues are collected from the uterine genital area using instruments such as cotton swabs, brushes, and small wooden sticks.
  • a diagnostic test is performed, it is a trend.
  • the cytodiagnosis test is generally performed by the Pap staining method.
  • Cervical cell abnormalities are broadly divided into dysplasia and cancer.
  • the dysplasia is classified into mild dysplasia, moderate dysplasia, and advanced dysplasia, and cancer is classified into five stages, stage 0, stage I, stage II, stage III and stage IV, depending on the degree of progression. Is done.
  • human papillomavirus Human Papillomavirus
  • HPV is involved in previous studies (Non-Patent Document 1).
  • HPV is a tumor virus with double-stranded DNA that infects human epithelium such as skin and mucous membranes.
  • HPV DNA is circular and 7.9 bp.
  • the gene composition of HPV DNA consists of six residues: El, E2, E4, E5, E6, and E7, called the URR (up-stream regulatory region), which is the gene expression regulatory region, and the early region involved in virus replication. It is classified into Ll and L2, which are called late regions, which code for genes and virus capsids. Of these genes, both E6 and E7 genes are known to be major genes involved in carcinogenesis.
  • HPV subtypes types are over 100 and are numbered in order of discovery.
  • HPV subtypes are 16 type, 18 type, 31 type, 33 type, 35 type, 39 type, 45 type, 51 type, 52 type, 56 type, 58 type, 59 type and 68 type.
  • the 52 type is the mainstream subtype of HPV in East Asia such as Japan, South Korea and China, so detection is said to be important.
  • HPV16, together with type 18, has been reported to account for about 70% of all uterine cancer cases.
  • HPV infects the squamous epithelium of the uterus and about 90% of HPV infections disappear spontaneously, while the remaining 10% are persistent infections. On average, about 75% of Japanese people are persistently infected.
  • uterine cells can dysplasia. If the virus is eliminated even if dysplasia occurs, the cells will return to normal, but if the infection continues without the virus being eliminated, the degree of cell dysplasia may progress. Mild dysplasia often heals spontaneously, but if it progresses to moderate or severe dysplasia, natural healing becomes difficult. If left untreated for a long time without treating advanced dysplasia, the lesions may progress, leading to cervical cancer. Therefore, detection of PV is important in screening for cervical cancer, and detection of HPV52 is particularly important in Japanese. It is also important to detect HPV16 because of its high incidence.
  • HPV can be confirmed by cytology of cells collected from the uterine genital area.
  • a method has been developed in which HPV is detected by extracting a gene from the collected cells and selectively amplifying the gene by PCR (Patent Document 1).
  • cytodiagnosis requires a psychologically resistant treatment for the subject, and even if it is mild, it may damage biological tissue. For this reason, cervical cancer screening is the least used because of its resistance to internal examinations, although it can be taken at public expense (no self-pay).
  • Patent Document 1 Japanese Unexamined Patent Application Publication No. 2004-121240
  • Non-Patent Document 2 “Winoless”, No. 52 ⁇ No. 2, pp. 287-293, 2002
  • an object of the present invention is to provide a detection technique that can detect HPV without causing pain if the subject feels resistance.
  • the detection method of the present invention is a method for detecting HPV from a human biological sample, wherein a human menstrual blood sample is used as the human biological sample, and the HPV gene in the human menstrual blood sample is detected. This is a method for detecting HPV.
  • the first primer set of the present invention is a primer set used for detecting an HP V52 type gene from a human menstrual blood sample by a gene amplification method, and includes a pair of forward primer and reverse primers,
  • the primer is an oligonucleotide (A1) or (A2) below, and the reverse primer, the oligonucleotide (B1) or (B2) below.
  • A2 An oligonucleotide having a nucleotide sequence in which one or several bases are substituted, deleted or added in the nucleotide sequence of SEQ ID NO: 1, and capable of amplifying an HPV52 type gene from a human menstrual blood sample.
  • the nucleotide sequence of SEQ ID NO: 2 consists of a nucleotide sequence in which one or several bases are substituted, deleted or added, and can amplify the HPV52 gene from human menstrual blood samples Oligonucleotide.
  • the second primer set of the present invention is a primer set used for detection of an HP V16 type gene from a human menstrual blood sample by a gene amplification method, comprising a pair of forward primer and reverse primers,
  • the primer is an oligonucleotide of the following (C1) or (C2)
  • the reverse primer is an oligonucleotide of the following (D1) or (D2).
  • C2 an oligonucleotide capable of amplifying an HPV16 gene from a human menstrual blood sample, wherein the nucleotide sequence of SEQ ID NO: 3 comprises a nucleotide sequence in which one or several bases are substituted, deleted, inserted or added .
  • a first detection kit of the present invention is a detection kit used for detection of an HPV52 type gene from a human menstrual blood sample by a gene amplification method, and includes the first primer set of the present invention.
  • the second detection kit of the present invention is a detection kit used for detection of an HPV16 type gene from a human menstrual blood sample by a gene amplification method, and includes the second primer set of the present invention.
  • the diagnostic method of the present invention is a diagnostic method of cervical cancer using a human biological sample, wherein the human biological sample is a human menstrual blood sample, and the human menstrual blood sample is detected by the HPV detection method of the present invention. It is a method for diagnosing cervical cancer by detecting HPV in blood samples.
  • the HPV gene can be detected using a menstrual blood sample, so that it is not necessary to collect tissue using an instrument as in conventional cytodiagnosis. There is little pain if the examiner feels resistance.
  • the first primer set of the present invention has high specificity for HPV52 type gene
  • the second primer set of the present invention has high specificity for HPV16 type gene. HPV The 52 type gene and the HPV16 type gene can be detected reliably.
  • FIG. 1 is an electrophoresis photograph of an amplification product obtained by a PCR reaction using a menstrual blood sample in one example of the present invention.
  • detection of HPV gene includes, for example, detection of presence or absence of HPV gene, detection of presence or absence of expression of HPV gene, detection of expression level of HPV gene, etc. It may be.
  • the detection of the HPV gene is not particularly limited, but is preferably performed by a gene amplification method.
  • the gene amplification method is not particularly limited. Polymerase chain reaction (PCR) method, NASBA (Nucleic acid sequence Dase amplification; 3 ⁇ 4 ⁇ TMA transcription—mediated amplification) method, SDA (Strand Displacement Amplifica tion) Among the strengths that can be raised, the PCR method is preferred.
  • the type (subtype) of HPV to be detected is not particularly limited, and can be applied to any type (subtype).
  • preferred HPV types (subtypes) to be detected are, for example, 16, 18, 31, 33, 35, 39, 45, 51, 52 56, 58, 59 or 68.
  • the detection target is HPV52 type
  • the detection method of the present invention the detection of the HPV gene is performed by the gene amplification method, and the primer set used in the gene amplification method is a pair of forward primer and reverse primer.
  • the forward primer is the oligonucleotide (A1) or (A2) and the reverse primer (B1) or (B2).
  • the gene amplification method is not limited, but PCR method is preferable.
  • the detection target is HPV16 type
  • the detection method of the present invention the detection of the HPV gene is performed by a gene amplification method, and the gene amplification method
  • the primer set used in the above includes a pair of forward primer and reverse primer, the forward primer is the oligonucleotide (C1) or (C2), and the reverse primer template ⁇ (D1) or (D2 It is preferable that the oligonucleotide is.
  • the gene amplification method is not limited, but the PCR method is preferred.
  • the detection kit of the present invention only needs to contain the primer set of the present invention as described above.
  • HPV52 type can be detected efficiently in the detection method of the present invention described above.
  • HPV16 type can be detected efficiently in the detection method of the present invention described above.
  • the gene amplification method is preferably a PCR method.
  • the detection method, primer set and detection kit of the present invention are preferably used for diagnosis of cervical cancer.
  • the detection method of the present invention can target all HPVs. Therefore, when detecting HPV genes by gene amplification, select a primer set according to the HPV type (subtype).
  • the first primer set of the present invention includes a pair of forward primer and reverse primer, and the forward primer is an oligonucleotide (A1) or (A2) below, and the reverse primer is The oligonucleotide is (B1) or (B2) below.
  • oligonucleotide comprising a nucleotide sequence in which one or several bases are substituted, deleted or added in the nucleotide sequence of SEQ ID NO: 2, and capable of detecting an HPV52 type gene from a human menstrual blood sample.
  • the number of bases to be substituted, deleted or added is, for example, in the range of:! To 5, preferably in the range of 1 to 3, More preferably, the number is 1 or 2, particularly preferably 1.
  • the second primer set of the present invention includes a pair of forward primer and reverse primer, and the forward primer is an oligonucleotide of (C1) or (C2) below, and the reverse primer is The following (D1) or (D2) oligonucleotide is characterized.
  • C2 An oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 3 with one or several nucleotide substitutions, deletions or additions, and capable of detecting an HPV16 gene from a human menstrual blood sample.
  • oligonucleotide comprising a nucleotide sequence in which one or several bases are substituted, deleted or added in the nucleotide sequence of SEQ ID NO: 4, and capable of detecting an HPV16 gene from a human menstrual blood sample.
  • the number of bases substituted, deleted or added in (C2) and (D2) is, for example, in the range of:! To 5 and preferably 1 to 3 Range, more preferably 1 or 2 The number is particularly preferably one.
  • each oligonucleotide of the primer set can be chemically synthesized using, for example, a general-purpose DNA synthesizer (for example, trade name: Model 394, manufactured by Applied Biosystems). Oligonucleotides may be synthesized using any other method well known in the art of the present invention.
  • a general-purpose DNA synthesizer for example, trade name: Model 394, manufactured by Applied Biosystems. Oligonucleotides may be synthesized using any other method well known in the art of the present invention.
