Oligonucleotides for detecting human papillomavirus

Abstract

To provide a means for highly sensitively detecting the presence of HPV high-risk candidate groups in a sample. [Solution] A method for detecting human papillomavirus (HPV) types 26, 53, 70, 73, and / or 82 that may be contained in a sample includes a step of performing a nucleic acid amplification reaction using a reaction solution that includes a primer set capable of amplifying (A) a region containing the 97th to 126th nucleotide sequence in a specific nucleotide sequence, (B) a region containing the 192nd to 213th nucleotide sequence in a specific nucleotide sequence, (C) a region containing the 40th to 59th nucleotide sequence in a specific nucleotide sequence, (D) a region containing the 386th to 407th nucleotide sequence in a specific nucleotide sequence, and / or (E) a region containing the 340th to 364th nucleotide sequence in a specific nucleotide sequence.

Description

Technical Field

[0001] The present invention relates to a method for detecting human papillomavirus (HPV) type 26, type 53, type 70, type 73, and / or type 82 that may be contained in a sample, and reagents therefor (for example, oligonucleotides such as primers and probes), kits, etc.

Background Art

[0002] Human papillomavirus (also abbreviated as HPV) is a non-enveloped double-stranded DNA virus that infects and proliferates in human skin and mucous membranes. When the DNA sequence of HPV differs by more than 10%, it is classified as a different type, and currently, more than 200 types of HPV have been identified. It has been found that the carcinogenic risk degree of HPV varies depending on the type. For example, HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 66, and 68 classified into the high-risk group are known to be the causes of cervical cancer, vaginal cancer / vulvar cancer, penile cancer, and head and neck cancer. For example, it is also said that about 90% of the HPV detected from cervical cancer patients account for types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 66, and 68 classified into this high-risk group.

[0003] Furthermore, in recent years, it has been reported that HPV types 26, 53, 70, 73, and 82 have been detected from the cervical cells of cervical cancer patients, and these HPV types are called the high-risk candidate group (Non-Patent Document 1). For example, it is also said that about 5% of the HPV detected from cervical cancer patients are HPV types of this high-risk candidate group.

[0004] When HPV infects the cervix, 90% of cases are eliminated by the innate immune system. However, if the infection persists for a long period, HPV can be incorporated into epithelial cells, immortalizing the cells or destabilizing their genome, potentially leading to cervical cancer. Therefore, regular testing of the cervix for HPV infection is important in preventing cervical cancer.

[0005] Various HPV detection methods have been developed to date. Among these testing methods, nucleic acid amplification technology, which can detect HPV with high sensitivity, is widely used.

[0006] However, conventionally, most HPV tests target high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 66, and 68, and there are very few known methods that can test for the aforementioned high-risk candidate groups at a practically sufficient level. As a result, even though high-risk candidate HPV types can also cause cervical cancer, commercially available HPV tests that target only high-risk HPV types will result in negative results for approximately 5% of patients infected with high-risk candidate groups, leading to delays in the prevention or treatment of cervical cancer.

[0007] Generally, when detecting multiple types of nucleic acids simultaneously using nucleic acid amplification reactions such as PCR, a single nucleic acid amplification reaction system will contain multiple sets of primers and probes. In this case, since nucleic acid amplification reactions for multiple target nucleic acids are carried out in a single solution, it is known that the multiple sets of primers and probes contained in the reaction solution mutually influence the amplification reactions of each nucleic acid. Furthermore, the fact that each primer has a different Tm value also affects the design of the optimal reaction system. As a result, nonspecific amplification or fundamental amplification failure is likely to occur in the nucleic acid amplification reaction. For this reason, selecting the amplification region in a primer set that can achieve high-sensitivity detection in nucleic acid amplification reactions targeting multiple nucleic acids is by no means easy. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] M Arbyn, et al., J Pathol 2014; 234: 431-435 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] An object of the present invention is to provide a means for highly sensitive and efficient detection of high-risk candidate HPV types (particularly HPV types 26, 53, 70, 73, and / or 82) that may be present in a sample. [Means for solving the problem]

[0010] As a result of diligent research, the inventors have found that by selecting and using specific oligonucleotides, it is possible to detect a group of HPV high-risk candidate strains (particularly HPV types 26, 53, 70, 73, and / or 82) that may be present in a sample with high sensitivity and efficiency. Based on this finding, the inventors further diligently conducted research and completed the present invention.

