WO2020050350A1 - Method for detecting human papillomavirus - Google Patents

Method for detecting human papillomavirus Download PDF

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WO2020050350A1
WO2020050350A1 PCT/JP2019/034908 JP2019034908W WO2020050350A1 WO 2020050350 A1 WO2020050350 A1 WO 2020050350A1 JP 2019034908 W JP2019034908 W JP 2019034908W WO 2020050350 A1 WO2020050350 A1 WO 2020050350A1
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adapter
seq
nos
sequences
primer pair
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PCT/JP2019/034908
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French (fr)
Japanese (ja)
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陽介 瀧本
啓太郎 萩原
雅武 金井
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株式会社ヘルスケアシステムズ
株式会社ダンテ
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Priority to JP2020541291A priority Critical patent/JPWO2020050350A1/en
Publication of WO2020050350A1 publication Critical patent/WO2020050350A1/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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for detecting human papillomavirus in a sample with high sensitivity.
  • Cervical cancer is a highly prevalent malignancy, involving human papillomavirus (HPV) in the carcinogenesis process, and HPV infection is a risk factor for cervical cancer progression.
  • HPV is a virus having double-stranded DNA and infects epithelia such as human skin and mucous membrane.
  • HPV6 HPV16
  • HPV18 may cause malignant cervical cancer.
  • HPV infection has been detected by PCR using a cell suspension collected from the cervix of a woman as a specimen.
  • Non-Patent Documents 1 to 3 Numerous primers have been developed for detection of HPV by PCR (see Non-Patent Documents 1 to 3).
  • HPV infection has not been detected in men.
  • An object of the present invention is to provide a method for detecting HPV in a sample with high sensitivity, and in particular, to provide a method for detecting HPV in semen or urine.
  • the present inventors have intensively studied a method for measuring HPV infection in men with high sensitivity and measuring HPV infection in women without causing pain.
  • the invention has been completed.
  • the present invention is as follows.
  • [1] A method for detecting HPV infection by amplifying HPV in a sample by a PCR reaction, (a) In the first PCR, a primer that specifically hybridizes to the HPV gene, and an adapter primer pair comprising an adapter consisting of a sequence that does not anneal to the HPV genome, the human genome, and the genomes of viruses and microorganisms that infect humans. Amplified by (b) in the second PCR, amplifying the amplification product of (a) with a pair of primers having a sequence complementary to the adapter, How to detect HPV infection. [2] The method of [1], wherein the copy number of HPV in the sample is 1000 copies / mL or less.
  • the combination of the adapter primer pair and the primer pair is any combination of the following (i) to (vi): (i) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 13 and 14, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 1 and 2; (ii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 15 and 16 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 3 and 4; (iii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 17 and 18, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 5 and 6; (iv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NO
  • Two-stage PCR of a first PCR using an adapter primer obtained by adding an adapter to a primer specifically hybridizing to the HPV gene of the present invention, and a second PCR using a primer having a sequence complementary to the adapter Thereby, it is possible to detect even when the copy number of HPV in the sample is only about 10 copies / mL.
  • the method of the present invention can detect HPV with high sensitivity even when using semen having a low copy number of HPV, cell suspension or urine collected from the cervix as a specimen. Further, it is possible to detect male HPV infection, which has not been conventionally performed by the present invention.
  • FIG. 3 is a view showing a sequence of a primer used in the present invention. It is a figure which shows the result of PCR amplification using the general-purpose primer set (primer set of GP5 + and GP6 + or primer set of MY11 and MY09).
  • FIG. 3 is a diagram showing the results of PCR amplification using an adapter primer set (a primer set of adaptor01-GP5 + and adaptor02-GP6 + or a primer set of adaptor03-MY11 and adaptor04-MY09) in 1st PCR. It is a figure which shows the result of having carried out PCR amplification using the primer set of adaptor05-GP5 + and adaptor06-GP6 + (FIG.
  • FIG. 7 is a diagram showing the results of PCR amplification using a primer set of adaptor09-MY11 and adaptor010-MY09 (left of FIG. 5) or a primer set of adaptor011-MY11 and adaptor012-MY09 (right of FIG. 5) as an adapter primer set. is there.
  • FIG. 4 is a diagram showing an agarose gel electrophoresis image showing the results of detection of HPV genomic DNA using a urine sample. The results of the detection of HPV Type 16 (FIG. 7A) and the results of the detection of HPV Type 18 (FIG.
  • FIG. 3 is a view showing the results of HPVHPType 16 detection using the HPV type-specific primers used in the present invention.
  • FIG. 3 is a view showing the results of HPVHPType 18 detection using the HPV type-specific primers used in the present invention.
  • FIG. 2 is a view showing a sequence of an HPV type-specific primer used in the present invention (part 2).
  • the present invention is a method for amplifying and detecting the gene of human papilloma virus (HPV) by PCR, and it is possible to detect HPV with high sensitivity by the method of the present invention. That is, it can be detected even when HPV is present in the sample at 1000 copies / mL or less, preferably 100 copies / mL or less, more preferably 10 copies / mL or less, or less than 10 copies / mL.
  • HPV human papilloma virus
  • HPV in a specimen can be detected even when a male semen having a small amount of HPV is used as the specimen. If HPV is present in male semen, symptoms due to infection are rarely manifested, but since sex partners are transmitted by sexual intercourse, detection of HPV in semen will prevent infection to others beforehand. It is significant to prevent.
  • women can be tested for HPV infection by rubbing the cervix or cervix with a swab or plastic brush, collecting cervical cells, and collecting the cell suspension. The presence of HPV in the solution is measured by PCR.
  • HPV in a specimen can be detected even when male or female urine is used as the specimen.
  • the HPV gene is amplified by adapter PCR using an adapter primer for HPV gene amplification, and the HPV is detected.
  • the specimen semen collected from a male subject, a cell suspension collected from the cervix of a female subject, and urine collected from a male or female subject can be used.
  • saliva, tissue fluid, cerebrospinal fluid, swabs, serum, plasma, blood, stool, etc. collected from a male or female subject can be used.
  • the sample volume is 5 ⁇ L to 10 mL, preferably 10 ⁇ L to 1 mL for semen or urine, for example, 5 ⁇ L to 100 ⁇ L, preferably 10 ⁇ L to 50 ⁇ L for semen, and 200 ⁇ L to 10 mL, preferably 200 ⁇ L for urine. If 5 mL, more preferably 500 ⁇ L to 1 mL, is used, the HPV gene in the sample can be amplified.
  • HPV is a virus having a circular double-stranded DNA of about 7.9 bp and infects human mucosa and skin.
  • the HPV DNA consists of URR (upstream regulatory region), a region that regulates gene expression, and E1, E2, E4, E5, E6, and E7, which are early genes that encode nonstructural proteins involved in viral replication. And two genes, L1 and L2, which are late genes that encode the viral capsid.
  • L1 and L2 are capsid proteins that constitute virus particles, and are involved in adsorption and entry into host cells.
  • HPV types that can be detected include 6b type, 16 type, 18 type, 31 type, 33 type, 35 type, 39 type, 45 type, 51 type, 52 type, 53 type, 56 type, Types 58, 59, 67, 68, 69, 70, 73, and 82 are among them, and among them, type 6b, which is likely to be infected with malignant cervical cancer in the future, In addition to types 16 and 18, type 53, which is highly likely to cause malignant cervical cancer in Japanese, is preferable.
  • the nucleotide sequences of the HPV 16 L1 gene and the HPV 6b L1 gene are shown in SEQ ID NOs: 32 and 33, respectively.
  • a primer sequence can be designed based on these sequence information or known HPV gene sequence information.
  • the first amplification using a primer pair of a forward primer and a reverse primer to which an adapter sequence is added to the 5 ′ side of a primer that specifically hybridizes to the HPV gene is performed.
  • (1st PCR) may be performed, and then a second amplification (2nd PCR) may be performed using a primer pair of a forward primer and a reverse primer that hybridize to the adapter sequence with respect to the amplification product.
  • a primer that has an adapter sequence added to the 5 ′ side of a primer that specifically hybridizes to an HPV gene is composed of an HPV region-specific sequence primer and an adapter, and is called an adapter primer.
  • the base length of the primer that specifically hybridizes to the HPV gene is 5 to 50 bases, preferably 10 to 40 bases, more preferably 15 to 35 bases, and still more preferably 20 to 30 bases.
  • a primer pair that specifically hybridizes to the HPV gene may be designed so as to amplify a region having a length of 40 to 1000 bases, preferably 200 to 300 bases, of the HPV gene. That is, it may be set so as to hybridize to the 3′-end and the 5′-end of the region of 40 to 1000 bases, preferably 100 to 500 bases, more preferably 150 to 450 bases of the HPV gene.
  • an amplification product having a length of 40 to 1000 bases, preferably 100 to 500 bases, more preferably 150 to 450 bases is obtained.
  • Primers that hybridize to the HPV gene have several, preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, More preferably, it may have one or two, more preferably one mismatch. As a result, not only a specific type of HPV but also a large number of types of HPV genes can be amplified with one set of primer pairs, and a large number of types of HPV can be detected.
  • L1 of HPV is a conserved region in many types (subtypes) of HPV, and the sequence identity between each type is high.
  • the L1-encoding DNA is preferably amplified using a primer pair that hybridizes with the L1-encoding DNA.
  • a known general-purpose primer pair for HPV detection can be used.
  • Such primer pairs include a primer pair of a forward primer GP5 + (SEQ ID NO: 25) and a reverse primer GP6 + (SEQ ID NO: 26), a primer pair of a forward primer MY11 (SEQ ID NO: 27) and a reverse primer MY09 (SEQ ID NO: 28), and a forward pair.
  • primer L1C1 SEQ ID NO: 29
  • reverse primer L1C2 SEQ ID NO: 30
  • reverse primer L1C2M SEQ ID NO: 31
  • the base length between GP5 + and GP6 + is 142 bases, the base length between MY11 and MY09 is 452 bases, and the base length between L1C1 and L1C2 or L1C2M is 253 bases.
  • the base length of the adapter is 10 to 40 bases, preferably 10 to 30 bases, and more preferably 15 to 20 bases.
  • the adapter sequence is preferably designed to have a GC content of 50 to 65%, since a high-order structure is likely to be formed when the CG content or AT content is high. Also, set so that both ends are not connected.
  • the Tm value may be designed to be 50 to 70 ° C., preferably 55 to 65 ° C., and more preferably 60 ⁇ 1 ° C.
  • the adapter sequence is designed so as not to anneal the HPV genome, human genome, and genomes of viruses and microorganisms that infect humans that may be present in the sample.
  • the sequence is artificially designed.
  • the base sequences of the forward primer and the reverse primer may be obtained from the sequence of the genome of a plant or a non-human animal whose sequence is being analyzed.
  • a PCR primer design tool such as Primer3web (http://primer3.ut.ee/).
  • an adapter sequence can be selected from the genomic sequence of a higher plant such as rice or Arabidopsis thaliana. That is, for example, the adapter sequence can be a partial sequence of the sequence of the Arabidopsis thaliana gene. At this time, it is preferable to design a pair of adapter sequences from sequences of different chromosomes of Arabidopsis thaliana in consideration of the possibility that the Arabidopsis thaliana genome is mixed in the sample and amplification occurs.
  • adapter examples include adaptor01 to adaptor12 consisting of the sequences represented by SEQ ID NOs: 1 to 12, adaptor01 and adaptor02, adaptor03 and adaptor04, adaptor05 and adaptor06, adaptor07 and adaptor08, adaptor09 and adaptor08, adaptor09 and adaptor10, and adaptor11 and adaptor12, respectively. , Forward primer and reverse primer.
  • the adapter consisting of the sequences shown in SEQ ID NOs: 1 to 12 is an adapter designed from the sequence of the Arabidopsis thaliana gene.
  • adapter13 to adapter24 consisting of the sequences represented by SEQ ID NOs: 36 to 47, respectively.
  • the combination may be added to the forward primer and the reverse primer, respectively.
  • the adapter consisting of the sequences shown in SEQ ID NOs: 36 to 47 is an adapter designed from the sequence of the Arabidopsis thaliana gene.
  • an adapter to a primer that specifically hybridizes to the HPV gene may be performed by ligation in which the ends of DNA strands are linked by a phosphodiester bond. Ligation can be performed by a known method using DNA ligase.
  • DNA ligase room-temperature DNA ligase such as T4-DNA ligase and E. coli DNA ligase can be used. Alternatively, thermostable DNA ligase may be used.
  • a primer and an adapter that specifically hybridize to the HPV gene may be linked via a linker.
  • the linker sequence is designed not to be complementary to the sequence in the HPV genome.
  • the base length of the linker is 10 bases or less, preferably 5 bases or less.
  • the adapter primer is a primer obtained by adding an adapter to a primer that specifically hybridizes to the HPV gene.
  • the adapter primer used in the present invention includes, for example, an adapter primer consisting of the base sequences represented by SEQ ID NOS: 13 to 24.
  • the adapter primers of SEQ ID NOs: 13 and 14 are a pair of a forward primer (adaptor01-GP5 +) in which adaptor01 is added to GP5 + and a reverse primer (adaptor02-GP6 +) in which adaptor02 is added to GP6 +.
  • a pair of a forward primer (adaptor05-GP5 +) and a reverse primer (adaptor06-GP6 +) obtained by adding an adaptor06 to GP6 +, and the adapter primers of SEQ ID NOs: 19 and 20 correspond to a forward primer (adaptor07-MY11) obtained by adding an adaptor07 to MY11.
  • the adapter primers of SEQ ID NOs: 23 and 24 are adaptor11 in MY11.
  • the adapter primer used in the present invention includes, for example, an adapter primer consisting of the nucleotide sequence represented by SEQ ID NO: 48 to 59.
  • the adapter primers of SEQ ID NOs: 48 and 49 are, respectively, a forward primer (adapter13-16_L1_F1) and a reverse primer (adapter14-16_L1_R1) each having an adapter 13 and an adapter 14 added to the head of the L1 primer targeting the region encoding the L1 protein.
  • the adapter primers of SEQ ID NOs: 50 and 51 are a forward primer (adapter15-16_L1_F2) and a reverse primer (adapter15-adapter) respectively having an adapter 15 and an adapter 16 added to the head of the L1 primer targeting the region encoding the L1 protein.
  • 16_L1_R2 the adapter primers of SEQ ID NOs: 52 and 53 are respectively a forward primer (adapter17-16_L1_F3) and a reverse primer with adapter17 and adapter18 added to the head of the L1 primer targeting the region encoding the L1 protein. (adapter18-16_L1_R3).
  • the adapter primers of SEQ ID NOs: 56 and 57 are, respectively, a forward primer (adapter21-18_L1_F2) and a reverse primer (adapter22-18_L1_R2) each having an adapter 21 and an adapter 22 added to the head of the L1 primer targeting the region encoding the L1 protein.
  • the adapter primers of SEQ ID NOs: 58 and 59 are respectively a forward primer (adapter23-18_L1_F3) and a reverse primer (adapter24) each having an adapter 23 and an adapter 24 added to the head of an L1 primer targeting a region encoding the L1 protein. -18_L1_R3).
  • a PCR extension reaction is performed using the above adapter primer to amplify the HPV gene in the sample.
  • the PCR reaction may be performed by a known method using a DNA polymerase under conditions usually used. Conditions for the PCR reaction can be appropriately set based on the Tm value of the primer and the like. For example, a PCR reagent containing a primer, a dNTP mixture, and a polymerase is added to a sample, heated at 94 ° C for 5 minutes, heated at 98 ° C for 10 seconds, cooled at 45 ° C for 30 seconds, and heated at 72 ° C for 30 seconds as one cycle. It may be repeated about 10 to 50 cycles. As the polymerase to be used, a known polymerase such as a limited Taq polymerase can be used.
  • the amplification product obtained by the first PCR has a sequence complementary to the adapter at both ends.
  • PCR may be performed using a polynucleotide that specifically hybridizes to a sequence complementary to the adapter in the amplification product as a primer.
  • the primer preferably has the same sequence as the adapter, but has several, preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and still more preferably May have one mismatch.
  • the primer is sometimes referred to as an adapter primer.
  • the reaction conditions of the second PCR can be set appropriately as in the case of the first PCR. For example, after heating at 94 ° C. for 5 minutes, heating at 98 ° C. for 10 seconds, cooling at 58 ° C. for 30 seconds, and heating at 72 ° C. for 30 seconds may be repeated as about 10 to 50 cycles.