  • the detection method of the present invention a method for detecting the HPV gene in a human menstrual blood sample by PCR will be described.
  • the present invention is not limited to this.
  • a menstrual blood sample is prepared.
  • a sample immediately after collection may be used, or a sample stored by refrigeration or frozen storage may be used.
  • a menstrual blood sample subjected to each drug treatment may be used.
  • DNA is extracted from the menstrual blood sample.
  • the extraction method of DNA is not limited at all, and conventionally known methods such as extraction method using phenol / chloroform can be employed. Moreover, you may implement using a commercially available DNA extraction reagent.
  • the primer set is preferably selected so that, for example, a sequence in the coding region of the HPV gene is amplified.
  • a primer set can be used in the present invention to detect HPV gene expression in menstrual blood samples.
  • the first primer set of the present invention is designed so that the sequence in the coding region of the HPV52 type gene can be amplified
  • the second primer set of the present invention is a coding for the HPV16 type gene. Designed so that the sequences in the region can be amplified.
  • the polymerase used for PCR is not particularly limited, and a conventionally known polymerase can be used, and examples thereof include Ex Taq (registered trademark) (manufactured by Takara).
  • the PCR reaction conditions are not particularly limited, and those skilled in the art can appropriately set based on, for example, the Tm of the primer. Specific examples include, for example, 94 ° C for 5 minutes, 94 ° C for 30 seconds, 55 Treat for 30 seconds at ° C and 1 minute at 72 ° C for a total of 25 cycles, then at 72 ° C for 5 minutes.
  • HPV gene eg, expression of mRNA
  • PCR a method for detecting the expression of HPV gene (eg, expression of mRNA) in a human menstrual blood sample by PCR.
  • the present invention is not limited to this.
  • RNA extraction method is not limited at all, and conventionally known methods such as guanidine isothiocyanate and phenol / chloroform extraction can be employed. A commercially available RNA extraction reagent may also be used.
  • RNA that will be a template of a PCR reaction described later is prepared.
  • the method for synthesizing cDNA using RNA as a cage is not particularly limited, and conventionally known methods such as reverse transcription PCR can be applied.
  • a commercially available random primer can be used as a primer, and cDNA can be synthesized using reverse transcriptase in the presence of dNTPs.
  • the reverse transcriptase for example, the trade name SuperScriptl Invitrogen
  • the trade name SuperScriptl Invitrogen can be used as the reverse transcriptase.
  • a PCR reaction is performed in the same manner as described above using a primer set for amplifying the HPV gene to be detected. This amplifies the HPV gene expressed in human menstrual blood.
  • the amplification of the HPV gene when the amplification of the HPV gene is confirmed, it means that the subject is infected with HPV.
  • a primer that specifically amplifies each HPV type (subtype) gene is used as the primer, it is also possible to discriminate the HPV type (subtype).
  • the detection of the presence or absence of amplification of the gene is not particularly limited.
  • the amplification product may be confirmed by electrophoresis, or may be confirmed by a reagent capable of detecting the amplification product.
  • the HPV detection method of the present invention when it is determined by the HPV detection method of the present invention that the patient is infected with HPV, for example, a subject with HPV infection has cervical cancer compared with a subject with no HPV infection. It can be judged that the risk of developing is high. Therefore, according to the HPV detection method of the present invention, for example, in diagnosis, prevention, treatment, etc. of uterine-related diseases including cervical cancer, non- A useful judgment index can always be obtained.
  • RNA was extracted from each menstrual blood sample by the acid phenol method.
  • CDNA was prepared from total RNA (1 ⁇ g) using random primers (trade name random primers; manufactured by Invitrogen). This cDNA was used as a saddle and PCR was performed using the forward primer (SEQ ID NO: 1) and the reverse primer (SEQ ID NO: 2). The PCR conditions were 94 ° C for 5 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute for a total of 25 cycles, followed by The treatment was performed at 72 ° C for 5 minutes. The composition of the PCR reaction solution is shown below.
  • HPV52 type nucleic acid IJ genomic DNA
  • Amplified by the primer set is the 81st to 228th region in the registered nucleic acid sequence.
  • FIG. 1 A part of the result of electrophoresis is shown in FIG.
  • the figure is an electrophoretogram of PCR reaction products from nine randomly selected menstrual blood samples.
  • Lane 6 is the marker ⁇ X174 / HaeIII.
  • Lanel-3, Lane5, and Lane7-ll are PCR reactions (9 samples in total) derived from each menstrual blood sample, respectively.
  • La ne4 is a positive control in which a PCR reaction was performed using a recombinant plasmid obtained by subcloning the HPV52 type gene as a sample.
  • HPV16 type i Using the cDNA prepared in Example 1 as a saddle, PCR was carried out using the forward primer (SEQ ID NO: 3) and the reverse primer (SEQ ID NO: 4). The PCR conditions are the same as described above. The PCR reaction product from each menstrual blood sample was subjected to electrophoresis, and the presence or absence of the amplification product of the desired size (about 86 bp) was confirmed. As a result, the target amplification product could be obtained from each menstrual blood sample of several subjects.
  • the nucleic acid sequence of HPV16 type (genome DNA) is registered in NCBI Accession No. AF534061.1. Amplified by the primer set is the 385th to 470th region in the registered nucleic acid sequence.
  • the detection method of the present invention uses a human menstrual blood sample, the presence or absence of HPV infection with almost no resistance and pain to the subject can be determined. Therefore, the present invention can be widely applied to diagnosis, treatment, prevention, academic research and the like of uterine-related diseases such as cervical cancer.

Abstract

A detecting technology that realizes detection of human papilloma virus (HPV) without the discomfort or pain of test subjects. There is provided a method of detecting HPV from a human biological sample, characterized in that a human menstrual blood sample is used as the human biological sample, and that detection of HPV is performed by detecting of an HPV gene from the human menstrual blood sample. The detection of an HPV gene is preferably carried out by the PCR method. In the event of detecting of HPV52 gene, preferably, an oligonucleotide of base sequence SEQ ID NO: 1 is used as the forward primer while an oligonucleotide of base sequence SEQ ID NO: 2 is used as the reverse primer.

Description

明 細 書  Specification
ヒトパピローマウィルス（HPV)の検出方法、それに用いるプライマーセット および検出キット  Human papillomavirus (HPV) detection method, primer set and detection kit used therefor
技術分野  Technical field
[0001] 本発明は、ヒトパピローマウィルス（HPV)の検出方法、 HPV52型の検出のための プライマーセットおよび検出キットに関する。  [0001] The present invention relates to a method for detecting human papillomavirus (HPV), a primer set for detecting HPV52, and a detection kit.
背景技術  Background
[0002] 子宮頸癌は、我が国で最も多い婦人科悪性腫瘍である。子宮頸癌の検診は、まず 、内診し、子宮の形状や大小を調べ、それから、綿棒、ブラシ、小さな木のスティック 等の器具を使って、子宮類部等から細胞ないし組織を採取し、細胞診テストを行うと レ、う流れである。前記細胞診テストでは、 Pap染色法により実施されるのが一般的で ある。前記 Pap染色法は、子宮類部等から採取した細胞をスライドグラスに塗布し、へ マトキシンゃェォジン等の染色液を用いて前記細胞を染色し、顕微鏡により、細胞の 形態や核の形態を観察して、異常を判定する方法である。子宮頸部細胞の異常は、 異形成と癌とに大別される。前記異形成は、軽度異形成、中等度異形成および高度 異形成に分類され、癌は、その進行度に応じ、 0期、 I期、 II期、 III期および IV期の 5 つのステージに分類される。  [0002] Cervical cancer is the most common gynecological malignant tumor in Japan. To check for cervical cancer, first, an internal examination is conducted to examine the shape and size of the uterus, and then cells or tissues are collected from the uterine genital area using instruments such as cotton swabs, brushes, and small wooden sticks. When a diagnostic test is performed, it is a trend. The cytodiagnosis test is generally performed by the Pap staining method. In the Pap staining method, cells collected from the uterine part, etc., are applied to a slide glass, the cells are stained with a staining solution such as hematoxin eosin, and the morphology of the cells and the morphology of the nucleus are observed with a microscope. Thus, it is a method of determining an abnormality. Cervical cell abnormalities are broadly divided into dysplasia and cancer. The dysplasia is classified into mild dysplasia, moderate dysplasia, and advanced dysplasia, and cancer is classified into five stages, stage 0, stage I, stage II, stage III and stage IV, depending on the degree of progression. Is done.
[0003] 一方、子宮頸癌の発癌過程に、ヒトパピローマウィルス（Human Papillomavirus  [0003] On the other hand, human papillomavirus (Human Papillomavirus)
： HPV)が関与することが、これまでの研究で明らかになつている（非特許文献 1)。 H PVは、 2本鎖 DNAを有する腫瘍ウィルスであり、ヒトの皮膚や粘膜等の上皮に感染 する。 HPVの DNAは、環状で、 7· 9bpである。 HPVの DNAにおける遺伝子構成 は、遺伝子発現調整領域である URR (up— stream regulatory region)、ウィル スの複製に関与する early regionと呼ばれる El、 E2、 E4、 E5、 E6、 E7の 6個の遺 伝子、ウィルスキヤプシドをコードする late regionと呼ばれる Ll、 L2に分類される。 これらの遺伝子の中で、 E6および E7の両遺伝子力 発癌に関与する主要な遺伝子 であることが分かっている。 HPVのサブタイプ（型）は、 100を超え、発見された順番 に番号が付されて分類されている。 HPVのサブタイプにおいて、癌との関連性が高 レヽの ίま、 16型、 18型、 31型、 33型、 35型、 39型、 45型、 51型、 52型、 56型、 58型 、 59型、 68型であるといわれている。これらのサブタイプの中でも、 52型は、 日本、 韓国および中国等の東アジア地域の HPVの主流のサブタイプであるため、その検 出が重要であるといわれている。また、 HPV16型は、 18型と共に、子宮類癌の全症 例の原因の約 70%を占めることが報告されている。 : HPV) is involved in previous studies (Non-Patent Document 1). HPV is a tumor virus with double-stranded DNA that infects human epithelium such as skin and mucous membranes. HPV DNA is circular and 7.9 bp. The gene composition of HPV DNA consists of six residues: El, E2, E4, E5, E6, and E7, called the URR (up-stream regulatory region), which is the gene expression regulatory region, and the early region involved in virus replication. It is classified into Ll and L2, which are called late regions, which code for genes and virus capsids. Of these genes, both E6 and E7 genes are known to be major genes involved in carcinogenesis. HPV subtypes (types) are over 100 and are numbered in order of discovery. Highly related to cancer among HPV subtypes It is said that they are 16 type, 18 type, 31 type, 33 type, 35 type, 39 type, 45 type, 51 type, 52 type, 56 type, 58 type, 59 type and 68 type. Among these subtypes, the 52 type is the mainstream subtype of HPV in East Asia such as Japan, South Korea and China, so detection is said to be important. HPV16, together with type 18, has been reported to account for about 70% of all uterine cancer cases.