[0011] The present invention encompasses embodiments described in the following sections. [Section 1] A method for detecting human papillomavirus (HPV) types 26, 53, 70, 73, and / or 82 that may be present in a sample, (A) A primer set for detecting HPV26 that can amplify the region containing the nucleotide sequence 97-126 in the nucleotide sequence shown in Sequence ID No. 1; (B) A primer set for detecting HPV type 53 that can amplify the region containing the nucleotide sequence 192-213 in the nucleotide sequence shown in Sequence ID No. 2; (C) A primer set for detecting HPV70 that can amplify the region containing the 40th to 59th nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 3; (D) A primer set for detecting HPV73 that can amplify the region containing the nucleotide sequence 386-407 in the nucleotide sequence shown in Sequence ID No. 4; and (E) A primer set for detecting HPV82 that can amplify the region containing the nucleotide sequence 340-364 in the nucleotide sequence shown in Sequence ID No. 5. A method comprising the step of carrying out a nucleic acid amplification reaction using a reaction solution containing at least one selected from the group consisting of the following.

[0012] [Section 2] The primer set (A) is a primer set comprising a primer having a sequence of bases including positions 19 to 38 in SEQ ID NO: 1, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences, and a primer having a sequence of bases including positions 169 to 188 in SEQ ID NO: 1, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; The primer set (B) is a primer set comprising a primer having a sequence of bases including positions 109 to 128 in SEQ ID NO: 2, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences, and a primer having a sequence of bases including positions 253 to 272 in SEQ ID NO: 2, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; The primer set (C) is a primer set comprising a primer having a sequence of bases including positions 3 to 22 in SEQ ID NO: 3, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences, and a primer having a sequence of bases including positions 169 to 188 in SEQ ID NO: 3, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; The primer set (D) is a primer set comprising a primer having a sequence of bases including positions 307 to 326 in SEQ ID NO: 4, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences, and a primer having a sequence of bases including positions 419 to 438 in SEQ ID NO: 4, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; and / or The primer set (E) is a primer set comprising a primer having a sequence of bases including positions 315 to 334 in SEQ ID NO: 5, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences, and a primer having a sequence of bases including positions 398 to 417 in SEQ ID NO: 5, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences, The method according to item 1, wherein in each primer set, one primer is complementary to the extension product of the other primer.

[0013] [Section 3] The primer set (A) is a primer set comprising a primer consisting of a nucleotide sequence of the 19th to 38th base sequence of Sequence ID No. 1, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those nucleotide sequences, and a primer consisting of a nucleotide sequence of the 169th to 188th base sequence of Sequence ID No. 1, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those nucleotide sequences; The primer set (B) is a primer set comprising a primer consisting of a nucleotide sequence of the nucleotide sequence 109 to 128 of SEQ ID NO: 2, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, and a primer consisting of a nucleotide sequence of the nucleotide sequence 253 to 272, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; The primer set (C) is a primer set comprising a primer consisting of a nucleotide sequence of the 3rd to 22nd base sequence of Sequence ID No. 3, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those nucleotide sequences, and a primer consisting of a nucleotide sequence of the 169th to 188th base sequence of Sequence ID No. 3, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those nucleotide sequences; The primer set (D) is a primer set comprising a primer consisting of a nucleotide sequence of the 307-326th nucleotide sequence of SEQ ID NO: 4, its complementary nucleotide sequence, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, and a primer consisting of a nucleotide sequence of the 419-438th nucleotide sequence of SEQ ID NO: 4, its complementary nucleotide sequence, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; and / or The primer set (E) is a primer set comprising a primer consisting of a nucleotide sequence of the 315th to 334th base sequence of SEQ ID NO: 5, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those nucleotide sequences, and a primer consisting of a nucleotide sequence of the 398th to 417th base sequence of SEQ ID NO: 5, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those nucleotide sequences. The method according to item 1 or 2, wherein in each primer set, one primer is complementary to the extension product of the other primer.

[0014] [Section 4] The method according to any one of items 1 to 3, wherein the reaction solution comprises all of the primer sets (A) to (E).

