Oligonucleotides for detecting human papillomavirus

Abstract

To provide a highly sensitive method for detecting the presence of high-risk individuals with human papillomavirus (HPV) in a sample. [Solution] To detect high-risk HPV that may be present in a sample, the present invention provides a method comprising the step of performing a nucleic acid amplification reaction using a reaction solution containing a primer set capable of amplifying specific base sequences derived from HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68.

Claims

1. A method for detecting two or more types of human papillomavirus (HPV) that may be present in a sample, namely types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, the following: (a) A primer set for detecting HPV type 16 that can amplify the region containing the nucleotide sequences 401 to 421 in the nucleotide sequence shown in Sequence ID No. 43; (b) A primer set for detecting HPV type 18 that can amplify the region containing the nucleotide sequences 550 to 573 in the nucleotide sequence shown in Sequence ID No. 44; (c) A primer set for detecting HPV type 31 that can amplify the region containing the nucleotide sequences 539 to 559 in the nucleotide sequence shown in Sequence ID No. 45; (d) A primer set for detecting HPV type 33 that can amplify the region containing the nucleotide sequences 542 to 567 in the nucleotide sequence shown in Sequence ID No. 46; (e) A primer set for detecting HPV type 35 that can amplify the region containing the nucleotide sequences 539 to 559 in the nucleotide sequence shown in Sequence ID No. 47; (f) A primer set for detecting HPV type 39 that can amplify the region containing the nucleotide sequences 751 to 776 in the nucleotide sequence shown in Sequence ID No. 48; (g) A primer set for detecting HPV type 45 that can amplify the region containing the nucleotide sequences 746 to 770 in the nucleotide sequence shown in Sequence ID No. 49; (h) A primer set for detecting HPV type 51 that can amplify the region containing the nucleotide sequences 723 to 742 in the nucleotide sequence shown in Sequence ID No. 50; (i) A primer set for detecting HPV type 52 that can amplify the region containing the nucleotide sequences 538 to 558 in the nucleotide sequence shown in Sequence ID No. 51; (j) A primer set for detecting HPV type 56 that can amplify the region containing the nucleotide sequences 751 to 775 in the nucleotide sequence shown in Sequence ID No. 52; (k) A primer set for detecting HPV type 58 that can amplify the region containing the nucleotide sequences 542 to 567 in the nucleotide sequence shown in Sequence ID No. 53; (l) A primer set for detecting HPV type 59 that can amplify the region containing the nucleotide sequence 750 to 775 in the nucleotide sequence shown in Sequence ID No. 54; (m) A primer set for detecting HPV type 66 that can amplify the region containing the nucleotide sequences 751 to 775 in the nucleotide sequence shown in Sequence ID No. 55; and (n) A primer set for detecting HPV68 that can amplify the region containing the nucleotide sequences 752 to 777 in the nucleotide sequence shown in Sequence ID No.

56. A method comprising the step of performing a nucleic acid amplification reaction using a reaction solution containing one or more primer sets selected from the group consisting of, which are commonly used for the detection of two or more HPV types, and / or two or more primer sets used in specific combinations for the detection of two or more HPV types.

2. The primer set (a) is A primer having a consecutive nucleotide sequence including positions 285-307 in Sequence ID No. 43, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 422-443 in Sequence ID No. 43, a complementary nucleotide sequence thereof, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those sequences; The primer set (b) is A primer having a consecutive nucleotide sequence including positions 488-509 in Sequence ID No. 44, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 621-642 in Sequence ID No. 44, a complementary nucleotide sequence thereof, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those sequences; The primer set (c) is A primer having a consecutive nucleotide sequence including positions 501 to 523 in Sequence ID No. 45, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 563 to 584 in Sequence ID No. 45, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; The aforementioned primer set (d) A primer having a consecutive nucleotide sequence including positions 513 to 535 in Sequence ID No. 46, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 575-596 in Sequence ID No. 46, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those sequences; The primer set (e) is A primer having a consecutive nucleotide sequence including positions 501 to 523 in Sequence ID No. 47, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 566 to 587 in Sequence ID No. 47, a complementary nucleotide sequence thereof, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those sequences; The aforementioned primer set (f) A primer having a consecutive nucleotide sequence including positions 599-620 in Sequence ID No. 48, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 807 to 827 in Sequence ID No. 48, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; The primer set (g) is A primer having a consecutive nucleotide sequence including positions 597-618 in Sequence ID No. 49, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 808 to 827 in Sequence ID No. 49, a complementary nucleotide sequence thereof, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those sequences; The aforementioned primer set (h) A primer having a consecutive nucleotide sequence including positions 656 to 677 in Sequence ID No. 50, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 741-762 in Sequence ID No. 50, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; The primer set (i) A primer having a consecutive nucleotide sequence including positions 500 to 522 in Sequence ID No. 51, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 568-589 in Sequence ID No. 51, a complementary nucleotide sequence thereof, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; The aforementioned primer set (j) A primer having a consecutive nucleotide sequence including positions 727 to 747 in Sequence ID No. 52, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 789 to 808 in Sequence ID No. 52, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; The aforementioned primer set (k) A primer having a consecutive nucleotide sequence including positions 513 to 535 in Sequence ID No. 53, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 578-599 in Sequence ID No. 53, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those sequences; The aforementioned primer set (l) A primer having a consecutive nucleotide sequence including positions 602-623 in Sequence ID No. 54, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 803 to 823 in Sequence ID No. 54, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; The aforementioned primer set (m) A primer having a consecutive nucleotide sequence including positions 727-747 in Sequence ID No. 55, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 789-808 in Sequence ID No. 55, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those sequences; and / or The aforementioned primer set (n) A primer having a consecutive nucleotide sequence including positions 597-618 in Sequence ID No. 56, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers having a consecutive nucleotide sequence including positions 804 to 826 in Sequence ID No. 56, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; The method according to claim 1, wherein in each primer set, one primer is complementary to the extension product of the other primer.