[0004] HPVは、子宮類部の扁平上皮に感染し、 HPV感染の約 90%は自然消失するが、 残りの約 10%が持続感染となる。 日本人は、平均して約 75%が持続感染になるとい われている。持続感染の場合、子宮類部細胞が異形成をおこす可能性がある。異形 成が起こってもウィルスが排除されれば、細胞は正常に戻るが、ウィルスが排除され ずに、感染が継続すると、細胞の異形成の程度が進行することがある。軽度異形成 の場合は、 自然治癒することが多いが、中等度異形成ないし高度異形成に進むと、 自然治癒が困難になる。そして、高度異形成を治療せずに長期間放置すると、病変 が進行し、子宮頸癌になる可能性がある。したがって、子宮頸癌の検診において、 Η PVの検出は重要であり、特に、 日本人では、 HPV52型の検出が重要である。また、 高い発症原因であることから、 HPV16型の検出も重要である。  [0004] HPV infects the squamous epithelium of the uterus and about 90% of HPV infections disappear spontaneously, while the remaining 10% are persistent infections. On average, about 75% of Japanese people are persistently infected. In the case of persistent infection, uterine cells can dysplasia. If the virus is eliminated even if dysplasia occurs, the cells will return to normal, but if the infection continues without the virus being eliminated, the degree of cell dysplasia may progress. Mild dysplasia often heals spontaneously, but if it progresses to moderate or severe dysplasia, natural healing becomes difficult. If left untreated for a long time without treating advanced dysplasia, the lesions may progress, leading to cervical cancer. Therefore, detection of PV is important in screening for cervical cancer, and detection of HPV52 is particularly important in Japanese. It is also important to detect HPV16 because of its high incidence.
[0005] HPVは、子宮類部から採取した細胞の細胞診でも、その感染を確認することがで きる。また、採取した細胞から遺伝子を抽出し、 PCR法により遺伝子を選択的に増幅 して、 HPVを検出する方法も開発されている（特許文献 1)。しかし、細胞診は、被検 者にとって心理的に抵抗のある処置を必要とし、また、細胞採取の際に、軽度とはい え生体組織を損傷する可能性もある。このため、子宮頸癌検診は、公費負担（自己 負担無し)で受診できるにも関わらず、内診に対する抵抗感等から、利用率が最も低 い検診である。  [0005] HPV can be confirmed by cytology of cells collected from the uterine genital area. In addition, a method has been developed in which HPV is detected by extracting a gene from the collected cells and selectively amplifying the gene by PCR (Patent Document 1). However, cytodiagnosis requires a psychologically resistant treatment for the subject, and even if it is mild, it may damage biological tissue. For this reason, cervical cancer screening is the least used because of its resistance to internal examinations, although it can be taken at public expense (no self-pay).
特許文献 1 :特開 2004— 121240号公報  Patent Document 1: Japanese Unexamined Patent Application Publication No. 2004-121240
非特許文献 2 :「ウイノレス」、第 52卷 第 2号、 pp. 287- 293, 2002  Non-Patent Document 2: “Winoless”, No. 52 卷 No. 2, pp. 287-293, 2002
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0006] そこで、本発明の目的は、被検者が抵抗感ゃ苦痛を伴うことなぐ HPVを検出でき る検出技術を提供することである。 課題を解決するための手段 [0006] Therefore, an object of the present invention is to provide a detection technique that can detect HPV without causing pain if the subject feels resistance. Means for solving the problem
[0007] 本発明者等は、前記目的を達成するために、一連の研究を重ねた。その過程で、 HPVウィルスの所在を中心に検討したところ、子宮体部に HPVが存在することを突 き止めた。そして、さらに研究を続けたところ、子宮内膜力も剥離した月経血中に HP Vが存在することを見出した。この知見は、前述の従来の常識を覆すものである。す なわち、前述のように、 HPVは、子宮類部の扁平上皮に感染することが常識であり、 子宮体部に HPVが存在すること、および、子宮内膜から剥離した月経血中に HPV が存在することは、常識に反することであった (非特許文献 1参照)。本発明者等によ る発見は、子宮頸癌等の子宮関連疾患において、学術的かつ臨床的に重大な発見 である。そして、この知見に基づき、さらに研究を進めたところ、下記の特定のフォヮ 一ドプライマ一とリバースプライマーを用いれば、遺伝子増幅法により月経血中から HPV52型遺伝子または HPV16型遺伝子を特異的に検出できることを見出し、本発 明に到達した。  [0007] In order to achieve the above object, the present inventors have made a series of studies. In the process, we focused on the location of the HPV virus and found out that HPV was present in the uterine body. After further research, they found that HP V was present in menstrual blood from which the endometrial force was also removed. This knowledge overturns the above-mentioned conventional common sense. In other words, as mentioned above, HPV is common sense that it infects the squamous epithelium of the uterus, the presence of HPV in the uterine body, and HPV The existence of is contrary to common sense (see Non-Patent Document 1). The discovery by the present inventors is a significant academic and clinical discovery in uterine-related diseases such as cervical cancer. Based on this finding, we further advanced our research. Using the following specific primer primer and reverse primer, we can detect the HPV52 gene or HPV16 gene in menstrual blood by gene amplification. And reached the present invention.
[0008] すなわち、本発明の検出方法は、ヒト生体試料からの HPVの検出方法であって、 前記ヒト生体試料としてヒト月経血試料を用い、前記ヒト月経血試料中の HPV遺伝子 を検出することにより、 HPVを検出する方法である。  That is, the detection method of the present invention is a method for detecting HPV from a human biological sample, wherein a human menstrual blood sample is used as the human biological sample, and the HPV gene in the human menstrual blood sample is detected. This is a method for detecting HPV.
[0009] 本発明の第 1のプライマーセットは、遺伝子増幅法によるヒト月経血試料からの HP V52型遺伝子の検出に用いるプライマーセットであって、一対のフォワードプライマ 一およびリバースプライマーを含み、前記フォワードプライマーが、下記 (A1)または（ A2)のオリゴヌクレオチドであり、前記リバースプライマーカ、下記（B1)または（B2) のオリゴヌクレオチドである。  [0009] The first primer set of the present invention is a primer set used for detecting an HP V52 type gene from a human menstrual blood sample by a gene amplification method, and includes a pair of forward primer and reverse primers, The primer is an oligonucleotide (A1) or (A2) below, and the reverse primer, the oligonucleotide (B1) or (B2) below.
(A1)配列番号 1の塩基配列からなるオリゴヌクレオチド。  (A1) An oligonucleotide having the base sequence of SEQ ID NO: 1.
(A2)配列番号 1の塩基配列において、 1若しくは数個の塩基が、置換、欠失または 付加された塩基配列からなり、かつヒト月経血試料から HPV52型遺伝子を増幅可能 なオリゴヌクレオチド。  (A2) An oligonucleotide having a nucleotide sequence in which one or several bases are substituted, deleted or added in the nucleotide sequence of SEQ ID NO: 1, and capable of amplifying an HPV52 type gene from a human menstrual blood sample.
(B1)配列番号 2の塩基配列からなるオリゴヌクレオチド。  (B1) An oligonucleotide having the base sequence of SEQ ID NO: 2.
(B2)配列番号 2の塩基配列において、 1若しくは数個の塩基が、置換、欠失または 付加された塩基配列からなり、かつヒト月経血試料から HPV52型遺伝子を増幅可能 なオリゴヌクレオチド。 (B2) The nucleotide sequence of SEQ ID NO: 2 consists of a nucleotide sequence in which one or several bases are substituted, deleted or added, and can amplify the HPV52 gene from human menstrual blood samples Oligonucleotide.
[0010] 本発明の第 2のプライマーセットは、遺伝子増幅法によるヒト月経血試料からの HP V16型遺伝子の検出に用いるプライマーセットであって、一対のフォワードプライマ 一およびリバースプライマーを含み、前記フォワードプライマーが、下記（C1)または（ C2)のオリゴヌクレオチドであり、前記リバースプライマーカ 下記（D1)または（D2) のオリゴヌクレオチドである。  [0010] The second primer set of the present invention is a primer set used for detection of an HP V16 type gene from a human menstrual blood sample by a gene amplification method, comprising a pair of forward primer and reverse primers, The primer is an oligonucleotide of the following (C1) or (C2), and the reverse primer is an oligonucleotide of the following (D1) or (D2).
(C1)配列番号 3の塩基配列からなるオリゴヌクレオチド。  (C1) An oligonucleotide having the base sequence of SEQ ID NO: 3.