[0015] [Section 5] The reaction solution is (I) HPV26 type detection probes having a sequence of bases including positions 97 to 126 in Sequence ID No. 1, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; (II) A continuous base sequence containing positions 192 to 213 in SEQ ID NO: 2, its complementary base sequence, or a base sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those base sequences, a probe for detecting HPV type 53; (III) A continuous base sequence containing positions 40 to 59 in SEQ ID NO: 3, its complementary base sequence, or a base sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those base sequences, a probe for detecting HPV type 70; (IV) A continuous base sequence containing positions 386 to 407 in SEQ ID NO: 4, its complementary base sequence, or a base sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those base sequences, a probe for detecting HPV type 73; and (V) A continuous base sequence containing positions 340 to 364 in SEQ ID NO: 5, its complementary base sequence, or a base sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those base sequences, a probe for detecting HPV type 82 The method according to any one of items 1 to 4, comprising at least one selected from the group consisting of

[0016] [Item 6] The probe (I) is a probe consisting of the base sequence of positions 97 to 126 in SEQ ID NO: 1, its complementary base sequence, or a base sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those base sequences; The probe (II) is a probe consisting of the base sequence of positions 192 to 213 in SEQ ID NO: 2, its complementary base sequence, or a base sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those base sequences; The probe (III) is a probe consisting of the 40th to 59th base sequence of Sequence ID No. 3, its complementary base sequence, or a base sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those base sequences; The probe (IV) is a probe consisting of the nucleotide sequence 386-407 of SEQ ID NO: 4, its complementary nucleotide sequence, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added; and / or The method according to claim 5, wherein the probe (V) is a probe comprising the base sequence of position 340 to 364 of sequence number 5, a complementary base sequence thereof, or a base sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) bases are substituted, deleted, inserted, or added in those base sequences.

[0017] [Section 7] The method according to item 5 or 6, wherein the reaction solution comprises all of the probes (I) to (V).

[0018] [Section 8] The method according to any one of items 5 to 7, wherein the probes (I) to (V) are fluorescently labeled probes.

[0019] [Section 9] The method according to item 8, wherein the fluorescently labeled probe is a TaqMan probe.

[0020] [Section 10] The method according to any one of claims 1 to 9, wherein the reaction solution contains a heat-stable DNA polymerase belonging to Family A.

[0021] [Section 11] The method according to claim 10, wherein the heat-stable DNA polymerase belonging to Family A is at least one selected from the group consisting of Taq polymerase, Tth polymerase, Hawk Z05 polymerase, and their variants.

[0022] [Section 12] The method according to any one of items 1 to 11, wherein the reaction solution contains 1 mM or more of divalent cations.

[0023] [Section 13] The method according to any one of items 1 to 12, wherein the nucleic acid amplification reaction is a PCR reaction.

[0024] [Section 14] The method according to any one of items 1 to 13, wherein the sample is a sample that has not undergone nucleic acid purification.

[0025] [Section 15] The method according to any one of items 1 to 14, wherein the sample is a cervical swab sample, a mouthwash sample, a urine sample, or a cervical canal swab sample.

[0026] [Section 16] The method according to any one of items 1 to 15, wherein the sample is a suspension or concentrate thereof suspended in liquid cytological preservation solution, water, physiological saline, or buffer solution.

[0027] [Section 17] A reagent or kit for use in the method described in any of items 1 to 16, comprising at least one selected from the group consisting of the primer sets (A) to (E).

[0028] [Section 18] The reagent or kit according to item 17, comprising at least one selected from the group consisting of probes (I) to (V) as described in item 5 or 6.

[0029] [Section 19] At least one primer set selected from the following groups: (A) A primer set for detecting HPV type 26, comprising: (A) a primer having a sequence of bases including positions 19 to 38 in SEQ ID NO: 1, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; and a primer having a sequence of bases including positions 169 to 188 in SEQ ID NO: 1, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; (B) A primer set for detecting HPV type 53, comprising a primer having a sequence of bases including positions 109 to 128 in SEQ ID NO: 2, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; and a primer having a sequence of bases including positions 253 to 272 in SEQ ID NO: 2, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; (C) A primer set for detecting HPV70 type comprising: a primer having a consecutive nucleotide sequence including positions 3 to 22 in SEQ ID NO: 3, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; and a primer having a consecutive nucleotide sequence including positions 169 to 188 in SEQ ID NO: 3, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences; (D) A primer set for detecting HPV73 type comprising a primer having a sequence of bases including positions 307 to 326 in SEQ ID NO: 4, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences, and a primer having a sequence of bases including positions 419 to 438 in SEQ ID NO: 4, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; and (E) A primer set for detecting HPV82 type comprising a primer having a sequence of bases including positions 315 to 334 in SEQ ID NO: 5, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added, and a primer having a sequence of bases including positions 398 to 417 in SEQ ID NO: 5, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added. (Here, in each primer set, one primer is complementary to the extension product of the other primer.)