3. The primer set (a) is A primer consisting of the nucleotide sequence shown in Sequence ID No. 15, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 16, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The primer set (b) is A primer consisting of the nucleotide sequence shown in Sequence ID No. 18, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 19, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The primer set (c) is A primer consisting of the nucleotide sequence shown in Sequence ID No. 21, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 22, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The aforementioned primer set (d) A primer consisting of the nucleotide sequence shown in Sequence ID No. 24, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 25, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The primer set (e) is A primer consisting of the nucleotide sequence shown in Sequence ID No. 21, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 22, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The aforementioned primer set (f) A primer consisting of the nucleotide sequence shown in Sequence ID No. 27, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 28, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The primer set (g) is A primer consisting of the nucleotide sequence shown in Sequence ID No. 30, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 31, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The aforementioned primer set (h) A primer consisting of the nucleotide sequence shown in Sequence ID No. 33, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 34, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The primer set (i) A primer consisting of the nucleotide sequence shown in Sequence ID No. 24, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 25, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The aforementioned primer set (j) A primer consisting of the nucleotide sequence shown in Sequence ID No. 36, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 37, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The aforementioned primer set (k) A primer consisting of the nucleotide sequence shown in Sequence ID No. 24, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 25, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The aforementioned primer set (l) A primer consisting of the nucleotide sequence shown in Sequence ID No. 30, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 28, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The aforementioned primer set (m) A primer consisting of the nucleotide sequence shown in Sequence ID No. 36, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 37, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; and / or The aforementioned primer set (n) A primer consisting of the nucleotide sequence shown in Sequence ID No. 27, its complementary nucleotide sequence, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set comprising primers consisting of the nucleotide sequence shown in Sequence ID No. 39, its complementary nucleotide sequence, or nucleotide sequences in which one to three nucleotides are substituted, deleted, inserted, or added; The method according to claim 1, wherein in each primer set, one primer is complementary to the extension product of the other primer.

4. The method according to claim 1, wherein the reaction solution comprises one, two, three, four, five, six, seven, or eight primers selected from the group consisting of the primer sets (c), (d), (e), (g), (i), (j), (k), and (m).

5. The method according to claim 1, wherein the reaction solution comprises the primer set (a) and / or (b).

6. The method according to claim 1, wherein the reaction solution comprises all of the primer sets (a) to (n).

7. below: (a) HPV16 detection probes having a continuous nucleotide sequence including positions 401 to 421 in Sequence ID No. 43, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (b) HPV18 detection probe having a consecutive nucleotide sequence including positions 550 to 573 in Sequence ID No. 44, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences. (c) HPV31 detection probes having a continuous nucleotide sequence including positions 539 to 559 in Sequence ID No. 45, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (d) HPV33 detection probes having a continuous nucleotide sequence including positions 542 to 567 in Sequence ID No. 46, a complementary nucleotide sequence thereof, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those sequences; (e) HPV35 detection probes having a continuous nucleotide sequence including positions 539 to 559 in Sequence ID No. 47, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (f) HPV 39 detection probes having a continuous nucleotide sequence including positions 751 to 776 in Sequence ID No. 48, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (g) HPV 45 detection probes having a continuous nucleotide sequence including positions 746 to 770 in Sequence ID No. 49, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (h) HPV51 detection probes having a continuous nucleotide sequence including positions 723 to 742 in Sequence ID No. 50, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (i) HPV52 detection probes having a continuous nucleotide sequence including positions 538 to 558 in Sequence ID No. 51, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (j) HPV 56 detection probes having a continuous nucleotide sequence including positions 751 to 775 in Sequence ID No. 52, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (k) HPV58 detection probes having a continuous nucleotide sequence including positions 542 to 567 in Sequence ID No. 53, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (l) HPV 59 detection probes having a continuous nucleotide sequence including positions 750 to 775 in Sequence ID No. 54, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (m) HPV66 detection probes having a continuous nucleotide sequence including positions 751 to 775 in Sequence ID No. 55, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; and (n) HPV68 detection probe having a consecutive nucleotide sequence including positions 752 to 777 in Sequence ID No. 56, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences. The method according to claim 1, comprising the step of detecting an HPV type using one or more probes selected from the group consisting of, which are commonly used for detecting two or more HPV types, and / or two or more probes which are used in a specific combination for detecting two or more HPV types.