(C2)配列番号 3の塩基配列において、 1若しくは数個の塩基が、置換、欠失、挿入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV16型遺伝子を 増幅可能なオリゴヌクレオチド。  (C2) an oligonucleotide capable of amplifying an HPV16 gene from a human menstrual blood sample, wherein the nucleotide sequence of SEQ ID NO: 3 comprises a nucleotide sequence in which one or several bases are substituted, deleted, inserted or added .
(D1)配列番号 4の塩基配列からなるオリゴヌクレオチド。  (D1) An oligonucleotide having the base sequence of SEQ ID NO: 4.
(D2)配列番号 4の塩基配列において、 1若しくは数個の塩基が、置換、欠失、揷入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV16型遺伝子を 増幅可能なオリゴヌクレオチド。  (D2) Oligo capable of amplifying an HPV16 gene from a human menstrual blood sample consisting of a base sequence in which one or several bases are substituted, deleted, inserted or added in the base sequence of SEQ ID NO: 4. nucleotide.
[0011] 本発明の第 1の検出キットは、遺伝子増幅法によるヒト月経血試料からの HPV52 型遺伝子の検出に用いる検出キットであって、前記本発明の第 1のプライマーセット を含む。また、本発明の第 2の検出キットは、遺伝子増幅法によるヒト月経血試料から の HPV16型遺伝子の検出に用いる検出キットであって、前記本発明の第 2のプライ マーセットを含む。 [0011] A first detection kit of the present invention is a detection kit used for detection of an HPV52 type gene from a human menstrual blood sample by a gene amplification method, and includes the first primer set of the present invention. The second detection kit of the present invention is a detection kit used for detection of an HPV16 type gene from a human menstrual blood sample by a gene amplification method, and includes the second primer set of the present invention.
[0012] 本発明の診断方法は、ヒト生体試料を用いた子宮頸癌の診断方法であって、ヒト生 体試料が、ヒト月経血試料であり、本発明の HPVの検出方法によって、ヒト月経血試 料中の HPVを検出することにより、子宮頸癌を診断する方法である。  [0012] The diagnostic method of the present invention is a diagnostic method of cervical cancer using a human biological sample, wherein the human biological sample is a human menstrual blood sample, and the human menstrual blood sample is detected by the HPV detection method of the present invention. It is a method for diagnosing cervical cancer by detecting HPV in blood samples.
発明の効果  The invention's effect
[0013] このように、本発明の検出方法によれば、月経血試料を用いて、 HPV遺伝子を検 出できるため、従来の細胞診のように器具を用いて組織を採取する必要がなぐ被検 者に抵抗感ゃ苦痛がほとんどない。また、前記本発明の第 1のプライマーセットは、 H PV52型遺伝子に対する特異性、前記本発明の第 2のプライマーセットは、 HPV16 型遺伝子に対する特異性が、それぞれ高いため、これらを用いることによって、 HPV 52型遺伝子および HPV16型遺伝子をそれぞれ確実に検出できる。 [0013] Thus, according to the detection method of the present invention, the HPV gene can be detected using a menstrual blood sample, so that it is not necessary to collect tissue using an instrument as in conventional cytodiagnosis. There is little pain if the examiner feels resistance. In addition, the first primer set of the present invention has high specificity for HPV52 type gene, and the second primer set of the present invention has high specificity for HPV16 type gene. HPV The 52 type gene and the HPV16 type gene can be detected reliably.
図面の簡単な説明  Brief Description of Drawings
[0014] [図 1]図 1は、本発明の一実施例における、月経血試料を用いた PCR反応により得ら れた増幅産物の電気泳動写真である。  FIG. 1 is an electrophoresis photograph of an amplification product obtained by a PCR reaction using a menstrual blood sample in one example of the present invention.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0015] 本発明において、「HPV遺伝子の検出」とは、例えば、 HPV遺伝子の有無の検出 、 HPV遺伝子の発現の有無の検出、 HPV遺伝子の発現量の検出等を含み、いず れの検出であってもよい。  In the present invention, “detection of HPV gene” includes, for example, detection of presence or absence of HPV gene, detection of presence or absence of expression of HPV gene, detection of expression level of HPV gene, etc. It may be.
[0016] 本発明の検出方法において、前記 HPV遺伝子の検出は、特に制限されないが、 遺伝子増幅法により実施されることが好ましい。前記遺伝子増幅法は、特に制限され ず、ポリメラーゼチェーンリアクション（Polymerase Chain Reaction； PCR)法、 N ASBA (Nucleic acid sequence Dase amplification; ¾^ TMA Transcrip tion— mediated amplification)法、 SDA (Strand Displacement Amplifica tion)法等があげられる力 中でも PCR法が好ましい。  [0016] In the detection method of the present invention, the detection of the HPV gene is not particularly limited, but is preferably performed by a gene amplification method. The gene amplification method is not particularly limited. Polymerase chain reaction (PCR) method, NASBA (Nucleic acid sequence Dase amplification; ¾ ^ TMA transcription—mediated amplification) method, SDA (Strand Displacement Amplifica tion) Among the strengths that can be raised, the PCR method is preferred.
[0017] 本発明の検出方法において、検出対象の HPVの型（サブタイプ）は、特に制限さ れず、あらゆる型（サブタイプ）に適用可能である。本発明の検出方法において、検 出対象となる好ましい HPVの型（サブタイプ）は、例えば、 16型、 18型、 31型、 33型 、 35型、 39型、 45型、 51型、 52型、 56型、 58型、 59型または 68型である。  In the detection method of the present invention, the type (subtype) of HPV to be detected is not particularly limited, and can be applied to any type (subtype). In the detection method of the present invention, preferred HPV types (subtypes) to be detected are, for example, 16, 18, 31, 33, 35, 39, 45, 51, 52 56, 58, 59 or 68.
[0018] 前記本発明の第 1のプライマーセットを使用する場合、 HPV52型を高い特異性で 検出できる。したがって、検出対象が HPV52型の場合、本発明の検出方法におい て、前記 HPV遺伝子の検出は、遺伝子増幅法により実施され、前記遺伝子増幅法 に使用されるプライマーセットが、一対のフォワードプライマーおよびリバースプライマ 一を含み、前記フォワードプライマーが、前記 (A1)または (A2)のオリゴヌクレオチド であり、前記リバースプライマーカ 前記（B1)または（B2)のオリゴヌクレオチドである ことが好ましい。また、前記遺伝子増幅法は、制限されないが、 PCR法が好ましい。  [0018] When the first primer set of the present invention is used, HPV52 type can be detected with high specificity. Therefore, when the detection target is HPV52 type, in the detection method of the present invention, the detection of the HPV gene is performed by the gene amplification method, and the primer set used in the gene amplification method is a pair of forward primer and reverse primer. Preferably, the forward primer is the oligonucleotide (A1) or (A2) and the reverse primer (B1) or (B2). The gene amplification method is not limited, but PCR method is preferable.
[0019] 前記本発明の第 2のプライマーセットを使用する場合、 HPV16型を高い特異性で 検出できる。したがって、検出対象が HPV16型の場合、本発明の検出方法におい て、前記 HPV遺伝子の検出は、遺伝子増幅法により実施され、前記遺伝子増幅法 に使用されるプライマーセットが、一対のフォワードプライマーおよびリバースプライマ 一を含み、前記フォワードプライマーが、前記（C1)または（C2)のオリゴヌクレオチド であり、前記リバースプライマーカ \前記（D1)または（D2)のオリゴヌクレオチドであ ることが好ましい。また、前記遺伝子増幅法は、制限されないが、 PCR法が好ましレ、 [0019] When the second primer set of the present invention is used, HPV16 type can be detected with high specificity. Therefore, when the detection target is HPV16 type, in the detection method of the present invention, the detection of the HPV gene is performed by a gene amplification method, and the gene amplification method The primer set used in the above includes a pair of forward primer and reverse primer, the forward primer is the oligonucleotide (C1) or (C2), and the reverse primer template \ (D1) or (D2 It is preferable that the oligonucleotide is. The gene amplification method is not limited, but the PCR method is preferred.
[0020] 本発明の検出キットは、前述のように本発明のプライマーセットを含んでいればよく[0020] The detection kit of the present invention only needs to contain the primer set of the present invention as described above.
、その他の構成等は何ら制限されない。 Other configurations are not limited at all.
[0021] 本発明の第 1のプライマーセットおよび第 1の検出キットによれば、前述の本発明の 検出方法において、 HPV52型の検出を、効率良く行うことができる。また、本発明の 第 2のプライマーセットおよび第 2の検出キットによれば、前述の本発明の検出方法 において、 HPV16型の検出を、効率良く行うことができる。 [0021] According to the first primer set and the first detection kit of the present invention, HPV52 type can be detected efficiently in the detection method of the present invention described above. Moreover, according to the second primer set and the second detection kit of the present invention, HPV16 type can be detected efficiently in the detection method of the present invention described above.
[0022] 本発明のプライマーセットおよび検出キットにおいて、前記遺伝子増幅法は、 PCR 法であることが好ましい。 [0022] In the primer set and detection kit of the present invention, the gene amplification method is preferably a PCR method.
[0023] 本発明の検出方法、プライマーセットおよび検出キットは、子宮頸癌の診断に用い ることが好ましい。 [0023] The detection method, primer set and detection kit of the present invention are preferably used for diagnosis of cervical cancer.
[0024] つぎに、本発明について、詳しく説明する。 [0024] Next, the present invention will be described in detail.
[0025] 前述のように、本発明の検出方法は、全ての HPVを対象とすることができる。このた め、遺伝子増幅法により HPV遺伝子の検出を行う場合は、 HPVの型（サブタイプ） に応じたプライマーセットを選択すればょレ、。  [0025] As described above, the detection method of the present invention can target all HPVs. Therefore, when detecting HPV genes by gene amplification, select a primer set according to the HPV type (subtype).