[0030] [Section 20] At least one probe selected from the following group: (I) HPV26 type detection probes having a sequence of bases including positions 97 to 126 in Sequence ID No. 1, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; (II) HPV53 type detection probes having a sequence of bases including positions 192-213 in Sequence ID No. 2, a complementary sequence thereof, or a sequence in which 1-3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added; (III) HPV70 type detection probes having a sequence of bases including positions 40 to 59 in Sequence ID No. 3, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added; (IV) HPV73 type detection probes having a consecutive nucleotide sequence including positions 386 to 407 in Sequence ID No. 4, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added; and (V) A probe for detecting HPV82 having a sequence of bases including positions 340 to 364 in Sequence ID No. 5, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added. [Effects of the Invention]

[0031] According to one aspect of the present invention, it is possible to efficiently and sensitively detect HPV types from samples that may contain high-risk candidate HPV types. Since it has been pointed out that long-term infection with high-risk candidate HPV types can lead to cervical cancer, the ability to efficiently and sensitively test for such high-risk candidate HPV types is of great significance. According to one aspect of the present invention, it is also possible to efficiently and sensitively detect high-risk candidate HPV types from samples suspended in liquid cytology preservation solution, water, physiological saline, or buffer solution. The present invention can be effectively used in life science research, clinical diagnosis, and the like. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 shows the amplification curve of real-time PCR for Test Example 1. [Figure 2] Figure 2 shows the amplification curve of real-time PCR for test example 4. [Modes for carrying out the invention]

[0033] The present invention will be described in more detail below with reference to embodiments, but the present invention is not limited to these embodiments.

[0034] All non-patent and patent documents described herein are incorporated herein by reference and are incorporated in their entirety into the specification. Furthermore, "~" in this specification means "greater than or equal to, less than or equal to," for example, "X~Y" in the specification means "greater than or equal to X, less than or equal to Y." "And / or" in this specification means any one or more possible combinations of the listed elements. "Includes" in this specification encompasses the concepts of "substantially consisting of" and "consisting only of."

[0035] One embodiment of the present invention is a method for detecting human papillomavirus (HPV) types 26, 53, 70, 73, and / or 82 that may be present in a sample, comprising the step of carrying out a nucleic acid amplification reaction using a reaction solution containing at least one primer set (also referred to as a "nucleic acid primer set," "primer pair," etc.) selected from the group consisting of (A), (B), (C), (D), and (E) below: (A) A primer set for detecting HPV26 that can amplify the region containing the nucleotide sequence 97-126 in the nucleotide sequence shown in Sequence ID No. 1; (B) A primer set for detecting HPV type 53 that can amplify the region containing the nucleotide sequence 192-213 in the nucleotide sequence shown in Sequence ID No. 2; (C) A primer set for detecting HPV70 that can amplify the region containing the 40th to 59th nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 3; (D) A primer set for detecting HPV73 that can amplify the region containing the nucleotide sequence 386-407 in the nucleotide sequence shown in Sequence ID No. 4; and (E) A primer set for detecting HPV82 that can amplify the region containing the nucleotide sequence 340-364 in the nucleotide sequence shown in Sequence ID No. 5.

[0036] The length of the region amplified by each of the primer sets (A) to (E) is not particularly limited as long as it is a length suitable for nucleic acid amplification, but for example it may be 50 to 250 nucleotides long, preferably 75 to 200 nucleotides long. The length of each primer may be, for example, 15 to 31 nucleotides long, preferably 16 to 30 nucleotides long, more preferably 17 to 29 nucleotides long, and even more preferably 18 to 28 nucleotides long.