  • the obtained PCR product can be confirmed by agarose gel electrophoresis. What is necessary is just to check whether the DNA of the target size has been amplified by agarose gel electrophoresis.
  • HPV in a sample can be detected with high sensitivity, and the number of copies of HPV in the sample is 1000 copies / mL, preferably 100 copies / mL, more preferably 10 copies / mL. It can be detected even if there is only a degree. This is 100 to 1000 times more sensitive than the conventional method of detecting HPV by PCR.
  • the present invention provides a primer-specific pair of the above-described adapter, which comprises an HPV genome, a human genome, and an adapter comprising a sequence that does not anneal to the genomes of viruses and microorganisms that infect humans, to the above-mentioned primer that specifically hybridizes to the HPV gene.
  • a kit for detecting HPV infection by amplifying HPV in a sample by a PCR reaction comprising a pair of primers having a sequence complementary to the above.
  • the kit may optionally contain a PCR reaction solution, an enzyme, a reagent for treating a sample, and the like.
  • Example 1 Development method of adapter PCR using adapter primer for gene amplification of HPV Preparation of plasmid containing HPV L1 sequence (1) 620 bases of L1 gene region of HPV Type6b, 16, 18, and 53 were introduced into pUC19 plasmid using In-Fusion (registered trademark) HD Cloning Kit (Takara Bio). .
  • This method involves two rounds of PCR.
  • the 1st PCR is performed using a primer that anneals to the L1 gene of HPV and adds a 20-base adapter sequence to the 5 ′ side of a known primer that amplifies DNA.
  • 2nd PCR is performed with primers consisting only of the adapter sequence.
  • the adapter sequence is designed from the genome of Arabidopsis thaliana, a higher plant, in order to make it a sequence that does not anneal to the genome of the HPV genome, human genome, viruses or microorganisms that infect humans, which are assumed to be included in the sample.
  • a pair of adapter sequences designed from different chromosomes were selected.
  • (1) 10 sets of forward primer and reverse primer sequences were obtained from each gene using the Primer3web (http://primer3.ut.ee/) from the genome sequences of the Arabidopsis thaliana genes AT1G62710, AT2G22770, AT3G15950, and AT4G28520.
  • the Primer was set to have a base length of 20, a Tm value of 60 ⁇ 1 ° C., and a GC% of 50 to 65.
  • Oligo DNA was prepared in which the forward primers obtained from the Arabidopsis thaliana genes AT1G62710 and AT3G15950 were added as adapter sequences to the GP5 + and GP5 + 5 'ends of the GP5 + and GP5 + primer sets (commonly used for HPV detection). Oligo DNA was prepared by adding a reverse primer obtained from the Arabidopsis thaliana genes AT2G22770 and AT4G28520 to the 5 'end of GP6 + as an adapter sequence.
  • an oligo DNA was prepared by adding a forward primer obtained from the Arabidopsis thaliana genes AT1G62710 and AT3G15950 to the 5 ′ end of MY11 of the MY11 and MY9 primer sets commonly used for HPV detection. Oligo DNAs were prepared by adding reverse primers obtained from AT2G22770 and AT4G28520 to the 5 ′ end of MY09 (adaptor01-GP5 +, adaptor02-GP6 +, etc.).
  • the nucleotide sequences of adaptor01 to adapter12 are shown in FIG. 1 and SEQ ID NOS: 1 to 12.
  • the adapter primers to which the adapter sequence was added to the HPV detection primer are adapter01-GP5 +, adapter02-GP6 +, adaptor03-MY11, adaptor04-MY09, adaptor05-GP5 +, adaptor06-GP6 +, adaptor07-MY11, adaptor08-MY09, adaptor09-.
  • the nucleotide sequences of GP5 +, adapter10-GP6 +, adapter11-MY11, and adapter12-MY09 are shown in FIG. 1 and SEQ ID NOs: 13 to 24.
  • the sequences of GP5 +, GP6 +, MY11 and MY09 are shown in SEQ ID NOS: 25 to 28.
  • the PCR apparatus used an Applied Biosystems (registered trademark) 2720 thermal cycler, and the PCR program was "94 ° C for 5 minutes, (98 ° C for 10 seconds, 45 ° C for 30 seconds, 72 ° C for 30 seconds) x 40 cycles”. (1st PCR conditions).
  • a primer set of adaptor01-GP5 + and adaptor02-GP6 + is used for 1st PCR, and a primer set of adaptor01 and adaptor02 corresponding to 2nd PCR is used.
  • a primer set of adaptor09 and adaptor10 and a primer set of adaptor11 and adaptor12 may be used.
  • the PCR program was “94 ° C for 5 minutes, (98 ° C for 10 seconds, 58 ° C for 30 seconds, 72 ° C for 30 seconds) ⁇ 40 cycles”.
  • FIG. 2 shows the results of PCR amplification using a general-purpose primer set (a primer set of GP5 + and GP6 + or a primer set of MY11 and MY09).
  • FIG. 2A shows the results of HPV type 6 and HPV type 16
  • FIG. 2B shows the results of HPV type 18 and HPV type 53.
  • FIG. 3 shows the results of HPV type 6 and HPV type 16
  • FIG. 3B shows the results of HPV type 18 and HPV type 53.
  • FIG. 4 shows the results of PCR amplification using a primer set of adaptor05-GP5 + and adaptor06-GP6 + (FIG. 4A) or a primer set of adaptor07-GP5 + and adaptor08-GP6 + (FIG. 4B) as the adapter primer set.
  • FIG. 5 shows the results of PCR amplification using the adapter set of adapter09-MY11 and adapter010-MY09 (FIG. 5A) or the set of adapter011-MY11 and adapter012-MY09 (FIG. 5B). Show.
  • the adapter (adaptor05-GP5 + and adaptor06-GP6 +) to which adapter05 and adapter06 were added to the GP5 + primer and GP6 + primer, respectively, or the adapter07 and adapter08 to the GP5 + primer and GP6 + primer, respectively.
  • primers (adaptor07-GP5 + and adaptor08-GP6 +), with HPV type 6,16,18 and 53, can be detected at a concentration of 10 1 copies / Tube, improvement in sensitivity was observed.
  • the MY11 and MY09 primers were respectively added with adaptor09 and adaptor010 (adaptor09-MY11 and adaptor010-MY09), or the MY11 and MY09 primers were respectively adapted to adaptor011 and adaptor012. in HPV type 6,16,18 and 53 even primers added (adaptor011-MY11 and adaptor012-MY09), can be detected at a concentration of 10 1 copies / Tube, improvement in sensitivity was observed.
  • Example 2 PCR reaction using a sample (semen) 80 ⁇ l of buffer 1 (20 mM Tris-HCl, 5 mM EDTA, 400 mM NaCl, 0.3% SDS, pH 8.0) was added to 20 ⁇ l of semen of 165 people and mixed. And heated at 95 ° C. for 5 minutes.
  • buffer 1 (20 mM Tris-HCl, 5 mM EDTA, 400 mM NaCl, 0.3% SDS, pH 8.0
  • DNA was extracted using NucleoSpin (registered trademark) Tissue XS (Machliner Gel) according to the instruction manual.
  • NucleoSpin registered trademark
  • Tissue XS Machine XS
  • a set of adapter01-GP5 + (SEQ ID NO: 13) and adapter02-GP6 + (SEQ ID NO: 14) was used in the first PCR, and a set of adapter01 (SEQ ID NO: 1) and adapter02 (SEQ ID NO: 2) in the second PCR.
  • PCR was performed using a primer set (GH20 / GH21) for amplifying the human ⁇ -globin gene region.
  • the PCR composition was the same as in Example 1 except for the amount of primer.
  • the primer sequences used were GH20: GAAGAGCCAAGGACAGGTAC (SEQ ID NO: 34) and GH21: GGAAAATAGACCAATAGGCAG (SEQ ID NO: 35).
  • the PCR program was “94 ° C for 5 minutes, (98 ° C for 10 seconds, 58 ° C for 30 seconds, 72 ° C for 30 seconds) ⁇ 30 cycles”.
  • HPV genomic DNA could be detected from sperm by the method described in Example 1.
  • Example 3 DNA extraction and detection from urine sample 200 ⁇ l of a 3M sodium acetate solution (Nippon Gene), 3 ml of ethanol (Fuji Film), and 50 ⁇ l of a 20 mg / ml glycogen solution (Nacalai Tesque) were added to 1 ml of the urine sample, and the mixture was overturned 10 times. Next, after incubation at minus 20 ° C for 16 hours, centrifugation was performed at 16,000 xg for 30 minutes at 4 ° C. The supernatant was discarded without sucking the pellet, and 4 ml of 80% ethanol was added. Thereafter, centrifugation was performed at 16,000 ⁇ g for 30 minutes at 4 ° C. The supernatant was discarded without sucking the pellet, and the pellet was subjected to NucleoSpin Tissue (registered trademark) Tissue (Takara Bio), and DNA was extracted according to the instruction manual.
  • NucleoSpin Tissue registered trademark
  • FIG. 6 An agarose gel electrophoresis photograph of the amplified DNA is shown in FIG. In FIG. 6, electrophoretic images of marker samples 1 to 20 are shown from the left. Nos. 2 and 9 were positive, others were negative.
  • Example 3 it was possible to detect HPV genomic DNA from urine by the method described in Example 1, using 1 ⁇ l of DNA extracted from urine.
  • the L1 protein coding region of HPV type 6, 18 was used as a DNA template. It was adjusted to 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 copy / ⁇ L. PCR was performed with EmeraldAmp® MAX PCR Master Mix. Thereafter, electrophoresis was performed on a 2.5% agarose gel (+ 0.002% MidoriGreen Advance).
  • composition of the 1st PCR reaction solution was as follows. 2 ⁇ M.Mix 10.00 ⁇ l ddH 2 O 5.00 ⁇ l MY11 (20 ⁇ M) 0.25 ⁇ l MY09 (20 ⁇ M) 0.25 ⁇ l 1.00 ⁇ l mold Total 20.00 ⁇ l
  • the conditions for ⁇ 1st ⁇ PCR were "94 ° C. for 5 minutes, ⁇ (98 ° C. for 10 seconds, ⁇ 45 ° C. for 30 seconds, ⁇ 72 ° C. for 30 seconds) ⁇ 40 cycles”.
  • FIG. 7 shows the results obtained when the commonly used MY11 and MY09 primer sets were used.
  • FIG. 7A shows the result of HPV Type 16 detection
  • FIG. 7B shows the result of HPV Type 18 detection.
  • the commonly used MY11 and MY09 primer sets were detectable from 10 copies / tube for HPV type 16 and 10 copies / tube for type 18.
  • HPV in the sample is present at 10 copies / mL, 10 copies / tube required for 1st PCR can be obtained. Therefore, according to the method of the present invention, if HPV in a sample is present at 10 copies / mL, it can be detected.
  • the second PCR was performed using 1 ⁇ l of the ⁇ 1st ⁇ PCR product as a template.
  • Adapters 13 to 24 were used as primers for 2nd PCR.
  • the sequences of the primers used are shown in FIG. 10 and SEQ ID NOs: 36 to 47.
  • composition of the 1st PCR reaction solution was as follows. 2 ⁇ M.Mix 10.00 ⁇ l ddH 2 O 8.50 ⁇ l primer F (20 ⁇ M) 0.25 ⁇ l primer R (20 ⁇ M) 0.25 ⁇ l 1.00 ⁇ l mold Total 20.00 ⁇ l
  • the conditions for ⁇ 1st ⁇ PCR were "94 ° C. for 5 minutes, ⁇ (98 ° C. for 10 seconds, ⁇ 45 ° C. for 30 seconds, ⁇ 72 ° C. for 30 seconds) ⁇ 30 cycles”.
  • composition of the reaction solution for 2nd PCR was as follows. 2 ⁇ M.Mix 10.00 ⁇ l ddH 2 O 8.50 ⁇ l primer F (20 ⁇ M) 0.25 ⁇ l primer R (20 ⁇ M) 0.25 ⁇ l 1st PCR product 1.00 ⁇ l Total 20.00 ⁇ l
  • the condition of the ⁇ 2nd ⁇ PCR was "94 ° C. for 5 minutes, ⁇ (98 ° C. for 10 seconds, ⁇ 45 ° C. for 30 seconds, ⁇ 72 ° C. for 30 seconds) ⁇ 30 cycles”.
  • FIG. 8 shows the detection result of Type 16, and FIG. 9 shows the detection result of Type 18.
  • FIG. 8A shows the result when adapter13-16_L1_F1 and adapter14-16_L1_R1 were used as the first primer (adapter primer) set, and adapter13 and adapter14 were used as the second primer set.
  • FIG. 8B shows the result when adapter15 was used as the first primer (adapter primer) set.
  • FIG. 8C shows 1st primer (adapter primer) set, adapter17-16_L1_F3 and adapter18-16_L1_R3 as 2nd primer set.
  • FIG. 9A shows the result when adapter19-18_L1_F1 and adapter20-18_L1_R1 were used as the first primer (adapter primer) set, and adapter19 and adapter20 were used as the second primer set, and FIG. 9B was the first primer (adapter primer) set.
  • FIG. 8C shows adapter23-18_L1_F3 and adapter24-18_L1_R3 as the first primer (adapter primer) set, and the second primer.
  • the results when adapter23 and adapter24 are used as a set are shown.
  • Adapter13-24 is a sequence obtained from the Arabidopsis AT2G22770.1 sequence.
  • FIG. 10 shows the sequence of a newly developed HPV type-specific primer.
  • the method of the present invention can be used for highly sensitive detection of HPV.
  • SEQ ID NO: 1 Primer (adaptor01) SEQ ID NO: 2: Primer (adaptor02) SEQ ID NO: 3: Primer (adaptor03) SEQ ID NO: 4: Primer (adaptor04) SEQ ID NO: 5: primer (adaptor05) SEQ ID NO: 6: primer (adaptor06) SEQ ID NO: 7: primer (adaptor07) SEQ ID NO: 8: primer (adaptor08) SEQ ID NO: 9: primer (adaptor09) SEQ ID NO: 10: primer (adaptor10) SEQ ID NO: 11: primer (adaptor11) SEQ ID NO: 12: primer (adaptor12) Sequence number 13: Primer (adaptor01-GP5 +) Sequence number 14: Primer (adaptor02-GP6 +) SEQ ID NO: 15: Primer (adaptor03-MY11) SEQ ID NO: 16: primer (adaptor04-MY09) SEQ ID NO: 17: Primer (adaptor05-GP5 +) SEQ ID NO: 18: primer (adaptor

Abstract

The purpose of the present invention is to provide a method for detecting HPV with high sensitivity in a subject, and particularly to provide a method for detecting HPV in semen or urine. A method for amplifying HPV in a subject by PCR reaction and detecting HPV infection, the method for detecting HPV infection including: (a) in a first PCR, performing amplification through use of an adapter-primer pair in which an adapter comprising a sequence which does not anneal with the HPV genome, a human genome, and the genome of a virus or microbe that infects a human is appended to a primer for hybridizing specifically with an HPV gene; and, (b) in a second PCR, amplifying the amplification product of (a) through use of a primer pair comprising a sequence that is complementary to the adapter.

Description

ヒトパピローマウイルスの検出方法Human papillomavirus detection method
　本発明は、検体中のヒトパピローマウイルスを高感度で検出する方法に関する。 (4) The present invention relates to a method for detecting human papillomavirus in a sample with high sensitivity.
　子宮頸がんは罹患率の高い悪性腫瘍であり、発がん過程にヒトパピローマウイルス（HPV)が関与し、HPVの感染は子宮頸がんの進行に対する危険因子である。HPVは２本鎖DNAを有するウイルスであり、ヒトの皮膚、粘膜等の上皮に感染する。HPVには多数の型（サブタイプ）が存在し、HPV6型、HPV16型、HPV18型等の型の感染により悪性の子宮頸がんに罹患するおそれがあることが報告されている。 Cervical cancer is a highly prevalent malignancy, involving human papillomavirus (HPV) in the carcinogenesis process, and HPV infection is a risk factor for cervical cancer progression. HPV is a virus having double-stranded DNA and infects epithelia such as human skin and mucous membrane. There are many types (subtypes) of HPV, and it has been reported that infection of types such as HPV6, HPV16, and HPV18 may cause malignant cervical cancer.
　従来より、女性の子宮頸部から採取した細胞懸濁液を検体として、PCR法によりHPVの感染が検出されていた。 Conventionally, HPV infection has been detected by PCR using a cell suspension collected from the cervix of a woman as a specimen.