[0026] 前述のように、検出対象が HPV52型の場合、前記プライマーセットとしては、前記 本発明の第 1のプライマーセットを使用することが好ましい。本発明の第 1のプライマ 一セットは、前述のように、一対のフォワードプライマーおよびリバースプライマーを含 み、前記フォワードプライマーが、下記（A1)または (A2)のオリゴヌクレオチドであり、 前記リバースプライマーが、下記（B1)または（B2)のオリゴヌクレオチドであることを 特徴とする。  [0026] As described above, when the detection target is HPV52 type, it is preferable to use the first primer set of the present invention as the primer set. As described above, the first primer set of the present invention includes a pair of forward primer and reverse primer, and the forward primer is an oligonucleotide (A1) or (A2) below, and the reverse primer is The oligonucleotide is (B1) or (B2) below.
(A1)配列番号 1の塩基配列からなるオリゴヌクレオチド。  (A1) An oligonucleotide having the base sequence of SEQ ID NO: 1.
gtgtagctaacgcacggcca ( ΰ歹 I [畨亏 1)  gtgtagctaacgcacggcca (ΰ 歹 I [畨 亏 1)
(Α2)配列番号 1の塩基配列において、 1若しくは数個の塩基が、置換、欠失または 付加された塩基配列からなり、かつ、ヒト月経血試料から HPV52型遺伝子を検出可 能なオリゴヌクレオチド。 (Note 2) In the nucleotide sequence of SEQ ID NO: 1, one or several bases are substituted, deleted or An oligonucleotide consisting of an added base sequence and capable of detecting the HPV52 gene from a human menstrual blood sample.
(B1)配列番号 2の塩基配列からなるオリゴヌクレオチド。  (B1) An oligonucleotide having the base sequence of SEQ ID NO: 2.
atacctctcttcgttgtagc (配歹' J番号 2)  atacctctcttcgttgtagc (Rice 'J number 2)
(B2)配列番号 2の塩基配列において、 1若しくは数個の塩基が、置換、欠失または 付加された塩基配列からなり、かつヒト月経血試料から HPV52型遺伝子を検出可能 なオリゴヌクレオチド。  (B2) An oligonucleotide comprising a nucleotide sequence in which one or several bases are substituted, deleted or added in the nucleotide sequence of SEQ ID NO: 2, and capable of detecting an HPV52 type gene from a human menstrual blood sample.
[0027] 前記 (A2)および（B2)において、置換、欠失または付加される塩基の数は、例え ば、：!〜 5個の範囲であり、好ましくは 1〜3個の範囲であり、より好ましくは 1もしくは 2 個であり、特に好ましくは 1個である。  [0027] In the above (A2) and (B2), the number of bases to be substituted, deleted or added is, for example, in the range of:! To 5, preferably in the range of 1 to 3, More preferably, the number is 1 or 2, particularly preferably 1.
[0028] 前述のように、検出対象が HPV16型の場合、前記プライマーセットとしては、前記 本発明の第 2のプライマーセットを使用することが好ましい。本発明の第 2のプライマ 一セットは、前述のように、一対のフォワードプライマーおよびリバースプライマーを含 み、前記フォワードプライマーが、下記（C1)または（C2)のオリゴヌクレオチドであり、 前記リバースプライマーが、下記（D1)または（D2)のオリゴヌクレオチドであることを 特徴とする。  [0028] As described above, when the detection target is HPV16 type, it is preferable to use the second primer set of the present invention as the primer set. As described above, the second primer set of the present invention includes a pair of forward primer and reverse primer, and the forward primer is an oligonucleotide of (C1) or (C2) below, and the reverse primer is The following (D1) or (D2) oligonucleotide is characterized.
(C1)配列番号 3の塩基配列からなるオリゴヌクレオチド。  (C1) An oligonucleotide having the base sequence of SEQ ID NO: 3.
accgttgtgtgatttgttaattaggt (目己歹 1J番号 3)  accgttgtgtgatttgttaattaggt (Meji 1J number 3)
(C2)配列番号 3の塩基配列において、 1若しくは数個の塩基力 置換、欠失または 付加された塩基配列からなり、かつ、ヒト月経血試料から HPV16型遺伝子を検出可 能なオリゴヌクレオチド。  (C2) An oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 3 with one or several nucleotide substitutions, deletions or additions, and capable of detecting an HPV16 gene from a human menstrual blood sample.
(D1)配列番号 4の塩基配列からなるオリゴヌクレオチド。  (D1) An oligonucleotide having the base sequence of SEQ ID NO: 4.
gctttttgtccagatgtctttgc (IB^[J#-^-4)  gctttttgtccagatgtctttgc (IB ^ [J #-^-4)
(D2)配列番号 4の塩基配列において、 1若しくは数個の塩基が、置換、欠失または 付加された塩基配列からなり、かつヒト月経血試料から HPV16型遺伝子を検出可能 なオリゴヌクレオチド。  (D2) An oligonucleotide comprising a nucleotide sequence in which one or several bases are substituted, deleted or added in the nucleotide sequence of SEQ ID NO: 4, and capable of detecting an HPV16 gene from a human menstrual blood sample.
[0029] 前記（C2)および（D2)におレ、て、置換、欠失または付加される塩基の数は、例え ば、：!〜 5個の範囲であり、好ましくは 1〜3個の範囲であり、より好ましくは 1もしくは 2 個であり、特に好ましくは 1個である。 [0029] The number of bases substituted, deleted or added in (C2) and (D2) is, for example, in the range of:! To 5 and preferably 1 to 3 Range, more preferably 1 or 2 The number is particularly preferably one.
[0030] 本発明において、プライマーセットの各オリゴヌクレオチドは、例えば、汎用の DNA 合成装置（例えば、 Applied Biosystems社製、商品名： Model 394)を用いて化 学的に合成することができる。オリゴヌクレオチドは、本発明の技術分野において、よ く知られる他の方法のいずれかを用いて合成してもよい。 In the present invention, each oligonucleotide of the primer set can be chemically synthesized using, for example, a general-purpose DNA synthesizer (for example, trade name: Model 394, manufactured by Applied Biosystems). Oligonucleotides may be synthesized using any other method well known in the art of the present invention.
[0031] 本発明の検出方法について、一例として、 PCRによりヒト月経血試料中の HPV遺 伝子を検出する方法を説明する。なお、本発明は、これには限定されない。 [0031] As an example of the detection method of the present invention, a method for detecting the HPV gene in a human menstrual blood sample by PCR will be described. The present invention is not limited to this.
[0032] まず、月経血試料を準備する。月経血試料は、例えば、採取直後のものを使用して もよいし、冷蔵保存や冷凍保存により保存したものを用いてもよい。また、各薬剤処 理を施した月経血試料を用いてもよい。 [0032] First, a menstrual blood sample is prepared. As the menstrual blood sample, for example, a sample immediately after collection may be used, or a sample stored by refrigeration or frozen storage may be used. In addition, a menstrual blood sample subjected to each drug treatment may be used.
[0033] つぎに、前記月経血試料から DNAを抽出する。 DNAの抽出方法は、何ら制限さ れず、例えば、フエノール'クロ口ホルムによる抽出法等、従来公知の方法が採用でき る。また、市販の DNA抽出試薬等を用いて実施してもよい。 Next, DNA is extracted from the menstrual blood sample. The extraction method of DNA is not limited at all, and conventionally known methods such as extraction method using phenol / chloroform can be employed. Moreover, you may implement using a commercially available DNA extraction reagent.
[0034] そして、抽出した DNAを铸型として、検出対象の HPV遺伝子を増幅するためのプ ライマーセットを用いて PCR反応を行い、前記 HPV遺伝子を増幅させる。 [0034] Then, using the extracted DNA as a saddle shape, a PCR reaction is performed using a primer set for amplifying the HPV gene to be detected, and the HPV gene is amplified.
[0035] 前記プライマーセットは、例えば、 HPV遺伝子のコーディング領域中の配列が増幅 されるよう選択することが好ましい。このようなプライマーセットは、本発明において、 月経血試料における HPV遺伝子の発現を検出するために用いることができる。具体 的には、例えば、検出対象の HPV遺伝子の型（サブタイプ）に応じて、前記型に特 有のコーディング領域中の配列が増幅されるように、プライマーセットを選択すること が好ましい。なお、本発明の第 1のプライマーセットは、 HPV52型遺伝子のコーディ ング領域中の配列が増幅可能なように設計されており、本発明の第 2のプライマーセ ットは、 HPV16型遺伝子のコーディング領域中の配列が増幅可能なように設計され ている。 [0035] The primer set is preferably selected so that, for example, a sequence in the coding region of the HPV gene is amplified. Such a primer set can be used in the present invention to detect HPV gene expression in menstrual blood samples. Specifically, for example, it is preferable to select a primer set according to the type (subtype) of the HPV gene to be detected so that the sequence in the coding region specific to the type is amplified. The first primer set of the present invention is designed so that the sequence in the coding region of the HPV52 type gene can be amplified, and the second primer set of the present invention is a coding for the HPV16 type gene. Designed so that the sequences in the region can be amplified.
[0036] PCRに使用するポリメラーゼは、特に制限されず、従来公知のポリメラーゼが使用 でき、例えば、例えば、 Ex Taq (登録商標）（Takara社製)等があげられる。 PCRの 反応条件は、特に制限されず、当業者であれば、例えば、プライマーの Tm等に基づ いて適宜設定できる。具体例としては、例えば、 94°Cで 5分間、 94°Cで 30秒間、 55 °Cで 30秒間、および 72°Cで 1分間を 1サイクルとして合計 25サイクル、次に 72°Cで 5 分間の処理を行う。 [0036] The polymerase used for PCR is not particularly limited, and a conventionally known polymerase can be used, and examples thereof include Ex Taq (registered trademark) (manufactured by Takara). The PCR reaction conditions are not particularly limited, and those skilled in the art can appropriately set based on, for example, the Tm of the primer. Specific examples include, for example, 94 ° C for 5 minutes, 94 ° C for 30 seconds, 55 Treat for 30 seconds at ° C and 1 minute at 72 ° C for a total of 25 cycles, then at 72 ° C for 5 minutes.
