WO2007148762A1 - Procédé de détection d'un papillomavirus humain (hpv) et ensemble d'amorces et trousses de détection utilisées pour ce procédé - Google Patents

Procédé de détection d'un papillomavirus humain (hpv) et ensemble d'amorces et trousses de détection utilisées pour ce procédé 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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WIPO (PCT)
Prior art keywords
gene
hpv
oligonucleotide
menstrual blood
human
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PCT/JP2007/062536
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English (en)
Japanese (ja)
Inventor
Masahiko Kuroda
Yoshinori Kosugi
Kosuke Oikawa
Masami Tanaka
Seiko Iizuka
Original Assignee
Masahiko Kuroda
Yoshinori Kosugi
Kosuke Oikawa
Masami Tanaka
Seiko Iizuka
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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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/ja
Publication of WO2007148762A1 publication Critical patent/WO2007148762A1/fr

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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

La présente invention concerne une technologie de détection qui permet d'identifier un papillomavirus humain (HPV) sans provoquer de malaises ou de douleur chez les sujets soumis au test. L'invention concerne un procédé de détection du HPV dans un échantillon biologique humain, caractérisé en ce qu'un échantillon sanguin menstruel humain est utilisé à titre d'échantillon biologique humain, et que la détection du HPV est obtenue grâce à l'identification d'un gène du HPV dans l'échantillon sanguin menstruel humain. L'identification d'un gène du HPV est de préférence mise en œuvre au moyen d'une PCR. Dans le cas d'une détection du gène HPV52, il est préférable d'utiliser un oligonucléotide correspondant à la séquence de base SEQ ID NO : 1 en tant qu'amorce sens et d'utiliser un oligonucléotide correspondant à la séquence de base SEQ ID NO : 2 en tant qu'amorce antisens.
PCT/JP2007/062536 2006-06-22 2007-06-21 Procédé de détection d'un papillomavirus humain (hpv) et ensemble d'amorces et trousses de détection utilisées pour ce procédé WO2007148762A1 (fr)

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JP2008522519A JPWO2007148762A1 (ja) 2006-06-22 2007-06-21 ヒトパピローマウイルス（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

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JP2006-173211 2006-06-22

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11148901A (ja) * 1991-02-06 1999-06-02 Igen Inc 改良されたルミネセンス検定のための装置
JP2000500342A (ja) * 1995-11-15 2000-01-18 ジェン−プローブ・インコーポレーテッド ヒトパピローマウイルス核酸に相補的な核酸プローブおよび関連する方法およびキット
JP2001197894A (ja) * 1987-02-26 2001-07-24 Biosearch Internatl Pty Ltd ヒト発癌性乳頭腫ウイルスの検出方法
JP2004121240A (ja) * 2002-09-12 2004-04-22 Geneticlab Co Ltd ヒトパピローマウイルスを検出するためのプライマーセット、検出方法および検出用ｄｎａアレイ
WO2006075245A2 (fr) * 2005-01-14 2006-07-20 The Regents Of The University Of Michigan Systemes, methodes et compositions pour la detection de papillomavirus humain dans des echantillons biologiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001197894A (ja) * 1987-02-26 2001-07-24 Biosearch Internatl Pty Ltd ヒト発癌性乳頭腫ウイルスの検出方法
JPH11148901A (ja) * 1991-02-06 1999-06-02 Igen Inc 改良されたルミネセンス検定のための装置
JP2000500342A (ja) * 1995-11-15 2000-01-18 ジェン−プローブ・インコーポレーテッド ヒトパピローマウイルス核酸に相補的な核酸プローブおよび関連する方法およびキット
JP2004121240A (ja) * 2002-09-12 2004-04-22 Geneticlab Co Ltd ヒトパピローマウイルスを検出するためのプライマーセット、検出方法および検出用ｄｎａアレイ
WO2006075245A2 (fr) * 2005-01-14 2006-07-20 The Regents Of The University Of Michigan Systemes, methodes et compositions pour la detection de papillomavirus humain dans des echantillons biologiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BORAI N.E. ET AL.: "Presence of HSV-1 DNA in semen and menstrual blood", J. REPROD. IMMUNOL., vol. 41, 1998, pages 137 - 147, XP003019882 *
SILVERMAN A.L. ET AL.: "HCV RNA is present in the menstrual blood of women with chronic hepatitis C infection", AM. J. GASTROENTEROL., vol. 89, no. 8, 1994, pages 1201 - 1202, XP003019881 *

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