8. The probe (a) is a probe consisting of the base sequence shown in Sequence ID No. 17, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (b) is a probe consisting of the base sequence shown in Sequence ID No. 20, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (c) is a probe consisting of the base sequence shown in Sequence ID No. 23, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (d) is a probe consisting of the base sequence shown in Sequence ID No. 26, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (e) is a probe consisting of the base sequence shown in Sequence ID No. 23, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (f) is a probe consisting of the base sequence shown in Sequence ID No. 29, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (g) is a probe consisting of the base sequence shown in Sequence ID No. 32, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (h) is a probe consisting of the base sequence shown in Sequence ID No. 35, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (i) is a probe consisting of the base sequence shown in Sequence ID No. 23, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (j) is a probe consisting of the base sequence shown in Sequence ID No. 38, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (k) is a probe consisting of the base sequence shown in Sequence ID No. 26, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (l) is a probe consisting of the base sequence shown in Sequence ID No. 29, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; The probe (m) is a probe consisting of the base sequence shown in Sequence ID No. 38, its complementary base sequence, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; and / or The method according to claim 7, wherein the probe (n) is a probe comprising the base sequence shown in Sequence ID No. 29, a complementary base sequence thereof, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences.

9. The method according to claim 7, wherein at least one, two, three, four, five, six, seven, or eight probes selected from the group consisting of (c), (d), (e), (g), (i), (j), (k), and (m) are used.

10. The method according to claim 7, wherein at least probe (a) and / or (b) is used.

11. The method according to claim 7, wherein all of the aforementioned probes (a) to (n) are used.

12. The method according to claim 7, wherein the probes (a) to (n) are fluorescently labeled probes.

13. The method according to claim 12, wherein the fluorescently labeled probe is a TaqMan probe.

14. The method according to claim 12, wherein the fluorescently labeled probe is labeled with a fluorescent compound detectable by the same fluorescent channel.

15. The method according to claim 1, wherein the reaction solution contains a heat-stable DNA polymerase belonging to Family A.

16. The method according to claim 15, wherein the heat-stable DNA polymerase belonging to Family A is at least one selected from the group consisting of Tth polymerase, Hawk Z05 polymerase, and their variants.

17. The method according to claim 1, wherein the reaction solution contains 1 mM or more of divalent cations.

18. The method according to claim 1, which is a one-step RT-PCR method.

19. The method according to claim 1, wherein the sample is a sample that has not undergone nucleic acid purification.

20. The method according to claim 1, wherein the sample is a cervical swab sample, a mouthwash sample, a urine sample, or a cervical canal swab sample.

21. The method according to claim 1, wherein the sample is a suspension or concentrate thereof suspended in a preserve site solution, water, physiological saline, or buffer solution.

22. A reagent or kit for use in the method according to any one of claims 1 to 21, comprising one or more primer sets selected from the primer sets (a) to (n) for common use in detecting two or more HPV types, and / or two or more primer sets used in specific combinations for detecting two or more HPV types.

23. The reagent or kit according to claim 22, comprising one or more probes selected from probes (a) to (n) described in claim 7, which are used in common for the detection of two or more HPV types, and / or two or more probes which are used in specific combinations for the detection of two or more HPV types.