[0037] また、本発明の検出方法について、一例として、 PCRによりヒト月経血試料中の HP V遺伝子の発現 (例えば、 mRNAの発現）を検出する方法を説明する。なお、本発 明は、これには限定されない。  [0037] As an example of the detection method of the present invention, a method for detecting the expression of HPV gene (eg, expression of mRNA) in a human menstrual blood sample by PCR will be described. The present invention is not limited to this.
[0038] まず、前述と同様にして準備した月経血試料力 RNAを抽出する。 RNAの抽出方 法は、何ら制限されず、例えば、グァニジンイソチオシァネートおよびフエノール'クロ 口ホルム抽出等、従来公知の方法が採用できる。また、市販の RNA抽出試薬を用い てもよい。  [0038] First, menstrual blood sample RNA prepared in the same manner as described above is extracted. The RNA extraction method is not limited at all, and conventionally known methods such as guanidine isothiocyanate and phenol / chloroform extraction can be employed. A commercially available RNA extraction reagent may also be used.
[0039] つぎに、抽出した RNAを錡型として、後述する PCR反応の铸型となる cDNAを調 製する。 RNAを錡型として cDNAを合成する方法は、特に制限されず、逆転写 PCR 法等の従来公知の方法を適用できる。例えば、プライマーとして市販のランダムブラ イマ一を用レ、、 dNTPsの存在下、逆転写酵素を用いて cDNAを合成することができ る。逆転写酵素としては、例えば、商品名 SuperScriptl lnvitrogen社製）等が使 用できる。  [0039] Next, using the extracted RNA as a template, cDNA that will be a template of a PCR reaction described later is prepared. The method for synthesizing cDNA using RNA as a cage is not particularly limited, and conventionally known methods such as reverse transcription PCR can be applied. For example, 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, the trade name SuperScriptl Invitrogen) can be used.
[0040] そして、得られた cDNAを铸型として、検出対象の HPV遺伝子を増幅するための プライマーセットを用いて、前述と同様に PCR反応を行う。これによつて、ヒト月経血 中で発現されている HPV遺伝子を増幅させる。  [0040] Then, using the obtained cDNA as a saddle, a PCR reaction is performed in the same manner as described above using a primer set for amplifying the HPV gene to be detected. This amplifies the HPV gene expressed in human menstrual blood.
[0041] 以上のような方法において、 HPV遺伝子の増幅が確認された場合は、被検者が H PVの感染者であることを意味する。また、前記プライマーとて、 HPVの各型（サブタ イブ)の遺伝子を特異的に増幅するプライマーを使用した場合、 HPVの型（サブタイ プ）を判別することも可能である。前記遺伝子の増幅の有無の検出は、特に制限され ず、例えば、増幅産物を電気泳動で確認してもよいし、増幅産物を検出し得る試薬 により確認、してもよレ、。  [0041] In the above method, when the amplification of the HPV gene is confirmed, it means that the subject is infected with HPV. In addition, when a primer that specifically amplifies each HPV type (subtype) gene is used as the primer, it is also possible to discriminate the HPV type (subtype). The detection of the presence or absence of amplification of the gene is not particularly limited. For example, the amplification product may be confirmed by electrophoresis, or may be confirmed by a reagent capable of detecting the amplification product.
[0042] また、本発明の HPVの検出方法により、 HPV感染であると判断された場合、例え ば、 HPV感染の被検者は、 HPV未感染の被検者と比較して、子宮頸癌を発症する 危険性が高いと判断することができる。このため、本発明の HPV検出方法によれば、 例えば、子宮頸癌をはじめとする子宮関連疾患の診断、予防、治療等において、非 常に有用な判断指標を得ることができる。 [0042] Further, when it is determined by the HPV detection method of the present invention that the patient is infected with HPV, for example, a subject with HPV infection has cervical cancer compared with a subject with no HPV infection. It can be judged that the risk of developing is high. Therefore, according to the HPV detection method of the present invention, for example, in diagnosis, prevention, treatment, etc. of uterine-related diseases including cervical cancer, non- A useful judgment index can always be obtained.
実施例 1  Example 1
[0043] HPV52職ィ云 の  [0043] HPV52
複数人の被検者から月経血試料を採取し、これらの各月経血試料から、酸フエノー ル法により全 RNAを抽出した。全 RNA(1 μ g)から、ランダムプライマー（商品名ラン ダムプライマー； Invitrogen社製）を用いて cDNAを調製した。この cDNAを錡型と して、前記フォワードプライマー（配列番号 1)および前記リバースプライマー（配列番 号 2)を用い、 PCRを実施した。 PCRの条件は、 94°Cで 5分間、 94°Cで 30秒間、 55 °Cで 30秒間、および 72°Cで 1分間を 1サイクルとして合計 25サイクルの処理を行レ、、 続いて、 72°Cで 5分間の処理を行った。 PCR反応液の組成を以下に示す。各月経 血試料由来の PCR反応物を電気泳動に供し、 目的の大きさ（約 150bp)の増幅産物 の有無を確認した。その結果、 10人の被検者の各月経血試料から、 目的の増幅産 物を得ることができた。 HPV52型の核酸配歹 IJ(ゲノム DNA)は、 NCBIァクセッション No. X74481.1に登録されている。前記プライマーセットによって増幅されるのは、 登録された核酸配列において、 81番目—228番目の領域である。  Menstrual blood samples were collected from several subjects, and total RNA was extracted from each menstrual blood sample by the acid phenol method. CDNA was prepared from total RNA (1 μg) using random primers (trade name random primers; manufactured by Invitrogen). This cDNA was used as a saddle and PCR was performed using the forward primer (SEQ ID NO: 1) and the reverse primer (SEQ ID NO: 2). The PCR conditions were 94 ° C for 5 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute for a total of 25 cycles, followed by The treatment was performed at 72 ° C for 5 minutes. The composition of the PCR reaction solution is shown below. The PCR reaction product from each menstrual blood sample was subjected to electrophoresis, and the presence or absence of the amplification product of the desired size (about 150 bp) was confirmed. As a result, the desired amplification product could be obtained from each menstrual blood sample of 10 subjects. HPV52 type nucleic acid IJ (genomic DNA) is registered in NCBI Accession No. X74481.1. Amplified by the primer set is the 81st to 228th region in the registered nucleic acid sequence.
[0044]  [0044]
(PCR反応液）  (PCR reaction solution)
TAKARA Ex T a q (5UZjU l、 TAKARA社製)  TAKARA Ex Taq (5UZjU l, manufactured by TAKARA)
10 E x T a q Bu f f e r  10 E x T a q Bu f f e r
d NTP M i x t u r e (各 2. 5mM)  d NTP M i x t u r e (2.5 mM each)
フォワード P r i me r (10 pmo I / \i I )  Forward P r i me r (10 pmo I / \ i I)
リ■/く一ス P r i me r (10 pmo I / μ I )  ■ Pri me r (10 pmo I / μ I)
滅菌蒸留水  Sterile distilled water
錶型 c DNA  Vertical cDNA
[0045] 電気泳動の結果の一部を図 1に示す。同図は、採取した複数の月経血試料のうち 無作為に選択した 9つの試料由来の PCR反応物の電気泳動写真である。同図にお いて、 Lane6は、マーカー φ X174/HaeIIIである。 Lanel〜3、 Lane5および Lan e7〜llは、それぞれ、各月経血試料由来の PCR反応物（計 9サンプル）である。 La ne4は、 HPV52型の遺伝子をサブクローニングした組換えプラスミドをサンプルとし て、 PCR反応を行ったポジティブコントロールである。このプラスミドは、プラスミドべク ター pGEMT— easy (Promega社製、以下同様）の A末端 PCR反応物挿入部位に 、 148塩基対（81番目—228番目）の HPV52型遺伝子の部分配列が揷入されてい る。同図に示すように、 Lanel、 3、 7は、ポジティブコントロールである Lane4と同じ 位置（約 150bp)にバンドが確認された。この結果から、前述のプライマーセットにより 、特異的に HPV52型遺伝子の部分配列が増幅されていることが示された。なお、 La ne2、 5、 8〜11および Lane3の下のバンドは、プライマーダイマーである。 [0045] A part of the result of electrophoresis is shown in FIG. The figure is an electrophoretogram of PCR reaction products from nine randomly selected menstrual blood samples. In the figure, Lane 6 is the marker φX174 / HaeIII. Lanel-3, Lane5, and Lane7-ll are PCR reactions (9 samples in total) derived from each menstrual blood sample, respectively. La ne4 is a positive control in which a PCR reaction was performed using a recombinant plasmid obtained by subcloning the HPV52 type gene as a sample. In this plasmid, a partial sequence of the HPV52 type gene of 148 base pairs (81st to 228th) was inserted into the insertion site of the A-terminal PCR reaction product of plasmid vector pGEMT-easy (Promega). ing. As shown in the figure, bands of Lanel 3, 3 and 7 were confirmed at the same position (about 150 bp) as Lane 4 which is a positive control. From this result, it was shown that the partial sequence of the HPV52 type gene was specifically amplified by the above primer set. The bands below Lane 2, 5, 8-11 and Lane 3 are primer dimers.