　PCR法によるHPVの検出のために多数のプライマーが開発されていた（非特許文献１～３を参照）。 多数 Numerous primers have been developed for detection of HPV by PCR (see Non-Patent Documents 1 to 3).
　従来の方法では、検体中に105～107コピー/ｍLのHPVが存在する必要があり、検体中に十分な数のHPVを含ませるためには、子宮頸部や頸管部を強くこする必要があり、苦痛が大きかった。 Conventional methods require the presence of 10 5 to 10 7 copies / mL of HPV in the sample, and rub the cervix and cervix strongly to include a sufficient number of HPV in the sample Needed and pain was great.
　また、男性もHPVに感染し、がんを発症することは稀であるが、性交渉によりセックスパートナーにHPVを感染させてしまうことがあった。HPVは精液中に存在しており、精液を検体として用いて、男性のHPV感染を測定することも可能であるが、精液中のHPVのコピー数は少ないので、従来のPCR法では、高感度で検出することはできなかった。
　そのため、従来は男性を対象にHPV感染の検出は行われていなかった。 Men are also rarely infected with HPV and develop cancer, but sexual intercourse can infect sex partners with HPV. HPV is present in semen, and it is possible to measure male HPV infection using semen as a sample, but since the copy number of HPV in semen is small, conventional PCR methods have high sensitivity. Could not be detected.
For this reason, HPV infection has not been detected in men.
　本発明は、高感度で検体中のHPVを検出する方法の提供、特に精液又は尿中のHPVを検出する方法の提供を目的とする。 (4) An object of the present invention is to provide a method for detecting HPV in a sample with high sensitivity, and in particular, to provide a method for detecting HPV in semen or urine.
　本発明者らは、男性のHPV感染を高感度で測定し、かつ女性のHPV感染を苦痛を与えずに測定する方法について鋭意検討を行った。 (4) The present inventors have intensively studied a method for measuring HPV infection in men with high sensitivity and measuring HPV infection in women without causing pain.
　その結果、HPV遺伝子に特異的にハイブリダイズするプライマーに、HPVゲノム、ヒトゲノム、ヒトに感染するウイルスや微生物のゲノムにアニーリングしない配列からなるアダプターを付加したアダプタープライマーを用いた第１のPCR、及びアダプターに相補的な配列からなるプライマーを用いた第２のPCRの２段階のPCRを行うことにより、検体中のHPVのコピー数が少なくても高感度でHPVを検出し得ることを見出し、本発明を完成させるに至った。 As a result, the first PCR using an adapter primer to which an adapter consisting of a sequence that does not anneal to the HPV genome, the human genome, and the genome of a virus or microorganism that infects humans is added to a primer that specifically hybridizes to the HPV gene, and It was found that HPV can be detected with high sensitivity even if the copy number of HPV in the sample is small by performing two-step PCR of the second PCR using a primer consisting of a sequence complementary to the adapter. The invention has been completed.
　すなわち、本発明は以下のとおりである。
[１]　検体中のHPVをPCR反応により増幅してHPVの感染を検出する方法であって、
(a)　第１のPCRにおいて、HPV遺伝子に特異的にハイブリダイズするプライマーに、HPVゲノム、ヒトゲノム、並びにヒトに感染するウイルス及び微生物のゲノムにアニーリングしない配列からなるアダプターを付加したアダプタープライマーの対により増幅し、
(b)　第２のPCRにおいて、(a)の増幅産物を前記アダプターに相補的な配列からなるプライマーの対により増幅することを含む、
HPVの感染を検出する方法。
[２]　検体中のHPVのコピー数が1000コピー/ｍL以下である、[１]の方法。
[３]　検体中のHPVのコピー数が100コピー/ｍL以下である、[１]の方法。
[４]　検体中のHPVのコピー数が10コピー/ｍL以下である、[１]の方法。
[５]　検体が精液又は尿である、[１]～[４]のいずれかの方法。
[６]　HPV遺伝子に特異的にハイブリダイズするプライマーがHPVのL1遺伝子に特異的にハイブリダイズするプライマーである、[１]～[５]のいずれかの方法。
[７]　アダプターが、シロイヌナズナの遺伝子の部分配列からなる、[１]～[６]のいずれかの方法。
[８]　アダプタープライマー対とプライマー対の組合せが以下の(i)～(vi)のいずれかの組合せである、[１]～[７]のいずれかの方法：
(i)　配列番号１３及び１４の配列からなるアダプタープライマー対、並びに配列番号１及び２の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(ii)　配列番号１５及び１６の配列からなるアダプタープライマー対、並びに配列番号３及び４の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(iii)　配列番号１７及び１８の配列からなるアダプタープライマー対、並びに配列番号５及び６の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(iv)　配列番号１９及び２０の配列からなるアダプタープライマー対、並びに配列番号７及び８の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(v)　配列番号２１及び２２の配列からなるアダプタープライマー対、並びに配列番号９及び１０の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(vi)　配列番号２３及び２４の配列からなるアダプタープライマー対、並びに配列番号１１及び１２の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ。
[９]　アダプタープライマー対とプライマー対の組合せが以下の(xi)～(xvi)のいずれかの組合せである、[１]～[７]のいずれかに記載の方法：
(xi)　配列番号４８及び４９の配列からなるアダプタープライマー対、並びに配列番号３６及び３７の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xii)　配列番号５０及び５１の配列からなるアダプタープライマー対、並びに配列番号３８及び３９の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xiii)　配列番号５２及び５３の配列からなるアダプタープライマー対、並びに配列番号４０及び４１の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xiv)　配列番号５４及び５５の配列からなるアダプタープライマー対、並びに配列番号４２及び４３の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xv)　配列番号５６及び５７の配列からなるアダプタープライマー対、並びに配列番号４４及び４５の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xvi)　配列番号５８及び５９の配列からなるアダプタープライマー対、並びに配列番号４６及び４７の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ。
[１０]　(a)　HPV遺伝子に特異的にハイブリダイズするプライマーに、HPVゲノム、ヒトゲノム、並びにヒトに感染するウイルス及び微生物のゲノムにアニーリングしない配列からなるアダプターを付加したアダプタープライマーの対;と
(b)　前記アダプターに相補的な配列からなるプライマーの対を含む、
検体中のHPVをPCR反応により増幅してHPVの感染を検出するためのキット。
[１１]　検体中のHPVのコピー数が1000コピー/ｍL以下である、[１０]のキット。
[１２]　検体中のHPVのコピー数が100コピー/ｍL以下である、[１０]のキット。
[１３]　検体中のHPVのコピー数が10コピー/ｍL以下である、[１０]のキット。
[１４]　検体が精液又は尿である、[１０]～[１３]のいずれかのキット。
[１５]　HPV遺伝子に特異的にハイブリダイズするプライマーがHPVのL1遺伝子に特異的にハイブリダイズするプライマーである、[１０]～[１４]のいずれかのキット。
[１６]　アダプターが、シロイヌナズナの遺伝子の部分配列からなる、[１０]～[１５]のいずれかのキット。
[１７]　アダプタープライマー対とプライマー対の組合せが以下の(i)～(vi)のいずれかの組合せである、[１０]～[１６]のいずれかのキット：
(i)　配列番号１３及び１４の配列からなるアダプタープライマー対、並びに配列番号１及び２の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(ii)　配列番号１５及び１６の配列からなるアダプタープライマー対、並びに配列番号３及び４の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(iii)　配列番号１７及び１８の配列からなるアダプタープライマー対、並びに配列番号５及び６の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(iv)　配列番号１９及び２０の配列からなるアダプタープライマー対、並びに配列番号７及び８の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(v)　配列番号２１及び２２の配列からなるアダプタープライマー対、並びに配列番号９及び１０の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(vi)　配列番号２３及び２４の配列からなるアダプタープライマー対、並びに配列番号１１及び１２の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ。
[１８]　アダプタープライマー対とプライマー対の組合せが以下の(xi)～(xvi)のいずれかの組合せである、[１０]～[１６]のいずれかのキット：
(xi)　配列番号４８及び４９の配列からなるアダプタープライマー対、並びに配列番号３６及び３７の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xii)　配列番号５０及び５１の配列からなるアダプタープライマー対、並びに配列番号３８及び３９の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xiii)　配列番号５２及び５３の配列からなるアダプタープライマー対、並びに配列番号４０及び４１の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xiv)　配列番号５４及び５５の配列からなるアダプタープライマー対、並びに配列番号４２及び４３の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xv)　配列番号５６及び５７の配列からなるアダプタープライマー対、並びに配列番号４４及び４５の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
(xvi)　配列番号５８及び５９の配列からなるアダプタープライマー対、並びに配列番号４６及び４７の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ。
　本明細書は本願の優先権の基礎となる日本国特許出願番号2018-167939号の開示内容を包含する。 That is, the present invention is as follows.
[1] A method for detecting HPV infection by amplifying HPV in a sample by a PCR reaction,
(a) In the first PCR, a primer that specifically hybridizes to the HPV gene, and an adapter primer pair comprising an adapter consisting of a sequence that does not anneal to the HPV genome, the human genome, and the genomes of viruses and microorganisms that infect humans. Amplified by
(b) in the second PCR, amplifying the amplification product of (a) with a pair of primers having a sequence complementary to the adapter,
How to detect HPV infection.
[2] The method of [1], wherein the copy number of HPV in the sample is 1000 copies / mL or less.
[3] The method of [1], wherein the copy number of HPV in the sample is 100 copies / mL or less.
[4] The method of [1], wherein the copy number of HPV in the sample is 10 copies / mL or less.
[5] The method according to any one of [1] to [4], wherein the specimen is semen or urine.
[6] The method according to any one of [1] to [5], wherein the primer that specifically hybridizes to the HPV gene is a primer that specifically hybridizes to the HPV L1 gene.
[7] The method of any of [1] to [6], wherein the adapter comprises a partial sequence of an Arabidopsis thaliana gene.
[8] The method according to any one of [1] to [7], wherein the combination of the adapter primer pair and the primer pair is any combination of the following (i) to (vi):
(i) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 13 and 14, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 1 and 2;
(ii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 15 and 16 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 3 and 4;
(iii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 17 and 18, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 5 and 6;
(iv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 19 and 20, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 7 and 8;
(v) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 21 and 22, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 9 and 10;
(vi) A combination of an adapter primer pair consisting of the sequences of SEQ ID NOS: 23 and 24 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 11 and 12.
[9] The method according to any of [1] to [7], wherein the combination of the adapter primer pair and the primer pair is any combination of the following (xi) to (xvi):
(xi) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 48 and 49 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 36 and 37;
(xii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 50 and 51 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 38 and 39;
(xiii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 52 and 53 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 40 and 41;
(xiv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 54 and 55 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 42 and 43;
(xv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 56 and 57 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 44 and 45;
(xvi) A combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 58 and 59 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 46 and 47.
[10] (a) A pair of an adapter primer which is obtained by adding an adapter comprising a sequence which does not anneal to the HPV genome, the human genome, and the genomes of viruses and microorganisms infecting humans, to a primer specifically hybridizing to the HPV gene;
(b) including a primer pair consisting of a sequence complementary to the adapter,
A kit for detecting HPV infection by amplifying HPV in a sample by PCR.
[11] The kit according to [10], wherein the copy number of HPV in the sample is 1000 copies / mL or less.
[12] The kit according to [10], wherein the copy number of HPV in the sample is 100 copies / mL or less.
[13] The kit of [10], wherein the copy number of HPV in the sample is 10 copies / mL or less.
[14] The kit of any of [10] to [13], wherein the specimen is semen or urine.
[15] The kit according to any of [10] to [14], wherein the primer specifically hybridizing to the HPV gene is a primer specifically hybridizing to the HPV L1 gene.
[16] The kit of any of [10] to [15], wherein the adapter comprises a partial sequence of an Arabidopsis thaliana gene.
[17] The kit according to any one of [10] to [16], wherein the combination of the adapter primer pair and the primer pair is any combination of the following (i) to (vi):
(i) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 13 and 14, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 1 and 2;
(ii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 15 and 16 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 3 and 4;
(iii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 17 and 18, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 5 and 6;
(iv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 19 and 20, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 7 and 8;
(v) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 21 and 22, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 9 and 10;
(vi) A combination of an adapter primer pair consisting of the sequences of SEQ ID NOS: 23 and 24 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 11 and 12.
[18] The kit according to any one of [10] to [16], wherein the combination of the adapter primer pair and the primer pair is any combination of the following (xi) to (xvi):
(xi) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 48 and 49 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 36 and 37;
(xii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 50 and 51 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 38 and 39;
(xiii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 52 and 53 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 40 and 41;
(xiv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 54 and 55 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 42 and 43;
(xv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 56 and 57 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 44 and 45;
(xvi) A combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 58 and 59 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 46 and 47.
This description includes the disclosure of Japanese Patent Application No. 2018-167939, which is a priority document of the present application.
　本発明のHPV遺伝子に特異的にハイブリダイズするプライマーにアダプターを付加したアダプタープライマーを用いた第１のPCR、及びアダプターに相補的な配列からなるプライマーを用いた第２のPCRの２段階のPCRにより、検体中のHPVのコピー数が10コピー/ｍL程度しか存在しない場合でも検出することが可能である。本発明の方法は、HPVのコピー数が少ない精液や子宮頸部から採取した細胞懸濁液や尿を検体として用いた場合でも高感度でHPVを検出することができる。また、本発明により従来行われていなかった、男性のHPV感染の検出を行うことができる。 Two-stage PCR of a first PCR using an adapter primer obtained by adding an adapter to a primer specifically hybridizing to the HPV gene of the present invention, and a second PCR using a primer having a sequence complementary to the adapter Thereby, it is possible to detect even when the copy number of HPV in the sample is only about 10 copies / mL. The method of the present invention can detect HPV with high sensitivity even when using semen having a low copy number of HPV, cell suspension or urine collected from the cervix as a specimen. Further, it is possible to detect male HPV infection, which has not been conventionally performed by the present invention.
本発明で用いたプライマーの配列を示す図である。FIG. 3 is a view showing a sequence of a primer used in the present invention. 汎用プライマーセット（GP5+とGP6+とのプライマーセット又はMY11とMY09とのプライマーセット）を用いてPCR増幅した結果を示す図である。It is a figure which shows the result of PCR amplification using the general-purpose primer set (primer set of GP5 + and GP6 + or primer set of MY11 and MY09). 1st PCRにアダプタープライマーセット（adaptor01-GP5+とadaptor02-GP6+とのプライマーセット又はadaptor03-MY11とadaptor04-MY09とのプライマーセット）を用いてPCR増幅した結果を示す図である。FIG. 3 is a diagram showing the results of PCR amplification using an adapter primer set (a primer set of adaptor01-GP5 + and adaptor02-GP6 + or a primer set of adaptor03-MY11 and adaptor04-MY09) in 1st PCR. アダプタープライマーセットとして、adaptor05-GP5+とadaptor06-GP6+とのプライマーセット（図４左）又はadaptor07-GP5+とadaptor08-GP6+とのプライマーセット（図４右）を用いてPCR増幅した結果を示す図である。It is a figure which shows the result of having carried out PCR amplification using the primer set of adaptor05-GP5 + and adaptor06-GP6 + (FIG. 4 left) or the adapter set of adaptor07-GP5 + and adaptor08-GP6 + (FIG. 4 right) as an adapter primer set. . アダプタープライマーセットとして、adaptor09-MY11とadaptor010-MY09とのプライマーセット（図５左）、又はadaptor011-MY11とadaptor012-MY09とのプライマーセット（図５右）を用いてPCR増幅した結果を示す図である。FIG. 7 is a diagram showing the results of PCR amplification using a primer set of adaptor09-MY11 and adaptor010-MY09 (left of FIG. 5) or a primer set of adaptor011-MY11 and adaptor012-MY09 (right of FIG. 5) as an adapter primer set. is there. 尿検体を用いたHPVゲノムDNAの検出の結果を示すアガロースゲル電気泳動像を示す図である。FIG. 4 is a diagram showing an agarose gel electrophoresis image showing the results of detection of HPV genomic DNA using a urine sample. 汎用されるMY11及びMY09プライマーセットを用いたときのHPV Type 16の検出の結果（図７Ａ）及び、HPV Type 18の検出の結果（図７Ｂ）を示す。The results of the detection of HPV Type 16 (FIG. 7A) and the results of the detection of HPV Type 18 (FIG. 7B) using the commonly used MY11 and MY09 primer sets are shown. 本発明で用いたHPVの型特異的プライマーを用いたHPV Type 16の検出結果を示す図である。FIG. 3 is a view showing the results of HPVHPType 16 detection using the HPV type-specific primers used in the present invention. 本発明で用いたHPVの型特異的プライマーを用いたHPV Type 18の検出結果を示す図である。FIG. 3 is a view showing the results of HPVHPType 18 detection using the HPV type-specific primers used in the present invention. 本発明で用いたHPVの型特異的プライマーの配列を示す図である（その２）。FIG. 2 is a view showing a sequence of an HPV type-specific primer used in the present invention (part 2).