[0037] One embodiment of the present invention is characterized by targeting specific regions in high-risk candidate HPV types (particularly HPV types 26, 53, 70, 73, and / or 82) and detecting those regions by nucleic acid amplification reaction. HPV is known to encode E1, E2, E4, E5, E6, E7, L1, and L2 genes in the ORF that encodes the viral protein. Among these, it is preferable to target specific regions in the E6 / E7 gene sequence to detect high-risk candidate HPV types, given their association with carcinogenicity. In this specification, Sequence ID 21 is the wild-type nucleotide sequence for HPV26, Sequence ID 1 is the nucleotide sequence encoding the E6 / E7 gene for HPV26, Sequence ID 22 is the wild-type nucleotide sequence for HPV53, Sequence ID 2 is the nucleotide sequence encoding the E6 / E7 gene for HPV53, Sequence ID 23 is the wild-type nucleotide sequence for HPV70, Sequence ID 3 is the nucleotide sequence encoding the E6 / E7 gene for HPV70, Sequence ID 24 is the wild-type nucleotide sequence for HPV73, Sequence ID 4 is the nucleotide sequence encoding the E6 / E7 gene for HPV73, Sequence ID 25 is the wild-type nucleotide sequence for HPV82, and Sequence ID 5 is the nucleotide sequence encoding the E6 / E7 gene for HPV82.

[0038] The primer set is not particularly limited as long as it is designed to amplify specific regions in each HPV type. For example, primer set (A) may be a combination of a primer designed to anneal to a position upstream of base sequences 97-126 in the sequence shown in Sequence ID No. 1 (for example, a position approximately 1-100 bases upstream) and a primer designed to anneal to a position downstream of base sequences 97-126 (for example, a position approximately 1-100 bases downstream), wherein one primer is designed to be complementary to the DNA extension product of the other primer. Primer sets (B) to (E) can be designed in the same manner.

[0039] In one preferred embodiment, primer set (A) is A primer having a consecutive nucleotide sequence including positions 19-38 of Sequence ID No. 1, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added, The primer set may include primers having a sequence of bases including positions 169 to 188 of Sequence ID No. 1, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added.

[0040] More preferably, primer set (A) A primer comprising the nucleotide sequence 19-38 of Sequence ID No. 1, its complementary nucleotide sequence, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, The primer set may include primers comprising the nucleotide sequences 169 to 188 of Sequence ID No. 1, their complementary nucleotide sequences, or nucleotide sequences in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added.

[0041] In particular, primer set (A) A primer consisting of the nucleotide sequence from positions 19 to 38 of SEQ ID NO: 1 (forward primer: SEQ ID NO: 6), It is preferable that the primer set includes a primer (reverse primer: SEQ ID NO: 7) consisting of a nucleotide sequence complementary to the nucleotide sequence of positions 169-188 of SEQ ID NO: 1.

[0042] By using the primer set (A) described above, cross-reactivity can be effectively suppressed even in samples containing multiple HPV types, making it easier to specifically detect HPV type 26, which is one of the high-risk candidate types.

[0043] In one preferred embodiment, primer set (B) is A primer having a consecutive nucleotide sequence including positions 109 to 128 of Sequence ID No. 2, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added, The primer set may include primers having a consecutive nucleotide sequence containing positions 253 to 272 of Sequence ID No. 2, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added.

[0044] More preferably, primer set (B) A primer comprising the nucleotide sequence 109-128 of Sequence ID No. 2, its complementary nucleotide sequence, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, The primer set may include primers comprising the nucleotide sequences 253 to 272 of Sequence ID No. 2, their complementary nucleotide sequences, or nucleotide sequences in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added.

[0045] In particular, primer set (B) A primer consisting of the nucleotide sequence from positions 109 to 128 of Sequence ID No. 2 (forward primer: Sequence ID No. 9), It is preferable that the primer set includes a primer (reverse primer: SEQ ID NO: 10) consisting of a nucleotide sequence complementary to the nucleotide sequence at positions 253-272 of SEQ ID NO: 2.

[0046] By using the primer set (B) described above, cross-reactivity can be effectively suppressed even in samples containing multiple HPV types, making it easier to specifically detect HPV type 53, which is one of the high-risk candidate types.

[0047] In one preferred embodiment, the primer set (C) is A primer having the nucleotide sequence of position 3 to 22 of Sequence ID No. 3, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, The primer set may include primers having the nucleotide sequences 169 to 188 of Sequence ID No. 3, their complementary nucleotide sequences, or nucleotide sequences in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added.