[0046] さらに、 目的のバンドが検出された PCR反応物について、実際に、 目的断片（HPV 52型遺伝子の部分配歹 1J)のみが増幅されているか否かを確認した。この確認は、 PC R増幅産物を TAクローニングにより pGEMTeasyベクターに入れ、ベクター内の配 歹 IJをプライマーとした塩基配列解析により行った。この結果、 目的のバンドが確認さ れた PCR反応物には、 目的断片のみが含まれていることが確認できた。つまり、前述 のプライマーセットにより、他の配列は増幅されておらず、 HPV52型遺伝子の目的 配列のみが増幅されていることが証明できた。  [0046] Furthermore, it was confirmed whether or not only the target fragment (partial partition 1H of HPV 52 type gene) was actually amplified in the PCR reaction product in which the target band was detected. This confirmation was performed by putting the PCR amplification product into the pGEMTeasy vector by TA cloning and analyzing the base sequence using the IJ in the vector as a primer. As a result, it was confirmed that the PCR reaction product in which the target band was confirmed contained only the target fragment. In other words, the other primer sequences were not amplified by the above-mentioned primer set, and it was proved that only the target sequence of the HPV52 type gene was amplified.
[0047] 続いて、同じ複数の被検者について、従来法により HPV52型の感染の有無を調 ベ、月経血試料を用いた本発明の実施例による結果の信頼性を確認した。すなわち 、前述の複数人の被検者について、子宮頸部から扁平上皮細胞を擦り取って、これ らを試料とした。これらの細胞試料について、液相（核酸)ハイブリダィゼーシヨンによ り、 HPVの検査をおこなった（SRL社)。その結果、月経血試料を用いた PCRによつ て HPV52型が陽性と判断された被検者は、同様に陽性を示した。一方、月経血試 料を用いた PCRによって HPV52型が陰性と判断された被検者は、従来方法により 同様に陰性を示した。これらの結果から、本発明の月経血試料を用いた方法によれ ば、扁平上皮細胞の採取を行うことなぐ且つ、扁平上皮細胞を用いた従来法と同様 の信頼性で、 HPV遺伝子の検出ならびに HPV感染の有無が判別可能であるといえ る。  [0047] Subsequently, the same plurality of subjects were examined for the presence or absence of HPV52 infection by a conventional method, and the reliability of the results of the example of the present invention using menstrual blood samples was confirmed. That is, for the above-mentioned multiple subjects, squamous epithelial cells were scraped from the cervix, and these were used as samples. These cell samples were examined for HPV by liquid phase (nucleic acid) hybridization (SRL). As a result, subjects who were judged positive for HPV52 by PCR using menstrual blood samples were also positive. On the other hand, subjects who were determined to be negative for HPV52 by PCR using menstrual blood samples were also negative by the conventional method. From these results, according to the method using the menstrual blood sample of the present invention, it is possible to detect HPV gene without collecting squamous epithelial cells and with the same reliability as conventional methods using squamous epithelial cells. It can be said that the presence or absence of HPV infection can be determined.
実施例 2  Example 2
[0048] HPV16型 i貴伝子の^^ 前記実施例 1で調製した cDNAを铸型として、前記フォワードプライマー（配列番号 3)および前記リバースプライマー（配列番号 4)を用レ、、 PCRを実施した。 PCRの条 件は、前述と同様である。各月経血試料由来の PCR反応物を電気泳動に供し、 目 的の大きさ（約 86bp)の増幅産物の有無を確認した。その結果、数人の被検者の各 月経血試料から、 目的増幅産物を得ることができた。 HPV16型の核酸配列 (ゲノム D NA)は、 NCBIァクセッション No. AF534061. 1に登録されている。前記プライマー セットによって増幅されるのは、登録された核酸配列において、 385番目—470番目 の領域である。 [0048] HPV16 type i Using the cDNA prepared in Example 1 as a saddle, PCR was carried out using the forward primer (SEQ ID NO: 3) and the reverse primer (SEQ ID NO: 4). The PCR conditions are the same as described above. The PCR reaction product from each menstrual blood sample was subjected to electrophoresis, and the presence or absence of the amplification product of the desired size (about 86 bp) was confirmed. As a result, the target amplification product could be obtained from each menstrual blood sample of several subjects. The nucleic acid sequence of HPV16 type (genome DNA) is registered in NCBI Accession No. AF534061.1. Amplified by the primer set is the 385th to 470th region in the registered nucleic acid sequence.
[0049] 続いて、同じ複数の被検者について、従来法により HPV16型の感染の有無を調 ベ、月経血試料を用いた本発明の実施例による結果の信頼性を確認した。すなわち 、前述の複数人の被検者について、子宮類部から扁平上皮細胞を擦り取って、これ らを試料とした。これらの細胞試料を用いて、前述と同様にして、全 RNAおよび cDN Aの調製、ならびに、 PCRによる HPVの検査を行った。その結果、月経血試料を用 レ、た PCRによって HPV16型が陽性と判断された被検者は、同様に陽性を示した。 一方、月経血試料を用いた PCRによって HPV16型が陰性と判断された被検者は、 従来法により同様に陰性を示した。これらの結果から、本発明の月経血試料を用い た方法によれば、扁平上皮細胞の採取を行うことなぐ且つ、扁平上皮細胞を用いた 従来法と同様の信頼性で、 HPV遺伝子の検出ならびに HPV感染の有無が判別可 能であるといえる。  [0049] Subsequently, the same plurality of subjects were examined for the presence or absence of HPV16 infection by a conventional method, and the reliability of the results of the examples of the present invention using menstrual blood samples was confirmed. That is, for the above-mentioned multiple subjects, squamous epithelial cells were scraped from the uterine part, and these were used as samples. Using these cell samples, total RNA and cDNA were prepared and HPV was examined by PCR in the same manner as described above. As a result, subjects who were judged positive for HPV type 16 by PCR using a menstrual blood sample were also positive. On the other hand, subjects who were judged negative for HPV type 16 by PCR using menstrual blood samples also showed negative results by the conventional method. From these results, according to the method using the menstrual blood sample of the present invention, it is possible to detect the HPV gene without collecting the squamous cells and with the same reliability as the conventional method using the squamous cells. It can be said that the presence or absence of HPV infection can be determined.
産業上の利用可能性  Industrial applicability
[0050] 以上のように、本発明の検出方法では、ヒト月経血試料を用いるため、被検者の抵 抗感および苦痛がほとんど無ぐ HPV感染の有無が判別できる。したがって、本発明 は、例えば、子宮頸癌等の子宮関連疾患の診断、治療、予防や学術研究等に広く 適用できる。 [0050] As described above, since the detection method of the present invention uses a human menstrual blood sample, the presence or absence of HPV infection with almost no resistance and pain to the subject can be determined. Therefore, the present invention can be widely applied to diagnosis, treatment, prevention, academic research and the like of uterine-related diseases such as cervical cancer.

Claims

請求の範囲 The scope of the claims
[1] ヒト生体試料からのヒトパピローマウィルス（HPV)の検出方法であって、  [1] A method for detecting human papillomavirus (HPV) from a human biological sample,
前記ヒト生体試料としてヒト月経血試料を用い、  Using a human menstrual blood sample as the human biological sample,
前記ヒト月経血試料中の HPV遺伝子を検出することにより、 HPVを検出する検出 方法。  A detection method for detecting HPV by detecting the HPV gene in the human menstrual blood sample.
[2] 前記 HPV遺伝子の検出が、遺伝子増幅法により実施される、請求の範囲 1記載の 検出方法。  [2] The detection method according to claim 1, wherein the HPV gene is detected by a gene amplification method.
[3] 前記遺伝子増幅法が、ポリメラーゼチェーンリアクション (PCR)法である、請求の範 囲 2記載の検出方法。  [3] The detection method according to claim 2, wherein the gene amplification method is a polymerase chain reaction (PCR) method.
[4] 検出対象の前記 HPVが、 HPV52型である、請求の範囲 1記載の検出方法。  [4] The detection method according to claim 1, wherein the HPV to be detected is HPV52 type.
[5] 検出対象の前記 HPVが、 HPV52型であり、 [5] The HPV to be detected is HPV52,
前記 HPV遺伝子の検出が、遺伝子増幅法により実施され、  The detection of the HPV gene is performed by a gene amplification method,
前記遺伝子増幅法に使用されるプライマーセットが、一対のフォワードプライマーお よびリバースプライマーを含み、  The primer set used for the gene amplification method includes a pair of forward primer and reverse primer,
前記フォワードプライマーが、下記（A1)または (A2)のオリゴヌクレオチドであり、前 記リバースプライマーが、下記（B1)または（B2)のオリゴヌクレオチドである、請求の 範囲 1記載の検出方法。  The detection method according to claim 1, wherein the forward primer is an oligonucleotide (A1) or (A2) below, and the reverse primer is an oligonucleotide (B1) or (B2) below.
(A1)配列番号 1の塩基配列からなるオリゴヌクレオチド。  (A1) An oligonucleotide having the base sequence of SEQ ID NO: 1.
(A2)配列番号 1の塩基配列において、 1若しくは数個の塩基が、置換、欠失、挿入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV52型遺伝子を 増幅可能なオリゴヌクレオチド。  (A2) an oligonucleotide that comprises a nucleotide sequence in which one or several bases are substituted, deleted, inserted or added in the nucleotide sequence of SEQ ID NO: 1 and can amplify an HPV52 gene from a human menstrual blood sample .
(B1)配列番号 2の塩基配列からなるオリゴヌクレオチド。  (B1) An oligonucleotide having the base sequence of SEQ ID NO: 2.
(B2)配列番号 2の塩基配列において、 1若しくは数個の塩基が、置換、欠失、揷入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV52型遺伝子を 増幅可能なオリゴヌクレオチド。  (B2) Oligo capable of amplifying the HPV52 gene from a human menstrual blood sample consisting of a base sequence in which one or several bases are substituted, deleted, inserted or added in the base sequence of SEQ ID NO: 2. nucleotide.