　以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
　本発明は、ヒトパピローマウイルス（HPV)の遺伝子をPCRにより増幅して検出する方法であり、本発明の方法により高感度でHPVを検出することができる。すなわち、検体中にHPVが1000コピー/ｍL以下、好ましくは100コピー/ｍL以下、さらに好ましくは10コピー/ｍL以下あるいは10コピー/ｍL未満しか存在しない場合でも検出することができる。 The present invention is a method for amplifying and detecting the gene of human papilloma virus (HPV) by PCR, and it is possible to detect HPV with high sensitivity by the method of the present invention. That is, it can be detected even when HPV is present in the sample at 1000 copies / mL or less, preferably 100 copies / mL or less, more preferably 10 copies / mL or less, or less than 10 copies / mL.
　このため、HPVの存在量が少ない男性の精液を検体として用いた場合も検体中のHPVを検出することができる。男性の精液中にHPVが存在する場合、感染による症状が現れることは稀であるが、性交渉によりセックスパートナーに感染してしまうので、精液中のHPVの検出は未然に他者への感染を防ぐという意義がある。また、女性のHPVに感染しているか否かの検査は、綿棒（スワブ）やプラスチック製のブラシ等で子宮頸部や頸管部をこすり、子宮頸部の細胞を採取し、採取した細胞懸濁液中にHPVが存在するかをPCRにより測定する。このとき、HPVを検出するためには、検体中に105～107コピー/ｍLのHPVが存在する必要があり、検体中に十分な数のHPVを含ませるためには、子宮頸部や頸管部を強くこする必要があり、苦痛が大きかった。 For this reason, HPV in a specimen can be detected even when a male semen having a small amount of HPV is used as the specimen. If HPV is present in male semen, symptoms due to infection are rarely manifested, but since sex partners are transmitted by sexual intercourse, detection of HPV in semen will prevent infection to others beforehand. It is significant to prevent. In addition, women can be tested for HPV infection by rubbing the cervix or cervix with a swab or plastic brush, collecting cervical cells, and collecting the cell suspension. The presence of HPV in the solution is measured by PCR. At this time, in order to detect HPV, it is necessary that 10 5 to 10 7 copies / mL of HPV be present in the sample, and in order to include a sufficient number of HPV in the sample, the cervix and She had to rub the neck strongly, which was painful.
　さらに、本発明においては、男性又は女性の尿を検体として用いた場合も検体中のHPVを検出することができる。 Furthermore, in the present invention, HPV in a specimen can be detected even when male or female urine is used as the specimen.
　本発明において、HPVの遺伝子増幅用アダプタープライマーを用いたアダプターPCRによりHPVの遺伝子を増幅し、HPVを検出する。 に お い て In the present invention, the HPV gene is amplified by adapter PCR using an adapter primer for HPV gene amplification, and the HPV is detected.
　本発明の方法において検体としては、男性被験体から採取した精液、女性被験体の子宮頸部から採取した細胞懸濁液、男性被験体又は女性被験体から採取した尿を用いることができる。また、男性被験体又は女性被験体から採取した唾液、組織液、髄液、拭い液、血清、血漿、血液、便等も用いることができる。検体量は精液または尿の場合、５μL～10ｍL、好ましくは10μ～１ｍLを用いれば、例えば、精液の場合、５μL～100μL、好ましくは10μL～50μL、尿の場合、200μL～10mL、好ましくは200μL～５mL、さらに好ましくは500μL～１mLを用いれば、検体中のHPV遺伝子を増幅することができる。 検 体 In the method of the present invention, as the specimen, semen collected from a male subject, a cell suspension collected from the cervix of a female subject, and urine collected from a male or female subject can be used. Also, saliva, tissue fluid, cerebrospinal fluid, swabs, serum, plasma, blood, stool, etc. collected from a male or female subject can be used. The sample volume is 5 μL to 10 mL, preferably 10 μL to 1 mL for semen or urine, for example, 5 μL to 100 μL, preferably 10 μL to 50 μL for semen, and 200 μL to 10 mL, preferably 200 μL for urine. If 5 mL, more preferably 500 μL to 1 mL, is used, the HPV gene in the sample can be amplified.
　HPVは約7.9bpの環状２本鎖DNAを有するウイルスであり、ヒトの粘膜や皮膚に感染する。HPVのDNAは、遺伝子発現を調節する領域であるURR（upstream regulatory region）、ウイルスの複製に関与する非構造タンパク質をコードする初期遺伝子群であるE1、E2、E4、E5、E6及びE7の６つの遺伝子、ウイルスキャプシドをコードする後期遺伝子群であるL1及びL2の２つの遺伝子を含む。L1及びL2はウイルス粒子を構成するキャプシドタンパク質であり、宿主細胞への吸着及び侵入に関与する。 HPV is a virus having a circular double-stranded DNA of about 7.9 bp and infects human mucosa and skin. The HPV DNA consists of URR (upstream regulatory region), a region that regulates gene expression, and E1, E2, E4, E5, E6, and E7, which are early genes that encode nonstructural proteins involved in viral replication. And two genes, L1 and L2, which are late genes that encode the viral capsid. L1 and L2 are capsid proteins that constitute virus particles, and are involved in adsorption and entry into host cells.
　本発明の方法で、検出し得るHPVの型として、6b型、16型、18型、31型、33型、35型、39型、45型、51型、52型、53型、56型、58型、59型、67型、68型、69型、70型、73型、82型が挙げられ、この中でも感染により将来的に悪性の子宮頸がんに罹患する可能性が高い6b型、16型、18型の他、日本人において、悪性の子宮頸がんに罹患する可能性が高い53型が好ましい。 In the method of the present invention, HPV types that can be detected include 6b type, 16 type, 18 type, 31 type, 33 type, 35 type, 39 type, 45 type, 51 type, 52 type, 53 type, 56 type, Types 58, 59, 67, 68, 69, 70, 73, and 82 are among them, and among them, type 6b, which is likely to be infected with malignant cervical cancer in the future, In addition to types 16 and 18, type 53, which is highly likely to cause malignant cervical cancer in Japanese, is preferable.
　HPV 16型のL1遺伝子の塩基配列及びHPV 6b型のL1遺伝子の塩基配列を、それぞれ、配列番号３２及び３３に示す。これらの配列情報あるいは公知のHPVの遺伝子配列情報に基づいてプライマーの配列を設計することができる。 The nucleotide sequences of the HPV 16 L1 gene and the HPV 6b L1 gene are shown in SEQ ID NOs: 32 and 33, respectively. A primer sequence can be designed based on these sequence information or known HPV gene sequence information.
　本発明の方法によりHPV遺伝子を増幅させるためには、HPVの遺伝子に特異的にハイブリダイズするプライマーの5'側にアダプター配列を付加したフォワードプライマーとリバースプライマーのプライマー対を用いて第１の増幅（1st PCR)を行い、次いで増幅産物に対してアダプター配列にハイブリダイズするフォワードプライマーとリバースプライマーのプライマー対を用いて第２の増幅（2nd PCR)を行えばよい。HPVの遺伝子に特異的にハイブリダイズするプライマーの5'側にアダプター配列を付加したプライマーは、HPVの領域特異的配列プライマーとアダプターからなり、アダプタープライマーと呼ぶ。 In order to amplify the HPV gene by the method of the present invention, the first amplification using a primer pair of a forward primer and a reverse primer to which an adapter sequence is added to the 5 ′ side of a primer that specifically hybridizes to the HPV gene is performed. (1st PCR) may be performed, and then a second amplification (2nd PCR) may be performed using a primer pair of a forward primer and a reverse primer that hybridize to the adapter sequence with respect to the amplification product. A primer that has an adapter sequence added to the 5 ′ side of a primer that specifically hybridizes to an HPV gene is composed of an HPV region-specific sequence primer and an adapter, and is called an adapter primer.
　HPVの遺伝子に特異的にハイブリダイズするプライマーの塩基長は、5～50塩基、好ましくは10～40塩基、さらに好ましくは15～35塩基、さらに好ましくは20～30塩基である。 プ ラ イ マ ー The base length of the primer that specifically hybridizes to the HPV gene is 5 to 50 bases, preferably 10 to 40 bases, more preferably 15 to 35 bases, and still more preferably 20 to 30 bases.
　HPVの遺伝子に特異的にハイブリダイズするプライマー対は、HPV遺伝子の40～1000塩基、好ましくは200～300塩基の長さの領域を増幅できるように設計すればよい。すなわち、HPV遺伝子の40～1000塩基、好ましくは100～500塩基、さらに好ましくは150～450塩基の領域の3'側と5'側の両端にハイブリダイズするように設定すればよい。このようなプライマー対を用いることにより、40～1000塩基、好ましくは100～500塩基、さらに好ましくは150～450塩基の長さの増幅産物が得られる。HPVの遺伝子にハイブリダイズするプライマーは各型のHPV遺伝子の塩基配列の連続した塩基配列において、数個、好ましくは1～5個、さらに好ましくは１～４個、さらに好ましくは１～３個、さらに好ましくは１個又は２個、さらに好ましくは１個のミスマッチを有していてもよい。この結果、１セットのプライマー対で特定の型のHPVだけではなく、多数の型のHPV遺伝子を増幅することができ、多数の型のHPVを検出することができる。 (4) A primer pair that specifically hybridizes to the HPV gene may be designed so as to amplify a region having a length of 40 to 1000 bases, preferably 200 to 300 bases, of the HPV gene. That is, it may be set so as to hybridize to the 3′-end and the 5′-end of the region of 40 to 1000 bases, preferably 100 to 500 bases, more preferably 150 to 450 bases of the HPV gene. By using such a primer pair, an amplification product having a length of 40 to 1000 bases, preferably 100 to 500 bases, more preferably 150 to 450 bases is obtained. Primers that hybridize to the HPV gene have several, preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, More preferably, it may have one or two, more preferably one mismatch. As a result, not only a specific type of HPV but also a large number of types of HPV genes can be amplified with one set of primer pairs, and a large number of types of HPV can be detected.
　HPVのL1はHPVの多数の型（サブタイプ）において、保存された領域であり、各型間における配列同一性が高い。本発明の方法においては、好ましくはL1をコードするDNAとハイブリダイズするプライマー対を用いてL1をコードするDNAを増幅すればよい。 (4) L1 of HPV is a conserved region in many types (subtypes) of HPV, and the sequence identity between each type is high. In the method of the present invention, the L1-encoding DNA is preferably amplified using a primer pair that hybridizes with the L1-encoding DNA.
　ただし、HPVの各型の間で100％の配列同一性で20塩基程度連続する配列は存在しない。そこで、上記のようにHPVの型によっては、プライマーの配列とHPVの遺伝子の配列の間にミスマッチがあってもよい。この場合、例えば、アニーリング温度を下げ、特異性を低下させればよい。また、複数のプライマー対を用いるmultiplex PCRにより増幅してもよい。 However, there is no sequence that is continuous for about 20 bases with 100% sequence identity between each type of HPV. Thus, as described above, depending on the type of HPV, there may be a mismatch between the sequence of the primer and the sequence of the HPV gene. In this case, for example, the annealing temperature may be lowered to lower the specificity. Alternatively, amplification may be performed by multiplex PCR using a plurality of primer pairs.
　HPVのL1遺伝子に特異的な配列として、例えば、公知のHPV検出の汎用プライマー対を用いることができる。このようなプライマー対として、フォワードプライマーGP5+（配列番号２５）とリバースプライマーGP6+（配列番号２６）のプライマー対、フォワードプライマーMY11（配列番号２７）とリバースプライマーMY09（配列番号２８）のプライマー対、フォワードプライマーL1C1（配列番号２９）とリバースプライマーL1C2（配列番号３０）若しくはリバースプライマーL1C2M（配列番号３１）等がある。これらのプライマーについては、例えば、Mori S et al., Cancer Sci, June 2011, vol.102, no.6, pp.1223-1227に記載されている。GP5+とGP6+の間の塩基長は142塩基であり、MY11とMY09の間の塩基長は452塩基であり、L1C1とL1C2若しくはL1C2Mの間の塩基長は253塩基である。 As a sequence specific to the HPV L1 gene, for example, a known general-purpose primer pair for HPV detection can be used. Such primer pairs include a primer pair of a forward primer GP5 + (SEQ ID NO: 25) and a reverse primer GP6 + (SEQ ID NO: 26), a primer pair of a forward primer MY11 (SEQ ID NO: 27) and a reverse primer MY09 (SEQ ID NO: 28), and a forward pair. There are primer L1C1 (SEQ ID NO: 29) and reverse primer L1C2 (SEQ ID NO: 30) or reverse primer L1C2M (SEQ ID NO: 31). These primers are described, for example, in Mori S et al., Cancer Sci, June 2011, vol. 102, no. 6, pp. 1223-1227. The base length between GP5 + and GP6 + is 142 bases, the base length between MY11 and MY09 is 452 bases, and the base length between L1C1 and L1C2 or L1C2M is 253 bases.
　アダプターの塩基長は、10～40塩基、好ましくは10～30塩基、さらに好ましくは15～20塩基である。 塩 基 The base length of the adapter is 10 to 40 bases, preferably 10 to 30 bases, and more preferably 15 to 20 bases.
　アダプターの配列はCG含量やAT含量が高いと高次構造をとりやすくなってしまうので、好ましくはGC含量が50～65％になるように設計する。また、両端が連結しないように設定する。また、Tm値は50～70℃、好ましくは55～65℃、さらに好ましくは60±1℃になるように設計すればよい。 (4) The adapter sequence is preferably designed to have a GC content of 50 to 65%, since a high-order structure is likely to be formed when the CG content or AT content is high. Also, set so that both ends are not connected. The Tm value may be designed to be 50 to 70 ° C., preferably 55 to 65 ° C., and more preferably 60 ± 1 ° C.
　アダプター配列は、検体に存在する可能性があるHPVゲノム、ヒトゲノム、ヒトに感染するウイルスや微生物のゲノム等のアニーリングしないように設計する。好ましくは、人工的に設計した配列とする。例えば、配列が解析されている植物やヒト以外の動物のゲノムの配列からフォワードプライマーとリバースプライマーの塩基配列を取得すればよい。人工的な配列や、植物やヒト以外の動物のゲノム配列から設計する場合、Primer3web（http://primer3.ut.ee/)等のPCRプライマー設計ツールを用いて設計することができる。 (4) The adapter sequence is designed so as not to anneal the HPV genome, human genome, and genomes of viruses and microorganisms that infect humans that may be present in the sample. Preferably, the sequence is artificially designed. For example, the base sequences of the forward primer and the reverse primer may be obtained from the sequence of the genome of a plant or a non-human animal whose sequence is being analyzed. When designing from an artificial sequence or the genome sequence of a plant or a non-human animal, it can be designed using a PCR primer design tool such as Primer3web (http://primer3.ut.ee/).
　例えば、高等植物であるイネ、シロイヌナズナ等の遺伝子のゲノム配列からアダプター配列を選択することができる。すなわち、例えば、アダプター配列はシロイヌナズナ遺伝子の配列の部分配列からなり得る。この際、検体中にシロイヌナズナゲノムが混入し増幅が起こるおそれを考慮し、対となるアダプター配列を、それぞれ、シロイヌナズナの異なる染色体の配列から設計することが好ましい。 For example, an adapter sequence can be selected from the genomic sequence of a higher plant such as rice or Arabidopsis thaliana. That is, for example, the adapter sequence can be a partial sequence of the sequence of the Arabidopsis thaliana gene. At this time, it is preferable to design a pair of adapter sequences from sequences of different chromosomes of Arabidopsis thaliana in consideration of the possibility that the Arabidopsis thaliana genome is mixed in the sample and amplification occurs.