[0048] More preferably, the primer set (C) is A primer comprising the nucleotide sequence from the 3rd to the 22nd base of Sequence ID No. 3, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those nucleotide sequences, The primer set may include primers comprising the nucleotide sequences 169 to 188 of Sequence ID No. 3, their complementary nucleotide sequences, or nucleotide sequences in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added.

[0049] In particular, Primer Set (C) A primer consisting of the nucleotide sequence from positions 3 to 22 of SEQ ID NO: 3 (forward primer: SEQ ID NO: 12), It is preferable that the primer set includes a primer (reverse primer: SEQ ID NO: 13) consisting of a nucleotide sequence complementary to the nucleotide sequence of positions 169-188 of SEQ ID NO: 3.

[0050] By using the primer set (C) described above, cross-reactivity can be effectively suppressed even in samples containing multiple HPV types, making it easier to specifically detect HPV type 70, which is one of the high-risk candidate types.

[0051] In one preferred embodiment, the primer set (D) is A primer having a consecutive nucleotide sequence including positions 307-326 of Sequence ID No. 4, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added, The primer set may include primers having a sequence of bases including positions 419 to 438 of Sequence ID No. 4, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added.

[0052] More preferably, the primer set (D) is A primer comprising the nucleotide sequence 307-326 of Sequence ID No. 4, its complementary nucleotide sequence, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, The primer set may include primers comprising the nucleotide sequences 419-438 of Sequence ID No. 4, their complementary nucleotide sequences, or nucleotide sequences in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added.

[0053] In particular, the primer set (D) A primer consisting of the nucleotide sequence 307-326 of SEQ ID NO: 4 (forward primer: SEQ ID NO: 15), It is preferable that the primer set includes a primer (reverse primer: SEQ ID NO: 16) consisting of a nucleotide sequence complementary to the nucleotide sequence at positions 419-438 of SEQ ID NO: 4.

[0054] By using the primer set (D) described above, cross-reactivity can be effectively suppressed even in samples containing multiple HPV types, making it easier to specifically detect HPV type 73, which is one of the high-risk candidate types.

[0055] In one preferred embodiment, the primer set (E) is A primer having a consecutive nucleotide sequence including positions 315-334 of Sequence ID No. 5, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added, The primer set may include primers having a consecutive nucleotide sequence containing positions 398 to 417 of Sequence ID No. 5, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added.

[0056] More preferably, the primer set (E) is A primer comprising the nucleotide sequence 315-334 of Sequence ID No. 5, its complementary nucleotide sequence, or a nucleotide sequence in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added in those nucleotide sequences, The primer set may include primers comprising the nucleotide sequences 398 to 417 of Sequence ID No. 5, their complementary nucleotide sequences, or nucleotide sequences in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added.

[0057] In particular, the primer set (E) A primer consisting of the nucleotide sequence 315-334 of SEQ ID NO: 5 (forward primer: SEQ ID NO: 18), It is preferable that the primer set includes a primer (reverse primer: SEQ ID NO: 19) consisting of a nucleotide sequence complementary to the nucleotide sequence at positions 398-417 of SEQ ID NO: 5.

[0058] By using the primer set (E) described above, cross-reactivity can be effectively suppressed even in samples containing multiple HPV types, making it easier to specifically detect HPV82, one of the high-risk candidate types.

[0059] The primers (nucleic acid primers) that make up primer sets (A) to (E) may be degenerate primers. When degenerate primers are used, the notation of the mixed bases is as follows, according to the conventions of the art: R=A or G, M=A or C, W=A or T, S=C or G, Y=C or T, K=G or T, H=A or T or C, B=G or T or C, D=G or A or T, V=A or C or G, N=A or C or G or T.

[0060] The primers constituting primer sets (A) to (E) are preferably designed to correspond to the base sequences of the E6 / E7 genes of HPV types 26, 53, 70, 73, and 82, respectively, as shown in SEQ ID NOs.1 to 5. When performing a PCR reaction (including real-time PCR reactions, etc.) using primer sets (A) to (E), the primer pair used in the PCR reaction may be a pair of primers in which one primer is complementary to the DNA extension product of the other primer. Furthermore, primer sets (A) to (E) may also include degenerate primers, and by using degenerate primers, it may be possible to amplify the nucleic acid and detect with sufficient sensitivity even if the HPV type to be detected is a subtype.