[6] 検出対象の前記 HPVが、 HPV16型である、請求の範囲 1記載の検出方法。  [6] The detection method according to claim 1, wherein the HPV to be detected is HPV16 type.
[7] 検出対象の前記 HPVが、 HPV16型であり、  [7] The HPV to be detected is HPV16 type,
前記 HPV遺伝子の検出が、遺伝子増幅法により実施され、 前記遺伝子増幅法に使用されるプライマーセットが、一対のフォワードプライマーお よびリバースプライマーを含み、 The detection of the HPV gene is performed by a gene amplification method, The primer set used for the gene amplification method includes a pair of forward primer and reverse primer,
前記フォワードプライマーが、下記（C1)または（C2)のオリゴヌクレオチドであり、前 記リバースプライマーが、下記（D1)または（D2)のオリゴヌクレオチドである、請求の 範囲 1記載の検出方法。  The detection method according to claim 1, wherein the forward primer is an oligonucleotide of (C1) or (C2) below, and the reverse primer is an oligonucleotide of (D1) or (D2) below.
(C1)配列番号 3の塩基配列からなるオリゴヌクレオチド。  (C1) An oligonucleotide having the base sequence of SEQ ID NO: 3.
(C2)配列番号 3の塩基配列において、 1若しくは数個の塩基が、置換、欠失、挿入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV16型遺伝子を 増幅可能なオリゴヌクレオチド。  (C2) an oligonucleotide capable of amplifying an HPV16 gene from a human menstrual blood sample, wherein the nucleotide sequence of SEQ ID NO: 3 comprises a nucleotide sequence in which one or several bases are substituted, deleted, inserted or added .
(D1)配列番号 4の塩基配列からなるオリゴヌクレオチド。  (D1) An oligonucleotide having the base sequence of SEQ ID NO: 4.
(D2)配列番号 4の塩基配列において、 1若しくは数個の塩基が、置換、欠失、揷入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV16型遺伝子を 増幅可能なオリゴヌクレオチド。  (D2) Oligo capable of amplifying an HPV16 gene from a human menstrual blood sample consisting of a base sequence in which one or several bases are substituted, deleted, inserted or added in the base sequence of SEQ ID NO: 4. nucleotide.
[8] 前記遺伝子増幅法が、 PCR法である、請求の範囲 5または 7記載の検出方法。 [8] The detection method according to claim 5 or 7, wherein the gene amplification method is a PCR method.
[9] 前記検出方法が、子宮頸癌の診断に使用される方法である、請求の範囲 1記載の 検出方法。 [9] The detection method according to claim 1, wherein the detection method is a method used for diagnosis of cervical cancer.
[10] 遺伝子増幅法によるヒト月経血試料からのヒトパピローマウィルス (HPV) 52型遺伝 子の検出に用いるプライマーセットであって、  [10] A primer set used for detecting human papillomavirus (HPV) type 52 gene from human menstrual blood samples by gene amplification method,
一対のフォワードプライマーおよびリバースプライマーを含み、  Including a pair of forward and reverse primers,
前記フォワードプライマーが、下記（A1)または (A2)のオリゴヌクレオチドであり、前 記リバースプライマーが、下記（B1)または（B2)のオリゴヌクレオチドであるプライマ A primer in which the forward primer is an oligonucleotide (A1) or (A2) below and the reverse primer is an oligonucleotide (B1) or (B2) below.
^— 、、 /ト ^ —,, /
(A1)配列番号 1の塩基配列からなるオリゴヌクレオチド。  (A1) An oligonucleotide having the base sequence of SEQ ID NO: 1.
(A2)配列番号 1の塩基配列において、 1若しくは数個の塩基が、置換、欠失、挿入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV52型遺伝子を 増幅可能なオリゴヌクレオチド。  (A2) an oligonucleotide that comprises a nucleotide sequence in which one or several bases are substituted, deleted, inserted or added in the nucleotide sequence of SEQ ID NO: 1 and can amplify an HPV52 gene from a human menstrual blood sample .
(B1)配列番号 2の塩基配列からなるオリゴヌクレオチド。  (B1) An oligonucleotide having the base sequence of SEQ ID NO: 2.
(B2)配列番号 2の塩基配列において、 1若しくは数個の塩基が、置換、欠失、揷入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV52型遺伝子を 増幅可能なオリゴヌクレオチド。 (B2) In the nucleotide sequence of SEQ ID NO: 2, one or several bases are substituted, deleted, or inserted Alternatively, an oligonucleotide comprising an added nucleotide sequence and capable of amplifying the HPV52 type gene from a human menstrual blood sample.
[11] 前記遺伝子増幅法が、ポリメラーゼチェーンリアクション (PCR)法である、請求の範 囲 10記載のプライマーセット。 [11] The primer set according to claim 10, wherein the gene amplification method is a polymerase chain reaction (PCR) method.
[12] 前記プライマーセットが、子宮頸癌の診断に用いるプライマーセットである、請求の 範囲 10記載のプライマーセット。 [12] The primer set according to claim 10, wherein the primer set is a primer set used for diagnosis of cervical cancer.
[13] 遺伝子増幅法によるヒト月経血試料からのヒトパピローマウィルス (HPV) 16型遺伝 子の検出に用いるプライマーセットであって、 [13] A primer set used for detection of human papillomavirus (HPV) type 16 gene from human menstrual blood samples by gene amplification method,
一対のフォワードプライマーおよびリバースプライマーを含み、  Including a pair of forward and reverse primers,
前記フォワードプライマーが、下記（C1)または（C2)のオリゴヌクレオチドであり、前 記リバースプライマーが、下記（D1)または（D2)のオリゴヌクレオチドであるプライマ The forward primer is an oligonucleotide (C1) or (C2) below, and the reverse primer is an oligonucleotide (D1) or (D2) below.
^— 、、 /ト ^ —,, /
(C1)配列番号 3の塩基配列からなるオリゴヌクレオチド。  (C1) An oligonucleotide having the base sequence of SEQ ID NO: 3.
(C2)配列番号 3の塩基配列において、 1若しくは数個の塩基が、置換、欠失、挿入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV16型遺伝子を 増幅可能なオリゴヌクレオチド。  (C2) an oligonucleotide capable of amplifying an HPV16 gene from a human menstrual blood sample, wherein the nucleotide sequence of SEQ ID NO: 3 comprises a nucleotide sequence in which one or several bases are substituted, deleted, inserted or added .
(D1)配列番号 4の塩基配列からなるオリゴヌクレオチド。  (D1) An oligonucleotide having the base sequence of SEQ ID NO: 4.
(D2)配列番号 4の塩基配列において、 1若しくは数個の塩基が、置換、欠失、挿入 または付加された塩基配列からなり、かつ、ヒト月経血試料から HPV16型遺伝子を 増幅可能なオリゴヌクレオチド。  (D2) an oligonucleotide capable of amplifying an HPV16 gene from a human menstrual blood sample, wherein the nucleotide sequence of SEQ ID NO: 4 comprises a nucleotide sequence in which one or several bases are substituted, deleted, inserted or added .
[14] 前記遺伝子増幅法が、ポリメラーゼチェーンリアクション (PCR)法である、請求の範 囲 13記載のプライマーセット。 [14] The primer set according to claim 13, wherein the gene amplification method is a polymerase chain reaction (PCR) method.
[15] 前記プライマーセットが、子宮頸癌の診断に用いるプライマーセットである、請求の 範囲 13記載のプライマーセット。 [15] The primer set according to claim 13, wherein the primer set is a primer set used for diagnosis of cervical cancer.
[16] 遺伝子増幅法によるヒト月経血試料からのヒトパピローマウィルス (HPV) 52型遺伝 子の検出に用いる検出キットであって、請求の範囲 10記載のプライマーセットを含む 検出キット。 [16] A detection kit used for detecting a human papillomavirus (HPV) type 52 gene from a human menstrual blood sample by a gene amplification method, comprising the primer set according to claim 10.
[17] 前記遺伝子増幅法が、 PCR法である、請求の範囲 16記載の検出キット。 [17] The detection kit according to claim 16, wherein the gene amplification method is a PCR method.
[18] 前記検出キットが、子宮頸癌の診断に用いるキットである、請求の範囲 16記載の検 出キット。 [18] The detection kit according to claim 16, wherein the detection kit is a kit used for diagnosis of cervical cancer.
[19] 遺伝子増幅法によるヒト月経血試料からのヒトパピローマウィルス (HPV) 16型遺伝 子の検出に用いる検出キットであって、請求の範囲 13記載のプライマーセットを含む 検出キット。  [19] A detection kit used for detecting a human papillomavirus (HPV) type 16 gene from a human menstrual blood sample by a gene amplification method, comprising the primer set according to claim 13.
[20] 前記遺伝子増幅法が、 PCR法である、請求の範囲 19記載の検出キット。  [20] The detection kit according to claim 19, wherein the gene amplification method is a PCR method.
[21] 前記検出キットが、子宮頸癌の診断に用いるキットである、請求の範囲 19記載の検 出キット。 [21] The detection kit according to claim 19, wherein the detection kit is a kit used for diagnosis of cervical cancer.
[22] ヒト生体試料を用いた子宮頸癌の診断方法であって、  [22] A diagnostic method for cervical cancer using a human biological sample,
ヒト生体試料が、ヒト月経血試料であり、  The human biological sample is a human menstrual blood sample,
請求の範囲 1記載の HPVの検出方法によって、ヒト月経血試料中の HPVを検出す ることにより、子宮頸癌を診断する診断方法。  A diagnostic method for diagnosing cervical cancer by detecting HPV in a human menstrual blood sample by the HPV detection method according to claim 1.
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