　アダプターとして、例えば、配列番号１～１２に表す配列からなるadaptor01～adaptor12が挙げられ、adaptor01とadaptor02、adaptor03とadaptor04、adaptor05とadaptor06、adaptor07とadaptor08、adaptor09とadaptor10、adaptor11とadaptor12の組み合わせで、それぞれ、フォワードプライマー及びリバースプライマーに付加すればよい。配列番号１～１２に表す配列からなるアダプターは、シロイヌナズナ遺伝子の配列から設計したアダプターである。 Examples of the adapter include adaptor01 to adaptor12 consisting of the sequences represented by SEQ ID NOs: 1 to 12, adaptor01 and adaptor02, adaptor03 and adaptor04, adaptor05 and adaptor06, adaptor07 and adaptor08, adaptor09 and adaptor08, adaptor09 and adaptor10, and adaptor11 and adaptor12, respectively. , Forward primer and reverse primer. The adapter consisting of the sequences shown in SEQ ID NOs: 1 to 12 is an adapter designed from the sequence of the Arabidopsis thaliana gene.
　さらに、アダプターとして、例えば、それぞれ、配列番号３６～４７に表す配列からなるadaptor13～adaptor24が挙げられ、adaptor13とadaptor14、adaptor15とadaptor16、adaptor17とadaptor18、adaptor19とadaptor20、adaptor21とadaptor22、adaptor23とadaptor24の組み合わせで、それぞれ、フォワードプライマー及びリバースプライマーに付加すればよい。配列番号３６～４７に表す配列からなるアダプターは、シロイヌナズナ遺伝子の配列から設計したアダプターである。 Further, as adapters, for example, adapter13 to adapter24 consisting of the sequences represented by SEQ ID NOs: 36 to 47, respectively, are exemplified. The combination may be added to the forward primer and the reverse primer, respectively. The adapter consisting of the sequences shown in SEQ ID NOs: 36 to 47 is an adapter designed from the sequence of the Arabidopsis thaliana gene.
　HPVの遺伝子に特異的にハイブリダイズするプライマーへのアダプターの付加は、DNA鎖の末端同士をリン酸ジエステル結合で連結するライゲーションにより行えばよい。ライゲーションは、DNAリガーゼを用いて公知の方法で行うことができる。DNAリガーゼとしては、T4 DNAリガーゼ、大腸菌DNAリガーゼ等の常温性DNAリガーゼを用いることができる。また、耐熱性DNAリガーゼを用いてもよい。 (4) The addition of an adapter to a primer that specifically hybridizes to the HPV gene may be performed by ligation in which the ends of DNA strands are linked by a phosphodiester bond. Ligation can be performed by a known method using DNA ligase. As the DNA ligase, room-temperature DNA ligase such as T4-DNA ligase and E. coli DNA ligase can be used. Alternatively, thermostable DNA ligase may be used.
　HPVの遺伝子に特異的にハイブリダイズするプライマーとアダプターはリンカーを介して連結していてもよい。リンカーの配列はHPVのゲノム中の配列とは相補的にならないように設計する。リンカーの塩基長は、10塩基以下、好ましくは５塩基以下である。リンカーが存在する場合であっても、アダプタープライマーは、HPVの遺伝子に特異的にハイブリダイズするプライマーにアダプターが付加されたプライマーであるという。 プ ラ イ マ ー A primer and an adapter that specifically hybridize to the HPV gene may be linked via a linker. The linker sequence is designed not to be complementary to the sequence in the HPV genome. The base length of the linker is 10 bases or less, preferably 5 bases or less. Even when a linker is present, the adapter primer is a primer obtained by adding an adapter to a primer that specifically hybridizes to the HPV gene.
　本発明で用いるアダプタープライマーとして、例えば、配列番号１３～２４で表される塩基配列からなるアダプタープライマーが挙げられる。配列番号１３及び１４のアダプタープライマーは、GP5+にadaptor01を付加したフォワードプライマー（adaptor01-GP5+)とGP6+にadaptor02を付加したリバースプライマー(adaptor02-GP6+)の対であり、配列番号１５及び１６のアダプタープライマーは、MY11にadaptor03を付加したフォワードプライマー(adaptor03-MY11)とMY09にadaptor04を付加したリバースプライマー（adaptor04-MY09)の対であり、配列番号１７及び１８のアダプタープライマーは、GP5+にadaptor05を付加したフォワードプライマー（adaptor05-GP5+)とGP6+にadaptor06を付加したリバースプライマー(adaptor06-GP6+)の対であり、配列番号１９及び２０のアダプタープライマーは、MY11にadaptor07を付加したフォワードプライマー(adaptor07-MY11)とMY09にadaptor08を付加したリバースプライマー（adaptor08-MY09)の対であり、配列番号２１及び２２のアダプタープライマーは、GP5+にadaptor09を付加したフォワードプライマー（adaptor09-GP5+)とGP6+にadaptor10を付加したリバースプライマー(adaptor10-GP6+)の対であり、配列番号２３及び２４のアダプタープライマーは、MY11にadaptor11を付加したフォワードプライマー(adaptor11-MY11)とMY09にadaptor12を付加したリバースプライマー（adaptor12-MY09)の対である。 ア ダ プ タ ー The adapter primer used in the present invention includes, for example, an adapter primer consisting of the base sequences represented by SEQ ID NOS: 13 to 24. The adapter primers of SEQ ID NOs: 13 and 14 are a pair of a forward primer (adaptor01-GP5 +) in which adaptor01 is added to GP5 + and a reverse primer (adaptor02-GP6 +) in which adaptor02 is added to GP6 +. Is a pair of a forward primer (adaptor03-MY11) in which adaptor03 is added to MY11 and a reverse primer (adaptor04-MY09) in which adaptor04 is added to MY09, and the adapter primers of SEQ ID NOs: 17 and 18 have adapter5 added to GP5 +. A pair of a forward primer (adaptor05-GP5 +) and a reverse primer (adaptor06-GP6 +) obtained by adding an adaptor06 to GP6 +, and the adapter primers of SEQ ID NOs: 19 and 20 correspond to a forward primer (adaptor07-MY11) obtained by adding an adaptor07 to MY11. This is a pair of reverse primers (adaptor08-MY09) in which adaptor08 is added to MY09. Is a pair of a forward primer (adaptor09-GP5 +) in which adaptor09 is added to GP5 + and a reverse primer (adaptor10-GP6 +) in which adaptor10 is added to GP6 +, and the adapter primers of SEQ ID NOs: 23 and 24 are adaptor11 in MY11. A pair of a forward primer (adaptor11-MY11) to which is added and a reverse primer (adaptor12-MY09) to which adaptor12 is added to MY09.
　さらに、本発明で用いるアダプタープライマーとして、例えば、配列番号４８～５９で表される塩基配列からなるアダプタープライマーが挙げられる。こ配列番号４８及び４９のアダプタープライマーは、L1タンパク質をコードする領域を標的とするL1プライマーの先頭に、それぞれadaptor13及びadapter14を付加したフォワードプライマー（adapter13-16_L1_F1)とリバースプライマー(adapter14-16_L1_R1)の対であり、配列番号５０及び５１のアダプタープライマーは、L1タンパク質をコードする領域を標的とするL1プライマーの先頭に、それぞれadaptor15及びadapter16を付加したフォワードプライマー（adapter15-16_L1_F2)とリバースプライマー(adapter15-16_L1_R2)の対であり、配列番号５２及び５３のアダプタープライマーは、L1タンパク質をコードする領域を標的とするL1プライマーの先頭に、それぞれadaptor17及びadapter18を付加したフォワードプライマー（adapter17-16_L1_F3)とリバースプライマー(adapter18-16_L1_R3)の対であり、配列番号５４及び５５のアダプタープライマーは、L1タンパク質をコードする領域を標的とするL1プライマーの先頭に、それぞれadaptor19及びadapter20を付加したフォワードプライマー（adapter19-18_L1_F1)とリバースプライマー(adapter20-18_L1_R1)の対であり、配列番号５６及び５７のアダプタープライマーは、L1タンパク質をコードする領域を標的とするL1プライマーの先頭に、それぞれadaptor21及びadapter22を付加したフォワードプライマー（adapter21-18_L1_F2)とリバースプライマー(adapter22-18_L1_R2)の対であり、配列番号５８及び５９のアダプタープライマーは、L1タンパク質をコードする領域を標的とするL1プライマーの先頭に、それぞれadaptor23及びadapter24を付加したフォワードプライマー（adapter23-18_L1_F3)とリバースプライマー(adapter24-18_L1_R3)の対である。 Furthermore, the adapter primer used in the present invention includes, for example, an adapter primer consisting of the nucleotide sequence represented by SEQ ID NO: 48 to 59. The adapter primers of SEQ ID NOs: 48 and 49 are, respectively, a forward primer (adapter13-16_L1_F1) and a reverse primer (adapter14-16_L1_R1) each having an adapter 13 and an adapter 14 added to the head of the L1 primer targeting the region encoding the L1 protein. The adapter primers of SEQ ID NOs: 50 and 51 are a forward primer (adapter15-16_L1_F2) and a reverse primer (adapter15-adapter) respectively having an adapter 15 and an adapter 16 added to the head of the L1 primer targeting the region encoding the L1 protein. 16_L1_R2), and the adapter primers of SEQ ID NOs: 52 and 53 are respectively a forward primer (adapter17-16_L1_F3) and a reverse primer with adapter17 and adapter18 added to the head of the L1 primer targeting the region encoding the L1 protein. (adapter18-16_L1_R3). The adapter primers of Nos. 54 and 55 are a pair of a forward primer (adapter19-18_L1_F1) and a reverse primer (adapter20-18_L1_R1) each having an adapter 19 and an adapter 20 added to the head of the L1 primer targeting the region encoding the L1 protein. Yes, the adapter primers of SEQ ID NOs: 56 and 57 are, respectively, a forward primer (adapter21-18_L1_F2) and a reverse primer (adapter22-18_L1_R2) each having an adapter 21 and an adapter 22 added to the head of the L1 primer targeting the region encoding the L1 protein. The adapter primers of SEQ ID NOs: 58 and 59 are respectively a forward primer (adapter23-18_L1_F3) and a reverse primer (adapter24) each having an adapter 23 and an adapter 24 added to the head of an L1 primer targeting a region encoding the L1 protein. -18_L1_R3).
　本発明のHPVの遺伝子増幅法では、第１のPCRにおいて、上記のアダプタープライマーを用いてPCR伸長反応を行い検体中のHPV遺伝子を増幅する。 で は In the HPV gene amplification method of the present invention, in the first PCR, a PCR extension reaction is performed using the above adapter primer to amplify the HPV gene in the sample.
　PCR反応は、DNAポリメラーゼを用いて通常用いる条件で公知の方法で行えばよい。PCR反応の条件は、プライマーのTm値等に基づいて適宜設定することができる。例えば、検体にプライマーとdNTP混合物やポリメラーゼを含むPCR試薬を添加し、94℃で５分間加熱後、98℃で10秒加熱、45℃で30秒冷却、72℃で30秒加熱を１サイクルとして10～50サイクル程度繰り返せばよい。用いるポリメラーゼは、限定される、Taqポリメラーゼ等の公知のポリメラーゼをもちることができる。 The PCR reaction may be performed by a known method using a DNA polymerase under conditions usually used. Conditions for the PCR reaction can be appropriately set based on the Tm value of the primer and the like. For example, a PCR reagent containing a primer, a dNTP mixture, and a polymerase is added to a sample, heated at 94 ° C for 5 minutes, heated at 98 ° C for 10 seconds, cooled at 45 ° C for 30 seconds, and heated at 72 ° C for 30 seconds as one cycle. It may be repeated about 10 to 50 cycles. As the polymerase to be used, a known polymerase such as a limited Taq polymerase can be used.
　第１のPCRにより得られる増幅産物は、両側末端にアダプターに相補的な配列を有する。 増 幅 The amplification product obtained by the first PCR has a sequence complementary to the adapter at both ends.
　第２のPCRにおいては、増幅産物中のアダプターに相補的な配列に特異的にハイブリダイズするポリヌクレオチドをプライマーとして用い、PCRを行えばよい。該プライマーは好ましくはアダプターと同じ配列であるが、数個、好ましくは1～5個、さらに好ましくは１～４個、さらに好ましくは１～３個、さらに好ましくは１個又は２個、さらに好ましくは１個のミスマッチを有していてもよい。該プライマーをアダプター用プライマーと呼ぶことがある。第２のPCRの結果、第１のPCRにより得られた増幅産物がさらに増幅される。 {Circle around (2)} In the second PCR, PCR may be performed using a polynucleotide that specifically hybridizes to a sequence complementary to the adapter in the amplification product as a primer. The primer preferably has the same sequence as the adapter, but has several, preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and still more preferably May have one mismatch. The primer is sometimes referred to as an adapter primer. As a result of the second PCR, the amplification product obtained by the first PCR is further amplified.
　第２のPCRの反応条件は、第１のPCRと同様に、適宜設定することができる。例えば、94℃で５分間加熱後、98℃で10秒加熱、58℃で30秒冷却、72℃で30秒加熱を１サイクルとして10～50サイクル程度繰り返せばよい。 反 応 The reaction conditions of the second PCR can be set appropriately as in the case of the first PCR. For example, after heating at 94 ° C. for 5 minutes, heating at 98 ° C. for 10 seconds, cooling at 58 ° C. for 30 seconds, and heating at 72 ° C. for 30 seconds may be repeated as about 10 to 50 cycles.
　得られたPCR産物は、アガロースゲル電気泳動により確認することができる。アガロースゲル電気泳動により目的のサイズのDNAが増幅されているか否かを調べればよい。 The obtained PCR product can be confirmed by agarose gel electrophoresis. What is necessary is just to check whether the DNA of the target size has been amplified by agarose gel electrophoresis.
　本発明の方法によれば、検体中のHPVを高感度で検出することができ、検体中のHPVのコピー数が、1000コピー/ｍL、好ましくは100コピー/ｍL、さらに好ましくは10コピー/ｍL程度しか存在しなくても検出することができる。これは、従来のPCRによるHPVの検出法よりも100～1000倍高い感度である。 According to the method of the present invention, HPV in a sample can be detected with high sensitivity, and the number of copies of HPV in the sample is 1000 copies / mL, preferably 100 copies / mL, more preferably 10 copies / mL. It can be detected even if there is only a degree. This is 100 to 1000 times more sensitive than the conventional method of detecting HPV by PCR.
　本発明は、上記のHPV遺伝子に特異的にハイブリダイズするプライマーに、HPVゲノム、ヒトゲノム、並びにヒトに感染するウイルス及び微生物のゲノムにアニーリングしない配列からなるアダプターを付加したアダプタープライマーの対と前記アダプターに相補的な配列からなるプライマーの対を含む、検体中のHPVをPCR反応により増幅してHPVの感染を検出するためのキットをも包含する。 The present invention provides a primer-specific pair of the above-described adapter, which comprises an HPV genome, a human genome, and an adapter comprising a sequence that does not anneal to the genomes of viruses and microorganisms that infect humans, to the above-mentioned primer that specifically hybridizes to the HPV gene. And a kit for detecting HPV infection by amplifying HPV in a sample by a PCR reaction, comprising a pair of primers having a sequence complementary to the above.
　また、該キットは適宜、PCR反応溶液、酵素、試料を処理するための試薬等を含んでいてもよい。 The kit may optionally contain a PCR reaction solution, an enzyme, a reagent for treating a sample, and the like.
　本発明を以下の実施例によって具体的に説明するが、本発明はこれらの実施例によって限定されるものではない。 The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
[実施例１]　HPVの遺伝子増幅用アダプタープライマーを用いたアダプターPCRの開発
方法
１．　HPV L1配列を含むプラスミドの調製
（１）　HPV Type6b, 16, 18, 53のL1遺伝子領域620塩基をそれぞれ、In-Fusion（登録商標） HD Cloning Kit（タカラバイオ）を用いてpUC19プラスミドへ導入した。 [Example 1] Development method of adapter PCR using adapter primer for gene amplification of HPV Preparation of plasmid containing HPV L1 sequence (1) 620 bases of L1 gene region of HPV Type6b, 16, 18, and 53 were introduced into pUC19 plasmid using In-Fusion (registered trademark) HD Cloning Kit (Takara Bio). .
（２）　それぞれのL1遺伝子領域が導入されたpUC19プラスミドを精製し、101、103、105、107copy/μlとなるように濃度調整した (2) The pUC19 plasmid into which each L1 gene region was introduced was purified, and the concentration was adjusted to 10 1 , 10 3 , 10 5 , and 10 7 copy / μl.