24. At least one primer set selected from the following groups: (a) A primer having a continuous nucleotide sequence including positions 285 to 307 in Sequence ID No. 43, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 16, comprising primers having a consecutive nucleotide sequence including positions 422-443 in sequence number 43, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those sequences; (b) A primer having a consecutive nucleotide sequence including positions 488 to 509 in Sequence ID No. 44, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 18, comprising primers having a consecutive nucleotide sequence including positions 621-642 in sequence number 44, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those sequences; (c) A primer having a continuous nucleotide sequence including positions 501 to 523 in Sequence ID No. 45, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 31, comprising primers having a consecutive nucleotide sequence including positions 563 to 584 in sequence number 45, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (d) A primer having a consecutive nucleotide sequence including positions 513 to 535 in Sequence ID No. 46, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 33, comprising primers having a continuous nucleotide sequence including positions 575-596 in sequence number 46, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those sequences; (e) A primer having a consecutive nucleotide sequence including positions 501 to 523 in Sequence ID No. 47, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 35, comprising primers having a consecutive nucleotide sequence including positions 566 to 587 in sequence number 47, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (f) A primer having a continuous nucleotide sequence including positions 599 to 620 in Sequence ID No. 48, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 39, comprising primers having a continuous nucleotide sequence including positions 807 to 827 in Sequence ID No. 48, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (g) A primer having a continuous nucleotide sequence including positions 597 to 618 in Sequence ID No. 49, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 45, comprising primers having a continuous nucleotide sequence including positions 808 to 827 in Sequence ID No. 49, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (h) A primer having a continuous nucleotide sequence including positions 656 to 677 in Sequence ID No. 50, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 51, comprising primers having a consecutive nucleotide sequence including positions 741 to 762 in Sequence ID No. 50, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (i) A primer having a consecutive nucleotide sequence including positions 500 to 522 in Sequence ID No. 51, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 52, comprising primers having a consecutive nucleotide sequence including positions 568-589 in Sequence ID No. 51, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those sequences; (j) A primer having a consecutive nucleotide sequence including positions 727 to 747 in Sequence ID No. 52, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 56, comprising primers having a continuous nucleotide sequence including positions 789 to 808 in Sequence ID No. 52, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (k) A primer having a continuous nucleotide sequence including positions 513 to 535 in Sequence ID No. 53, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 58, comprising primers having a continuous nucleotide sequence including positions 578-599 in Sequence ID No. 53, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added in those sequences; (l) A primer having a consecutive nucleotide sequence including positions 602 to 623 in Sequence ID No. 54, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 59, comprising primers having a consecutive nucleotide sequence including positions 803 to 823 in Sequence ID No. 54, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (m) A primer having a continuous nucleotide sequence including positions 727 to 747 in Sequence ID No. 55, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, A primer set for detecting HPV type 66, comprising primers having a continuous nucleotide sequence including positions 789 to 808 in Sequence ID No. 55, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; and (n) A primer having a continuous nucleotide sequence including positions 597 to 618 in Sequence ID No. 56, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, HPV68 detection primer set comprising primers having a consecutive nucleotide sequence including positions 804-826 in Sequence ID No. 56, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 nucleotides are substituted, deleted, inserted, or added to those sequences. (Here, in each primer set, one primer is complementary to the extension product of the other primer.)

25. At least one probe selected from the following group: (a) HPV16 detection probes having a continuous nucleotide sequence including positions 401 to 421 in Sequence ID No. 43, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (b) Numbers 550-573 in Sequence ID No. 44 HPV-18 detection probes having a continuous base sequence including the nucleotide sequence, a complementary base sequence thereof, or a base sequence in which one to three bases are substituted, deleted, inserted, or added in those base sequences; (c) HPV31 detection probes having a continuous nucleotide sequence including positions 539 to 559 in Sequence ID No. 45, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (d) HPV33 detection probes having a continuous nucleotide sequence including positions 542 to 567 in Sequence ID No. 46, a complementary nucleotide sequence thereof, or a nucleotide sequence in which one to three nucleotides are substituted, deleted, inserted, or added in those sequences; (e) HPV35 detection probes having a continuous nucleotide sequence including positions 539 to 559 in Sequence ID No. 47, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (f) HPV 39 detection probes having a continuous nucleotide sequence including positions 751 to 776 in Sequence ID No. 48, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (g) HPV 45 detection probes having a continuous nucleotide sequence including positions 746 to 770 in Sequence ID No. 49, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (h) HPV51 detection probes having a continuous nucleotide sequence including positions 723 to 742 in Sequence ID No. 50, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (i) HPV52 detection probes having a continuous nucleotide sequence including positions 538 to 558 in Sequence ID No. 51, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (j) HPV 56 detection probes having a continuous nucleotide sequence including positions 751 to 775 in Sequence ID No. 52, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (k) HPV58 detection probes having a continuous nucleotide sequence including positions 542 to 567 in Sequence ID No. 53, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (l) HPV 59 detection probes having a continuous nucleotide sequence including positions 750 to 775 in Sequence ID No. 54, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (m) HPV66 detection probes having a continuous nucleotide sequence including positions 751 to 775 in Sequence ID No. 55, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; and (n) A probe for detecting HPV type 68 having a continuous nucleotide sequence including positions 752 to 777 in sequence number 56, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences.

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