WO2020050350A1 - Méthode de dépistage du virus du papillome humain - Google Patents

Méthode de dépistage du virus du papillome humain 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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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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    • 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

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Abstract

La présente invention a pour but de fournir une méthode de dépistage du VPH avec une sensibilité élevée chez un sujet, et plus particulièrement, de fournir une méthode de dépistage du VPH dans le sperme ou l'urine. L'Invention concerne également une méthode d'amplification du VPH chez un sujet par réaction PCR et le dépistage d'une infection par le VPH, ladite méthode de dépistage d'une infection par le VPH comprenant: (a) dans une première PCR, la mise en oeuvre d'une amplification à travers l'utilisation d'une paire adaptateur-amorce dans laquelle un adaptateur comprenant une séquence qui ne s'hybride pas avec le génome du VPH, un génome humain, et le génome d'un virus ou d'un microbe qui infecte un être humain est accolé à une amorce pour s'hybrider spécifiquement avec un gène VPH; et, (b) dans une seconde PCR, l'amplification du produit d'amplification de (a) à travers l'utilisation d'une paire d'amorces comprenant une séquence qui est complémentaire de l'adaptateur.
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