２．　プライマーにおけるアダプター配列の設計
　本法は2回のPCRを行う。1st PCRはHPVのL1遺伝子とアニーリングし、DNAを増幅する既知のプライマーの5’側に20塩基のアダプター配列を付加したプライマーを用いて行う。2nd PCRはアダプター配列のみのプライマーで行う。アダプター配列は検体に含まれることが想定されるHPVゲノム、ヒトゲノム、ヒトに感染するウイルスや微生物のゲノムとアニーリングしない配列とするため、高等植物であるシロイヌナズナ（Arabidopsis thaliana）のゲノムから設計し、さらにシロイヌナズナゲノムの混入によるPCR増幅を避けるためにペアとなるアダプター配列を、それぞれ異なる染色体から設計したものを選択した。 2. Design of Adapter Sequence in Primers This method involves two rounds of PCR. The 1st PCR is performed using a primer that anneals to the L1 gene of HPV and adds a 20-base adapter sequence to the 5 ′ side of a known primer that amplifies DNA. 2nd PCR is performed with primers consisting only of the adapter sequence. The adapter sequence is designed from the genome of Arabidopsis thaliana, a higher plant, in order to make it a sequence that does not anneal to the genome of the HPV genome, human genome, viruses or microorganisms that infect humans, which are assumed to be included in the sample. In order to avoid PCR amplification due to the contaminating Arabidopsis thaliana genome, a pair of adapter sequences designed from different chromosomes were selected.
（１）　シロイヌナズナ遺伝子AT1G62710、AT2G22770、AT3G15950、AT4G28520のゲノム配列からPrimer3web（http://primer3.ut.ee/）を用いて、各遺伝子から10セットのフォワードプライマーとリバースプライマー配列を取得した。Primerの塩基長は20、Tm値は60±1℃、GC％は50から65となるよう設定した。 (1) 10 sets of forward primer and reverse primer sequences were obtained from each gene using the Primer3web (http://primer3.ut.ee/) from the genome sequences of the Arabidopsis thaliana genes AT1G62710, AT2G22770, AT3G15950, and AT4G28520. The Primer was set to have a base length of 20, a Tm value of 60 ± 1 ° C., and a GC% of 50 to 65.
（２）　シロイヌナズナ遺伝子AT1G62710、AT3G15950から取得したフォワードプライマーを、アダプター配列として（HPVの検出で汎用される）GP5+, GP6+プライマーセットのGP5＋の5’末端に付加したオリゴDNAを作製した。シロイヌナズナ遺伝子AT2G22770、AT4G28520から取得したリバースプライマーを、アダプター配列としてGP6+の5’末端に付加したオリゴDNAを作製した。 (2) Oligo DNA was prepared in which the forward primers obtained from the Arabidopsis thaliana genes AT1G62710 and AT3G15950 were added as adapter sequences to the GP5 + and GP5 + 5 'ends of the GP5 + and GP5 + primer sets (commonly used for HPV detection). Oligo DNA was prepared by adding a reverse primer obtained from the Arabidopsis thaliana genes AT2G22770 and AT4G28520 to the 5 'end of GP6 + as an adapter sequence.
（３）　（２）と同様に、シロイヌナズナ遺伝子AT1G62710、AT3G15950から取得したフォワードプライマーをHPVの検出で汎用される MY11, MY9プライマーセットのMY11の5’末端に付加したオリゴDNAを作成し、シロイヌナズナ遺伝子AT2G22770、AT4G28520から取得したリバースプライマーをMY09の5’末端に付加したオリゴDNAを作製した（adaptor01-GP5+, adaptor02-GP6+など）。 (3) In the same manner as in (2), an oligo DNA was prepared by adding a forward primer obtained from the Arabidopsis thaliana genes AT1G62710 and AT3G15950 to the 5 ′ end of MY11 of the MY11 and MY9 primer sets commonly used for HPV detection. Oligo DNAs were prepared by adding reverse primers obtained from AT2G22770 and AT4G28520 to the 5 ′ end of MY09 (adaptor01-GP5 +, adaptor02-GP6 +, etc.).
　adaptor01～adaptor12の塩基配列を図１及び配列番号１～１２に表す。また、HPV検出用プライマーにadaptor配列を付加したアダプタープライマーであるadaptor01-GP5+、adaptor02-GP6+、adaptor03-MY11、adaptor04-MY09、adaptor05-GP5+、adaptor06-GP6+、adaptor07-MY11、adaptor08-MY09、adaptor09-GP5+、adaptor10-GP6+、adaptor11-MY11、adaptor12-MY09の塩基配列を図１及び配列番号１３～２４に表す。
　GP5+、GP6+、MY11及びMY09の配列を、配列番号２５～２８に表す。 The nucleotide sequences of adaptor01 to adapter12 are shown in FIG. 1 and SEQ ID NOS: 1 to 12. The adapter primers to which the adapter sequence was added to the HPV detection primer are adapter01-GP5 +, adapter02-GP6 +, adaptor03-MY11, adaptor04-MY09, adaptor05-GP5 +, adaptor06-GP6 +, adaptor07-MY11, adaptor08-MY09, adaptor09-. The nucleotide sequences of GP5 +, adapter10-GP6 +, adapter11-MY11, and adapter12-MY09 are shown in FIG. 1 and SEQ ID NOs: 13 to 24.
The sequences of GP5 +, GP6 +, MY11 and MY09 are shown in SEQ ID NOS: 25 to 28.
３．　設計したオリゴDNAをプライマーとして使用したPCRの検証
（１）　汎用されているGP5+とGP6+とのプライマーセット、MY11とMY09とのプライマーセット、作成したオリゴDNAプライマーセット（adaptor01-GP5+とadaptor02-GP6+とのプライマーセット、adaptor03-MY11とadaptor04-MY09とのプライマーセット、adaptor05-GP5+とadaptor06-GP6+とのプライマーセット、adaptor07-MY11とadaptor08-MY09とのプライマーセット、adaptor09-GP5+とadaptor10-GP6+とのプライマーセット、adaptor11-MY11とadaptor12-MY09とのプライマーセット）を用いてPCRを行った。PCR酵素はタカラバイオ社のEmeraldAmp（登録商標） MAX PCR Master Mixを用い、１．で調製したプラスミドを鋳型とした。
　PCR反応液の組成は以下のとおりであった。 3. Verification of PCR using designed oligo DNA as primer (1) General-purpose primer set of GP5 + and GP6 +, primer set of MY11 and MY09, and prepared oligo DNA primer set (adaptor01-GP5 + and adaptor02-GP6 + Primer set, adaptor03-MY11 and adaptor04-MY09, primer set with adaptor05-GP5 + and adaptor06-GP6 +, primer set with adaptor07-MY11 and adaptor08-MY09, primer with adaptor09-GP5 + and adaptor10-GP6 + PCR was performed using the primer set (adaptor11-MY11 and the adapter12-MY09). As the PCR enzyme, Takara Bio's EmeraldAmp (registered trademark) MAX PCR Master Mix was used. The plasmid prepared in step was used as a template.
The composition of the PCR reaction solution was as follows.
GP5+とGP6+とのプライマーセットを用いた場合
　２×M.Mix　　10.00μl
　ddH2O　　　　 8.50μl
　GP5+(20μM)　 0.25μl
　GP6+(20μM)　 0.25μl
　鋳型　　　　　1.00μl
　トータル　　 20.00μl
MY11とMY09とのプラーマーセットを用いた場合
　２×M.Mix　　10.00μl
　ddH2O　　　　 5.00μl
　GP5+(20μM)　 2.00μl
　GP6+(20μM)　 2.00μl
　鋳型　　　　　1.00μl
　トータル　　 20.00μl When using the primer set of GP5 + and GP6 + 2 × M.Mix 10.00μl
ddH 2 O 8.50μl
GP5 + (20μM) 0.25μl
GP6 + (20μM) 0.25μl
1.00μl mold
Total 20.00μl
2 × M.Mix 10.00μl when using MY11 and MY09 Plummer Set
ddH 2 O 5.00μl
GP5 + (20μM) 2.00μl
GP6 + (20μM) 2.00μl
1.00μl mold
Total 20.00μl
　PCR装置はApplied Biosystems（登録商標） 2720サーマルサイクラーを用い、PCRプログラムは「94℃-5分, (98℃-10秒, 45℃-30秒, 72℃-30秒)×40サイクル」とした(1st PCR条件）。 The PCR apparatus used an Applied Biosystems (registered trademark) 2720 thermal cycler, and the PCR program was "94 ° C for 5 minutes, (98 ° C for 10 seconds, 45 ° C for 30 seconds, 72 ° C for 30 seconds) x 40 cycles”. (1st PCR conditions).
（２）　（１）の1st PCR産物を1μl分取し、アダプター配列のみのプライマー（adaptor01～adaptor12）を用いて2回目のPCR(2nd PCR)を行った。PCR酵素はタカラバイオ社のEmeraldAmp（登録商標） MAX PCR Master Mixを用いた。
　2nd PCR反応液の組成は以下のとおりであった。 (2) 1 μl of the 1st PCR product of (1) was taken, and a second PCR (2nd PCR) was performed using primers (adaptor01 to adapter12) having only an adapter sequence. As the PCR enzyme, Takara Bio's EmeraldAmp (registered trademark) MAX PCR Master Mix was used.
The composition of the 2nd PCR reaction solution was as follows.
adaptor1とadaptor2とのプライマーセットを用いた場合
　２×M.Mix　　　　10.00μl
　ddH2O　　　　　　 8.50μl
　adaptor01(20μM)　 0.25μl
　adaptor02(20μM)　 0.25μl
　鋳型　　　　　　　1.00μl
　トータル　　　　 20.00μl When using the primer set of adaptor1 and adaptor2 2 × M.Mix 10.00μl
ddH 2 O 8.50μl
adaptor01 (20μM) 0.25μl
adaptor02 (20μM) 0.25μl
1.00μl mold
Total 20.00μl
　上の例は、1st PCRにadaptor01-GP5+とadaptor02-GP6+とのプライマーセットを用い、2nd PCRに対応するadaptor01とadaptor02とのプライマーセットを用いている。1st PCRにadaptor03-MY11とadaptor04-MY09とのプライマーセット、adaptor05-GP5+とadaptor06-GP6+とのプライマーセット、adaptor07-MY11とadaptor08-MY09とのプライマーセット、adaptor09-GP5+とadaptor10-GP6+とのプライマーセット、又はadaptor11-MY11とadaptor12-MY09とのプライマーセットを用いた場合には、2nd PCRには、それぞれ、adaptor03とadaptor04とのプライマーセット、adaptor05とadaptor06とのプライマーセット、adaptor07とadaptor08とのプライマーセット、adaptor09とadaptor10とのプライマーセット、adaptor11とadaptor12とのプライマーセットを用いればよい。 In the above example, a primer set of adaptor01-GP5 + and adaptor02-GP6 + is used for 1st PCR, and a primer set of adaptor01 and adaptor02 corresponding to 2nd PCR is used. 1st PCR primer set with adaptor03-MY11 and adapter04-MY09, primer set with adaptor05-GP5 + and adaptor06-GP6 +, primer set with adaptor07-MY11 and adaptor08-MY09, primer set with adaptor09-GP5 + and adaptor10-GP6 + Or, when using a primer set of adaptor11-MY11 and adaptor12-MY09, the 2nd PCR, respectively, a primer set of adaptor03 and adaptor04, a primer set of adaptor05 and adaptor06, a primer set of adaptor07 and adaptor08 And a primer set of adaptor09 and adaptor10 and a primer set of adaptor11 and adaptor12 may be used.
　PCRプログラムは「94℃-5分, (98℃-10秒, 58℃-30秒, 72℃-30秒)×40サイクル」とした。 The PCR program was “94 ° C for 5 minutes, (98 ° C for 10 seconds, 58 ° C for 30 seconds, 72 ° C for 30 seconds) × 40 cycles”.
（３）　（２）のPCR産物5μlを2％アガロースゲル（蛍光物質として0.002％ MidoriGreen Advance（日本ジェネティクス社）を添加）にアプライし、100Vで25分間電気泳動した。 (3) 5 μl of the PCR product of (2) was applied to a 2% agarose gel (0.002% {MidoriGreen} Advance (Nippon Genetics) was added as a fluorescent substance) and electrophoresed at 100 V for 25 minutes.
（４）　UVトランスイルミネーターでDNAの検出を行った。 (4) DNA was detected using a UV transilluminator.
結果
（１）汎用プライマーセットを用いた場合
　汎用プライマーセット（GP5+とGP6+とのプライマーセット又はMY11とMY09とのプライマーセット）を用いてPCR増幅した結果を図２に示す。図２ＡはHPV type 6及びHPV type 16の結果を示し、図２ＢはHPV type 18及びHPV type 53の結果を示す。 Results (1) When a general-purpose primer set was used FIG. 2 shows the results of PCR amplification using a general-purpose primer set (a primer set of GP5 + and GP6 + or a primer set of MY11 and MY09). FIG. 2A shows the results of HPV type 6 and HPV type 16, and FIG. 2B shows the results of HPV type 18 and HPV type 53.
　図２Ａ及び図２Ｂに示すように、汎用されているGP5+とGP6+とのプライマーセット及びMY11とMY09とのプライマーセットを用いた場合、HPV type 6、16、18及び53を5x104コピー/tubeの濃度で検出することができたが、5x102コピー/tubeの濃度では検出することができなかった。 As shown in FIG. 2A and FIG. 2B, when a primer set of GP5 + and GP6 + and a primer set of MY11 and MY09, which are widely used, were used, HPV types 6, 16, 18, and 53 were used at 5 × 10 4 copies / tube. It could be detected at a concentration of 5 × 10 2 copies / tube, but not at a concentration of 5 × 10 2 copies / tube.
（２）アダプタープライマーセットを用いた場合
　1st PCRにアダプタープライマーセット（adaptor01-GP5+とadaptor02-GP6+とのプライマーセット又はadaptor03-MY11とadaptor04-MY09とのプライマーセット）を用いてPCR増幅した結果を図３に示す。図３ＡはHPV type 6及びHPV type 16の結果を示し、図３ＢはHPV type 18及びHPV type 53の結果を示す。 (2) In the case of using an adapter primer set The results of PCR amplification using the adapter primer set (a primer set of adaptor01-GP5 + and adaptor02-GP6 + or a primer set of adaptor03-MY11 and adaptor04-MY09) are shown in FIG. 3 is shown. FIG. 3A shows the results of HPV type 6 and HPV type 16, and FIG. 3B shows the results of HPV type 18 and HPV type 53.
　図３Ａ及び図３Ｂに示すように、GP5+プライマー及びGP6+プライマーに、それぞれ、adaptor01及びadaptor02を付加したプライマー（adaptor01-GP5+とadaptor02-GP6+）を用いることにより、HPV type 6、16及び18では、101コピー/tubeの濃度で検出することができた。 As shown in FIGS. 3A and 3B, by using primers (adaptor01-GP5 + and adaptor02-GP6 +) to which adapter01 and adapter02 were added to the GP5 + primer and GP6 + primer, respectively, HPV types 6, 16 and 18 had 10%. Detection was possible at a density of 1 copy / tube.
　一方、MY11プライマー及びMY09プライマーに、それぞれ、adaptor03及びadaptor04を付加したプライマー（adaptor03-MY11とadaptor04-MY09）を用いた場合は、感度の向上は認められなかった。 On the other hand, when primers (adaptor03-MY11 and adaptor04-MY09) obtained by adding adaptor03 and adaptor04 to the MY11 primer and MY09 primer, respectively, no improvement in sensitivity was observed.
　同様に、図４にアダプタープライマーセットとして、adaptor05-GP5+とadaptor06-GP6+とのプライマーセット（図４Ａ）又はadaptor07-GP5+とadaptor08-GP6+とのプライマーセット（図４Ｂ）を用いてPCR増幅した結果を示す。また、図５にアダプタープライマーセットとして、adaptor09-MY11とadaptor010-MY09とのプライマーセット（図５Ａ）、又はadaptor011-MY11とadaptor012-MY09とのプライマーセット（図５Ｂ）を用いてPCR増幅した結果を示す。 Similarly, FIG. 4 shows the results of PCR amplification using a primer set of adaptor05-GP5 + and adaptor06-GP6 + (FIG. 4A) or a primer set of adaptor07-GP5 + and adaptor08-GP6 + (FIG. 4B) as the adapter primer set. Show. FIG. 5 shows the results of PCR amplification using the adapter set of adapter09-MY11 and adapter010-MY09 (FIG. 5A) or the set of adapter011-MY11 and adapter012-MY09 (FIG. 5B). Show.
　図４Ａ及び図４Ｂが示すように、GP5+プライマー及びGP6+プライマーに、それぞれ、adaptor05及びadaptor06を付加したプライマー（adaptor05-GP5+とadaptor06-GP6+）、あるいはGP5+プライマー及びGP6+プライマーに、それぞれ、adaptor07及びadaptor08を付加したプライマー（adaptor07-GP5+とadaptor08-GP6+）では、HPV type 6、16、18及び53で、101コピー/tubeの濃度で検出することができ、感度の向上が認められた。 As shown in FIG. 4A and FIG. 4B, the adapter (adaptor05-GP5 + and adaptor06-GP6 +) to which adapter05 and adapter06 were added to the GP5 + primer and GP6 + primer, respectively, or the adapter07 and adapter08 to the GP5 + primer and GP6 + primer, respectively. in addition primers (adaptor07-GP5 + and adaptor08-GP6 +), with HPV type 6,16,18 and 53, can be detected at a concentration of 10 1 copies / Tube, improvement in sensitivity was observed.
　また、図５Ａ及び図５Ｂが示すように、MY11プライマー及びMY09プライマーにそれぞれ、adaptor09及びadaptor010を付加したプライマー（adaptor09-MY11とadaptor010-MY09）、あるいはMY11プライマー及びMY09プライマーに、それぞれ、adaptor011及びadaptor012を付加したプライマー（adaptor011-MY11とadaptor012-MY09）でもHPV type 6、16、18及び53で、101コピー/tubeの濃度で検出することができ、感度の向上が認められた。 As shown in FIGS. 5A and 5B, the MY11 and MY09 primers were respectively added with adaptor09 and adaptor010 (adaptor09-MY11 and adaptor010-MY09), or the MY11 and MY09 primers were respectively adapted to adaptor011 and adaptor012. in HPV type 6,16,18 and 53 even primers added (adaptor011-MY11 and adaptor012-MY09), can be detected at a concentration of 10 1 copies / Tube, improvement in sensitivity was observed.
[実施例２]　検体（精液）を用いたPCR反応
　165名の精液20μlに80μlのバッファ1（20 mM Tris-HCl, 5 mM EDTA, 400mM NaCl, 0.3% SDS, pH8.0）を加えて混和し、95℃で5分間加熱した。 [Example 2] PCR reaction using a sample (semen) 80 µl of buffer 1 (20 mM Tris-HCl, 5 mM EDTA, 400 mM NaCl, 0.3% SDS, pH 8.0) was added to 20 µl of semen of 165 people and mixed. And heated at 95 ° C. for 5 minutes.
　次いで、10μlのプロティナーゼK溶液（20 mg/ml, 富士フィルム）、2μlの1M DTT（富士フィルム）を加えて混和し、50℃で10分間加熱した。その後、25℃で16時間インキュベートした。 Next, 10 μl of proteinase K solution (20 mg / ml, Fuji film) and 2 μl of 1M DTT (Fuji film) were added, mixed, and heated at 50 ° C. for 10 minutes. Then, it incubated at 25 degreeC for 16 hours.
　NucleoSpin(登録商標） Tissue XS（マッハライナーゲル社）を用いて、取説に従いDNAを抽出した。HPVの検出のために、1st PCRではadpter01-GP5+（配列番号１３）とadapter02-GP6+（配列番号１４）のセットを用い、2nd PCRでadapter01（配列番号１）とadapter02（配列番号２）のセットを用いた。DNA抽出を確認するために、ヒトβ-globin遺伝子領域を増幅するプライマーセット（GH20/GH21）を用いてPCRを行った。 DNA was extracted using NucleoSpin (registered trademark) Tissue XS (Machliner Gel) according to the instruction manual. For detection of HPV, a set of adapter01-GP5 + (SEQ ID NO: 13) and adapter02-GP6 + (SEQ ID NO: 14) was used in the first PCR, and a set of adapter01 (SEQ ID NO: 1) and adapter02 (SEQ ID NO: 2) in the second PCR. Was used. To confirm DNA extraction, PCR was performed using a primer set (GH20 / GH21) for amplifying the human β-globin gene region.
　PCR組成はプライマーの量を除いて実施例１と同様であった。用いたプライマー配列は、GH20：GAAGAGCCAAGGACAGGTAC（配列番号３４）、及びGH21：GGAAAATAGACCAATAGGCAG（配列番号３５）であった。 The PCR composition was the same as in Example 1 except for the amount of primer. The primer sequences used were GH20: GAAGAGCCAAGGACAGGTAC (SEQ ID NO: 34) and GH21: GGAAAATAGACCAATAGGCAG (SEQ ID NO: 35).
　PCRプログラムは「94℃-5分, (98℃-10秒, 58℃-30秒, 72℃-30秒)×30サイクル」であった。 The PCR program was “94 ° C for 5 minutes, (98 ° C for 10 seconds, 58 ° C for 30 seconds, 72 ° C for 30 seconds) × 30 cycles”.
　すべての検体において特異的増幅を確認した。 特異 Specific amplification was confirmed in all samples.
　精子から抽出したDNA 1μlを用いて、実施例１に記載の方法で精子からHPVゲノムDNAを検出することができた。 Using 1 μl of DNA extracted from sperm, HPV genomic DNA could be detected from sperm by the method described in Example 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　結果を表１に示す。表１に示すように、陽性率が大幅に改善している。 The results are shown in Table 1. As shown in Table 1, the positive rate is significantly improved.
[実施例３]　尿検体からのDNA抽出と検出
1 mlの尿検体に200μlの3M 酢酸ナトリウム溶液（ニッポンジーン）、3mlのエタノール（富士フィルム）、50μlの20mg/mlグリコーゲン溶液（ナカライテスク）を加え、転倒混和を10回行った。次いで、マイナス20℃で16時間インキュベートした後、4℃下で16,000×g、30分間遠心分離を行った。ペレットを吸わないように上清を捨て、4 mlの80％エタノールを加えた。その後、4℃下で16,000×g、30分間遠心分離を行った。ペレットを吸わないように上清を捨て、ペレットをNucleoSpin Tissue（登録商標）Tissue（タカラバイオ）に供し、取説に従いDNAを抽出した。 [Example 3] DNA extraction and detection from urine sample
200 μl of a 3M sodium acetate solution (Nippon Gene), 3 ml of ethanol (Fuji Film), and 50 μl of a 20 mg / ml glycogen solution (Nacalai Tesque) were added to 1 ml of the urine sample, and the mixture was overturned 10 times. Next, after incubation at minus 20 ° C for 16 hours, centrifugation was performed at 16,000 xg for 30 minutes at 4 ° C. The supernatant was discarded without sucking the pellet, and 4 ml of 80% ethanol was added. Thereafter, centrifugation was performed at 16,000 × g for 30 minutes at 4 ° C. The supernatant was discarded without sucking the pellet, and the pellet was subjected to NucleoSpin Tissue (registered trademark) Tissue (Takara Bio), and DNA was extracted according to the instruction manual.
　以降は、実施例２の精液検体と同じ方法で検出した。 Thereafter, detection was performed by the same method as the semen sample of Example 2.
　増幅したDNAのアガロースゲル電気泳動写真を図６に示す。図６中、左からマーカー検体１～20の電気泳動像を示す。２番と９番の検体が陽性であり、他は陰性であった。 (6) An agarose gel electrophoresis photograph of the amplified DNA is shown in FIG. In FIG. 6, electrophoretic images of marker samples 1 to 20 are shown from the left. Nos. 2 and 9 were positive, others were negative.
　実施例３の結果が示すように、尿から抽出したDNA 1μlを用いて、実施例１に記載の方法で尿からHPVゲノムDNAを検出することができた。 {As shown in the results of Example 3, it was possible to detect HPV genomic DNA from urine by the method described in Example 1, using 1 μl of DNA extracted from urine.
[実施例４]　HPVの型特異的プライマーの検証 [Example 4] Verification of HPV type-specific primer
　HPV type6, 18のL1タンパク質コード領域をDNA鋳型として使用した。
　101, 102, 103, 104, 105, 106, 107, 108 copy/μLに調整した。
　EmeraldAmp（登録商標） MAX PCR Master MixでPCRを行った。
　その後、2.5%アガロースゲル（+0.002% MidoriGreen Advance）で電気泳動を行った。 The L1 protein coding region of HPV type 6, 18 was used as a DNA template.
It was adjusted to 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 copy / μL.
PCR was performed with EmeraldAmp® MAX PCR Master Mix.
Thereafter, electrophoresis was performed on a 2.5% agarose gel (+ 0.002% MidoriGreen Advance).
　1st PCRの反応液の組成は以下のとおりであった。
　２×M.Mix　　　　10.00μl
　ddH2O　　　　　　 5.00μl
　MY11(20μM)　　　 0.25μl
　MY09(20μM)　　　 0.25μl
　鋳型　　　　　　　1.00μl
　トータル　　　　 20.00μl The composition of the 1st PCR reaction solution was as follows.
2 × M.Mix 10.00μl
ddH 2 O 5.00μl
MY11 (20 μM) 0.25 μl
MY09 (20 μM) 0.25 μl
1.00μl mold
Total 20.00μl
　1st PCRの条件は、「94℃-5分, (98℃-10秒, 45℃-30秒, 72℃-30秒)×40サイクル」であった。 The conditions for {1st} PCR were "94 ° C. for 5 minutes, Δ (98 ° C. for 10 seconds, Δ45 ° C. for 30 seconds, Δ72 ° C. for 30 seconds) × 40 cycles”.
（１）汎用プライマーの検証
　汎用されるMY11及びMY09プライマーセットを用いたときの結果を図７に示す。図７ＡはHPV Type 16の検出の結果、図７ＢはHPV Type 18の検出の結果を示す。
　汎用されるMY11,MY09プライマーセットはHPV type 16に関しては10コピー/tubeから検出可能であり、type 18に関しては10コピー/tubeから検出可能であった。 (1) Verification of General-Purpose Primers FIG. 7 shows the results obtained when the commonly used MY11 and MY09 primer sets were used. FIG. 7A shows the result of HPV Type 16 detection, and FIG. 7B shows the result of HPV Type 18 detection.
The commonly used MY11 and MY09 primer sets were detectable from 10 copies / tube for HPV type 16 and 10 copies / tube for type 18.
　なお、検体中のHPVが10コピー/ｍL存在すれば、1st PCRに必要な10コピー/tubeが得られる。従って、本発明の方法では、検体中のHPVが10コピー/ｍL存在すれば検出することができる。 If HPV in the sample is present at 10 copies / mL, 10 copies / tube required for 1st PCR can be obtained. Therefore, according to the method of the present invention, if HPV in a sample is present at 10 copies / mL, it can be detected.
（２）新規プライマーの検証
　1st PCRは、5'末端用に開発したアダプタープライマー配列（adapter13～24が先頭に付くL1プライマー）を使用した。用いたアダプタープライマーの配列を図１０及び配列番号４８～５９に示す。 (2) Verification of new primers For the 1st PCR, an adapter primer sequence (L1 primer with adapters 13 to 24 at the head) developed for the 5 'end was used. The sequence of the adapter primer used is shown in FIG. 10 and SEQ ID NOs: 48 to 59.
　1st PCR産物１μlを鋳型とし、2nd PCRを行った。2nd PCRのプライマーはadapter13～adapter24を使用した。用いたプライマーの配列を図１０及び配列番号３６～４７に示す。 The second PCR was performed using 1 μl of the {1st} PCR product as a template. Adapters 13 to 24 were used as primers for 2nd PCR. The sequences of the primers used are shown in FIG. 10 and SEQ ID NOs: 36 to 47.
　1st PCRの反応液の組成は以下のとおりであった。
　２×M.Mix　　　　10.00μl
　ddH2O　　　　　　 8.50μl
　primer F(20μM)　 0.25μl
　primer R(20μM)　 0.25μl
　鋳型　　　　　　　1.00μl
　トータル　　　　 20.00μl The composition of the 1st PCR reaction solution was as follows.
2 × M.Mix 10.00μl
ddH 2 O 8.50μl
primer F (20 μM) 0.25 μl
primer R (20 μM) 0.25 μl
1.00μl mold
Total 20.00μl
　1st PCRの条件は、「94℃-5分, (98℃-10秒, 45℃-30秒, 72℃-30秒)×30サイクル」であった。 The conditions for {1st} PCR were "94 ° C. for 5 minutes, Δ (98 ° C. for 10 seconds, Δ45 ° C. for 30 seconds, Δ72 ° C. for 30 seconds) × 30 cycles”.
　2nd PCRの反応液の組成は以下のとおりであった。
　２×M.Mix　　　　10.00μl
　ddH2O　　　　　　 8.50μl
　primer F(20μM)　 0.25μl
　primer R(20μM)　 0.25μl
　1st PCR産物　　　 1.00μl
　トータル　　　　 20.00μl The composition of the reaction solution for 2nd PCR was as follows.
2 × M.Mix 10.00μl
ddH 2 O 8.50μl
primer F (20 μM) 0.25 μl
primer R (20 μM) 0.25 μl
1st PCR product 1.00μl
Total 20.00μl
　2nd PCRの条件は、「94℃-5分, (98℃-10秒, 45℃-30秒, 72℃-30秒)×30サイクル」であった。 The condition of the {2nd} PCR was "94 ° C. for 5 minutes, Δ (98 ° C. for 10 seconds, Δ45 ° C. for 30 seconds, Δ72 ° C. for 30 seconds) × 30 cycles”.
　Type 16の検出結果を図８に、Type 18の検出結果を図９に示す。図８Ａは1stプライマー（アダプタープライマー）セットとして、adapter13-16_L1_F1及びadapter14-16_L1_R1を用い、2ndプライマーセットとして、adapter13及びadapter14を用いたときの結果、図８Ｂは1stプライマー（アダプタープライマー）セットとして、adapter15-16_L1_F2及びadapter16-16_L1_R2を用い、2ndプライマーセットとして、adapter15及びadapter16を用いたときの結果、図８Ｃは1stプライマー（アダプタープライマー）セットとして、adapter17-16_L1_F3及びadapter18-16_L1_R3を用い、2ndプライマーセットとして、adapter17及びadapter18を用いたときの結果を示す。また、図９Ａは1stプライマー（アダプタープライマー）セットとして、adapter19-18_L1_F1及びadapter20-18_L1_R1を用い、2ndプライマーセットとして、adapter19及びadapter20を用いたときの結果、図９Ｂは1stプライマー（アダプタープライマー）セットとして、adapter21-18_L1_F2及びadapter22-18_L1_R2を用い、2ndプライマーセットとして、adapter21及びadapter22を用いたときの結果、図８Ｃは1stプライマー（アダプタープライマー）セットとして、adapter23-18_L1_F3及びadapter24-18_L1_R3を用い、2ndプライマーセットとして、adapter23及びadapter24を用いたときの結果を示す。　adapter13-24はシロイヌナズナAT2G22770.1配列から取得した配列である。 FIG. 8 shows the detection result of Type 16, and FIG. 9 shows the detection result of Type 18. FIG. 8A shows the result when adapter13-16_L1_F1 and adapter14-16_L1_R1 were used as the first primer (adapter primer) set, and adapter13 and adapter14 were used as the second primer set. FIG. 8B shows the result when adapter15 was used as the first primer (adapter primer) set. Using -16_L1_F2 and adapter16-16_L1_R2, as a 2nd primer set, using adapter15 and adapter16, FIG. 8C shows 1st primer (adapter primer) set, adapter17-16_L1_F3 and adapter18-16_L1_R3 as 2nd primer set. , Adapter17 and adapter18 are shown. FIG. 9A shows the result when adapter19-18_L1_F1 and adapter20-18_L1_R1 were used as the first primer (adapter primer) set, and adapter19 and adapter20 were used as the second primer set, and FIG. 9B was the first primer (adapter primer) set. Using adapter21-18_L1_F2 and adapter22-18_L1_R2, and using adapter21 and adapter22 as the second primer set, FIG. 8C shows adapter23-18_L1_F3 and adapter24-18_L1_R3 as the first primer (adapter primer) set, and the second primer. The results when adapter23 and adapter24 are used as a set are shown. Adapter13-24 is a sequence obtained from the Arabidopsis AT2G22770.1 sequence.
　図８及び９に示すように、すべてのプライマーで従来法では検出できなかった101コピー/tubeの検出が可能であった。 As shown in FIGS. 8 and 9, it was possible to detect 10 1 copies / tube can not be detected by the conventional method in all the primers.
　図１０に新規に開発したHPVの型特異的プライマーの配列を示す。 FIG. 10 shows the sequence of a newly developed HPV type-specific primer.
　本発明の方法は高感度でのHPVの検出に利用することができる。 方法 The method of the present invention can be used for highly sensitive detection of HPV.
配列番号１：プライマー（adaptor01)
配列番号２：プライマー（adaptor02)
配列番号３：プライマー（adaptor03)
配列番号４：プライマー（adaptor04)
配列番号５：プライマー（adaptor05)
配列番号６：プライマー（adaptor06)
配列番号７：プライマー（adaptor07)
配列番号８：プライマー（adaptor08)
配列番号９：プライマー（adaptor09)
配列番号１０：プライマー（adaptor10)
配列番号１１：プライマー（adaptor11)
配列番号１２：プライマー（adaptor12)
配列番号１３：プライマー（adaptor01-GP5+）
配列番号１４：プライマー（adaptor02-GP6+）
配列番号１５：プライマー（adaptor03-MY11)
配列番号１６：プライマー（adaptor04-MY09)
配列番号１７：プライマー（adaptor05-GP5+）
配列番号１８：プライマー（adaptor06-GP6+）
配列番号１９：プライマー（adaptor07-MY11)
配列番号２０：プライマー（adaptor08-MY09)
配列番号２１：プライマー（adaptor09-GP5+）
配列番号２２：プライマー（adaptor10-GP6+）
配列番号２３：プライマー（adaptor11-MY11)
配列番号２４：プライマー（adaptor12-MY09)
配列番号２５：プライマー（GP5+）
配列番号２６：プライマー（GP6+）
配列番号２７：プライマー（MY11)
配列番号２８：プライマー（MY09)
配列番号２９：プライマー（L1C1)
配列番号３０：プライマー（L1C2)
配列番号３１：プライマー（L1C2M)
配列番号３４：プライマー（GH20）
配列番号３５：プライマー（GH21)
配列番号３６：プライマー（adapter13)
配列番号３７：プライマー（adapter14)
配列番号３８：プライマー（adapter15)
配列番号３９：プライマー（adapter16)
配列番号４０：プライマー（adapter17)
配列番号４１：プライマー（adapter18)
配列番号４２：プライマー（adapter19)
配列番号４３：プライマー（adapter20)
配列番号４４：プライマー（adapter21)
配列番号４５：プライマー（adapter22)
配列番号４６：プライマー（adapter23)
配列番号４７：プライマー（adapter24)
配列番号４８：プライマー（adapter13-16_L1_F1)
配列番号４９：プライマー（adapter14-16_L1_R1)
配列番号５０：プライマー（adapter15-16_L1_F2)
配列番号５１：プライマー（adapter16-16_L1_R2)
配列番号５２：プライマー（adapter17-16_L1_F3)
配列番号５３：プライマー（adapter18-16_L1_R3)
配列番号５４：プライマー（adapter19-18_L1_F1)
配列番号５５：プライマー（adapter20-18_L1_R1)
配列番号５６：プライマー（adapter21-18_L1_F2)
配列番号５７：プライマー（adapter22-18_L1_R2)
配列番号５８：プライマー（adapter23-18_L1_F3)
配列番号５９：プライマー（adapter24-18_L1_R3)
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 SEQ ID NO: 1: Primer (adaptor01)
SEQ ID NO: 2: Primer (adaptor02)
SEQ ID NO: 3: Primer (adaptor03)
SEQ ID NO: 4: Primer (adaptor04)
SEQ ID NO: 5: primer (adaptor05)
SEQ ID NO: 6: primer (adaptor06)
SEQ ID NO: 7: primer (adaptor07)
SEQ ID NO: 8: primer (adaptor08)
SEQ ID NO: 9: primer (adaptor09)
SEQ ID NO: 10: primer (adaptor10)
SEQ ID NO: 11: primer (adaptor11)
SEQ ID NO: 12: primer (adaptor12)
Sequence number 13: Primer (adaptor01-GP5 +)
Sequence number 14: Primer (adaptor02-GP6 +)
SEQ ID NO: 15: Primer (adaptor03-MY11)
SEQ ID NO: 16: primer (adaptor04-MY09)
SEQ ID NO: 17: Primer (adaptor05-GP5 +)
SEQ ID NO: 18: primer (adaptor06-GP6 +)
SEQ ID NO: 19: primer (adaptor07-MY11)
SEQ ID NO: 20: Primer (adaptor08-MY09)
SEQ ID NO: 21: primer (adaptor09-GP5 +)
SEQ ID NO: 22: Primer (adaptor10-GP6 +)
SEQ ID NO: 23: Primer (adaptor11-MY11)
SEQ ID NO: 24: Primer (adaptor12-MY09)
SEQ ID NO: 25: Primer (GP5 +)
SEQ ID NO: 26: Primer (GP6 +)
SEQ ID NO: 27: Primer (MY11)
SEQ ID NO: 28: Primer (MY09)
SEQ ID NO: 29: Primer (L1C1)
SEQ ID NO: 30: Primer (L1C2)
SEQ ID NO: 31: Primer (L1C2M)
SEQ ID NO: 34: Primer (GH20)
SEQ ID NO: 35: Primer (GH21)
SEQ ID NO: 36: Primer (adapter13)
SEQ ID NO: 37: primer (adapter14)
SEQ ID NO: 38: Primer (adapter15)
SEQ ID NO: 39: Primer (adapter16)
SEQ ID NO: 40: Primer (adapter17)
SEQ ID NO: 41: primer (adapter18)
SEQ ID NO: 42: primer (adapter19)
SEQ ID NO: 43: Primer (adapter20)
SEQ ID NO: 44: Primer (adapter21)
SEQ ID NO: 45: Primer (adapter22)
SEQ ID NO: 46: Primer (adapter23)
SEQ ID NO: 47: Primer (adapter24)
SEQ ID NO: 48: Primer (adapter13-16_L1_F1)
SEQ ID NO: 49: Primer (adapter14-16_L1_R1)
SEQ ID NO: 50: Primer (adapter15-16_L1_F2)
SEQ ID NO: 51: Primer (adapter16-16_L1_R2)
SEQ ID NO: 52: Primer (adapter17-16_L1_F3)
SEQ ID NO: 53: Primer (adapter18-16_L1_R3)
SEQ ID NO: 54: Primer (adapter19-18_L1_F1)
Sequence number 55: Primer (adapter20-18_L1_R1)
SEQ ID NO: 56: Primer (adapter21-18_L1_F2)
SEQ ID NO: 57: Primer (adapter22-18_L1_R2)
SEQ ID NO: 58: Primer (adapter23-18_L1_F3)
SEQ ID NO: 59: Primer (adapter24-18_L1_R3)
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (18)

  1. 　検体中のHPVをPCR反応により増幅してHPVの感染を検出する方法であって、
    (a)　第１のPCRにおいて、HPV遺伝子に特異的にハイブリダイズするプライマーに、HPVゲノム、ヒトゲノム、並びにヒトに感染するウイルス及び微生物のゲノムにアニーリングしない配列からなるアダプターを付加したアダプタープライマーの対により増幅し、
    (b)　第２のPCRにおいて、(a)の増幅産物を前記アダプターに相補的な配列からなるプライマーの対により増幅することを含む、
    HPVの感染を検出する方法。     A method for detecting HPV infection by amplifying HPV in a sample by a PCR reaction,
    (a) In the first PCR, a primer that specifically hybridizes to the HPV gene, and an adapter primer pair comprising an adapter consisting of a sequence that does not anneal to the HPV genome, the human genome, and the genomes of viruses and microorganisms that infect humans. Amplified by
    (b) in the second PCR, amplifying the amplification product of (a) with a pair of primers having a sequence complementary to the adapter,
    How to detect HPV infection.
  2. 　検体中のHPVのコピー数が1000コピー/ｍL以下である、請求項１記載の方法。 方法 The method according to claim 1, wherein the number of copies of HPV in the sample is 1000 copies / mL or less.
  3. 　検体中のHPVのコピー数が100コピー/ｍL以下である、請求項１記載の方法。 方法 The method according to claim 1, wherein the copy number of HPV in the sample is 100 copies / mL or less.
  4. 　検体中のHPVのコピー数が10コピー/ｍL以下である、請求項１記載の方法。 方法 The method according to claim 1, wherein the copy number of HPV in the sample is 10 copies / mL or less.
  5. 　検体が精液又は尿である、請求項１～４のいずれか１項に記載の方法。 (5) The method according to any one of (1) to (4), wherein the specimen is semen or urine.
  6. 　HPV遺伝子に特異的にハイブリダイズするプライマーがHPVのL1遺伝子に特異的にハイブリダイズするプライマーである、請求項１～５のいずれか１項に記載の方法。 The method according to any one of claims 1 to 5, wherein the primer that specifically hybridizes to the HPV gene is a primer that specifically hybridizes to the HPV L1 gene.
  7. 　アダプターが、シロイヌナズナの遺伝子の部分配列からなる、請求項１～６のいずれか１項に記載の方法。 (7) The method according to any one of (1) to (6), wherein the adapter comprises a partial sequence of an Arabidopsis thaliana gene.
  8. 　アダプタープライマー対とプライマー対の組合せが以下の(i)～(vi)のいずれかの組合せである、請求項１～７のいずれか１項に記載の方法：
    (i)　配列番号１３及び１４の配列からなるアダプタープライマー対、並びに配列番号１及び２の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (ii)　配列番号１５及び１６の配列からなるアダプタープライマー対、並びに配列番号３及び４の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (iii)　配列番号１７及び１８の配列からなるアダプタープライマー対、並びに配列番号５及び６の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (iv)　配列番号１９及び２０の配列からなるアダプタープライマー対、並びに配列番号７及び８の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (v)　配列番号２１及び２２の配列からなるアダプタープライマー対、並びに配列番号９及び１０の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (vi)　配列番号２３及び２４の配列からなるアダプタープライマー対、並びに配列番号１１及び１２の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ。     The method according to any one of claims 1 to 7, wherein the combination of the adapter primer pair and the primer pair is any combination of the following (i) to (vi):
    (i) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 13 and 14, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 1 and 2;
    (ii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 15 and 16 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 3 and 4;
    (iii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 17 and 18, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 5 and 6;
    (iv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 19 and 20, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 7 and 8;
    (v) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 21 and 22, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 9 and 10;
    (vi) A combination of an adapter primer pair consisting of the sequences of SEQ ID NOS: 23 and 24 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 11 and 12.
  9. 　アダプタープライマー対とプライマー対の組合せが以下の(xi)～(xvi)のいずれかの組合せである、請求項１～７のいずれか１項に記載の方法：
    (xi)　配列番号４８及び４９の配列からなるアダプタープライマー対、並びに配列番号３６及び３７の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xii)　配列番号５０及び５１の配列からなるアダプタープライマー対、並びに配列番号３８及び３９の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xiii)　配列番号５２及び５３の配列からなるアダプタープライマー対、並びに配列番号４０及び４１の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xiv)　配列番号５４及び５５の配列からなるアダプタープライマー対、並びに配列番号４２及び４３の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xv)　配列番号５６及び５７の配列からなるアダプタープライマー対、並びに配列番号４４及び４５の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xvi)　配列番号５８及び５９の配列からなるアダプタープライマー対、並びに配列番号４６及び４７の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ。     The method according to any one of claims 1 to 7, wherein the combination of the adapter primer pair and the primer pair is any one of the following (xi) to (xvi):
    (xi) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 48 and 49 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 36 and 37;
    (xii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 50 and 51 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 38 and 39;
    (xiii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 52 and 53 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 40 and 41;
    (xiv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 54 and 55 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 42 and 43;
    (xv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 56 and 57 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 44 and 45;
    (xvi) A combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 58 and 59 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 46 and 47.
  10. 　(a)　HPV遺伝子に特異的にハイブリダイズするプライマーに、HPVゲノム、ヒトゲノム、並びにヒトに感染するウイルス及び微生物のゲノムにアニーリングしない配列からなるアダプターを付加したアダプタープライマーの対;と
    (b)　前記アダプターに相補的な配列からなるプライマーの対を含む、
    検体中のHPVをPCR反応により増幅してHPVの感染を検出するためのキット。     (A) a primer that specifically hybridizes to the HPV gene, an HPV genome, a human genome, and an adapter primer pair to which an adapter consisting of a sequence that does not anneal to the genomes of viruses and microorganisms that infect humans is added; and
    (b) including a primer pair consisting of a sequence complementary to the adapter,
    A kit for detecting HPV infection by amplifying HPV in a sample by PCR.
  11. 　検体中のHPVのコピー数が1000コピー/ｍL以下である、請求項１０記載のキット。 11. The kit according to claim 10, wherein the copy number of HPV in the sample is 1000 copies / mL or less.
  12. 　検体中のHPVのコピー数が100コピー/ｍL以下である、請求項１０記載のキット。 11. The kit according to claim 10, wherein the copy number of HPV in the sample is 100 copies / mL or less.
  13. 　検体中のHPVのコピー数が10コピー/ｍL以下である、請求項１０記載のキット。 The kit according to claim 10, wherein the copy number of HPV in the sample is 10 copies / mL or less.
  14. 　検体が精液又は尿である、請求項１０～１３のいずれか１項に記載のキット。 The kit according to any one of claims 10 to 13, wherein the specimen is semen or urine.
  15. 　HPV遺伝子に特異的にハイブリダイズするプライマーがHPVのL1遺伝子に特異的にハイブリダイズするプライマーである、請求項１０～１４のいずれか１項に記載のキット。 The kit according to any one of claims 10 to 14, wherein the primer that specifically hybridizes to the HPV gene is a primer that specifically hybridizes to the HPV L1 gene.
  16. 　アダプターが、シロイヌナズナの遺伝子の部分配列からなる、請求項１０～１５のいずれか１項に記載のキット。 The kit according to any one of claims 10 to 15, wherein the adapter comprises a partial sequence of an Arabidopsis thaliana gene.
  17. 　アダプタープライマー対とプライマー対の組合せが以下の(i)～(vi)のいずれかの組合せである、請求項１０～１６のいずれか１項に記載のキット：
    (i)　配列番号１３及び１４の配列からなるアダプタープライマー対、並びに配列番号１及び２の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (ii)　配列番号１５及び１６の配列からなるアダプタープライマー対、並びに配列番号３及び４の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (iii)　配列番号１７及び１８の配列からなるアダプタープライマー対、並びに配列番号５及び６の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (iv)　配列番号１９及び２０の配列からなるアダプタープライマー対、並びに配列番号７及び８の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (v)　配列番号２１及び２２の配列からなるアダプタープライマー対、並びに配列番号９及び１０の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (vi)　配列番号２３及び２４の配列からなるアダプタープライマー対、並びに配列番号１１及び１２の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ。     The kit according to any one of claims 10 to 16, wherein the combination of the adapter primer pair and the primer pair is any combination of the following (i) to (vi):
    (i) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 13 and 14, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 1 and 2;
    (ii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 15 and 16 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 3 and 4;
    (iii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 17 and 18, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 5 and 6;
    (iv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 19 and 20, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 7 and 8;
    (v) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 21 and 22, and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 9 and 10;
    (vi) A combination of an adapter primer pair consisting of the sequences of SEQ ID NOS: 23 and 24 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 11 and 12.
  18. 　アダプタープライマー対とプライマー対の組合せが以下の(xi)～(xvi)のいずれかの組合せである、請求項１０～１６のいずれか１項に記載のキット：
    (xi)　配列番号４８及び４９の配列からなるアダプタープライマー対、並びに配列番号３６及び３７の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xii)　配列番号５０及び５１の配列からなるアダプタープライマー対、並びに配列番号３８及び３９の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xiii)　配列番号５２及び５３の配列からなるアダプタープライマー対、並びに配列番号４０及び４１の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xiv)　配列番号５４及び５５の配列からなるアダプタープライマー対、並びに配列番号４２及び４３の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xv)　配列番号５６及び５７の配列からなるアダプタープライマー対、並びに配列番号４４及び４５の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ；
    (xvi)　配列番号５８及び５９の配列からなるアダプタープライマー対、並びに配列番号４６及び４７の配列からなるアダプターに相補的な配列からなるプライマー対の組合せ。     The kit according to any one of claims 10 to 16, wherein the combination of the adapter primer pair and the primer pair is any combination of the following (xi) to (xvi):
    (xi) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 48 and 49 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 36 and 37;
    (xii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 50 and 51 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 38 and 39;
    (xiii) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 52 and 53 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOS: 40 and 41;
    (xiv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 54 and 55 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 42 and 43;
    (xv) a combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 56 and 57 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 44 and 45;
    (xvi) A combination of an adapter primer pair consisting of the sequences of SEQ ID NOs: 58 and 59 and a primer pair consisting of a sequence complementary to the adapter consisting of the sequences of SEQ ID NOs: 46 and 47.
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