[0061] The detection method of the present invention may use all of the primer sets (A) to (E), or may select and use one, two, three, or four of these primer sets. For example, primer set (A) may be used to detect HPV type 26, and primer sets capable of amplifying different regions from primer sets (B) to (E) may be used to detect HPV types 53, 70, 73, and / or 82. Also, in a particular embodiment of the present invention, for example, if it is already known that the suspected HPV type is HPV type 26, primer set (A) may be used to detect HPV type 26 that may be present in the sample, and in this case, it is not necessary to use primer sets for detecting HPV types 53, 70, 73, and / or 82. From the viewpoint of being able to detect infection of high-risk candidate HPV types with higher accuracy, it is preferable to use a combination of two or more, more preferably three or more, and even more preferably four or more primer sets (A) to (E), and it is particularly preferable to use a combination of all of primer sets (A) to (E). By using primer sets (A) to (E) in combination, even when performing multiplex PCR in cases where multiple HPV types may be present, cross-reactivity or nonspecific reactions can be suppressed, making it possible to detect high-risk candidate HPV types with greater specificity.

[0062] When detecting HPV types 26, 53, 70, 73, and 82 using primer sets (A) to (E), the concentrations of the primers constituting the primer set used are not particularly limited as long as the effects of the present invention are achieved. However, for example, when nucleic acid amplification is performed by a PCR reaction (including, for example, a real-time PCR reaction), it is preferable that the concentration of each forward primer is 0.1 μM to 3 μM and the concentration of each reverse primer is 0.1 μM to 3 μM relative to the entire PCR reaction solution. More preferably, by setting the concentration of each forward primer to 0.1 μM to 2 μM and the concentration of each reverse primer to 0.1 μM to 2 μM, it becomes possible to detect high-risk candidate HPV types with higher sensitivity.

[0063] In one preferred embodiment, a method for testing for the presence of HPV types 26, 53, 70, 73, and / or 82 that may be present in a sample may include at least the following steps: (1) A step of preparing one or more primer sets selected from the group consisting of primer sets (A) to (E); (2) A step of preparing a mixture by mixing the sample with one or more primer sets and a PCR reaction solution (including a real-time PCR reaction solution, etc.) that contains heat-resistant DNA polymerase prepared in step (1); and (3) A step of sealing the reaction vessel containing the mixture and then carrying out a PCR reaction (including a real-time PCR reaction, etc.). Here, the step of preparing the mixture in step (2) can be carried out, for example, by adding a PCR reaction solution containing one or more primer sets selected from primer sets (A) to (E) and heat-stable DNA polymerase, etc., to the sample, but the order of mixing is not important.

[0064] In a further embodiment, the testing method (detection method) of the present invention may further include, as step (1'), a pretreatment of a sample that may contain HPV, such as nucleic acid isolation and purification or chemical / thermodynamic treatment, to expose DNA from the HPV capsid structure in the sample. Step (1') may be performed simultaneously with the PCR reaction in step (3).

[0065] In a further embodiment of the detection method of the present invention, it is preferable to perform detection using at least one probe (particularly a labeled hybridization probe) or a double-stranded DNA-binding fluorescent compound. This allows the analysis of the amplified product to be monitored by monitoring the fluorescence signal rather than by electrophoresis, reducing the effort required for analysis. Furthermore, it is possible to reduce the risk of contamination as there is no need to open the reaction vessel. It is also possible to identify the HPV type by labeling each hybridization probe corresponding to HPV types 26, 53, 70, 73, and / or 82 with different fluorescent dyes. Alternatively, if it is necessary to detect whether or not a person is infected with any of the high-risk candidate HPV types without identifying the HPV type, some or all of the hybridization probes corresponding to these HPV types may be labeled with the same fluorescent dye. From the viewpoint of being able to exhibit better specificity, although not limited, it is preferable in the present invention to use hybridization probes, and it is more preferable to label each hybridization probe corresponding to HPV types 26, 53, 70, 73, and 82 with different fluorescent dyes.

[0066] Examples of double-stranded DNA-binding fluorescent compounds include SYBR® Green I, SYBR® Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), EvaGreen® (Biotium), LCGreen (Idaho), and LightCycler® 480 ResoLight (Roche Applied Science).

[0067] Examples of hybridization probes include TaqMan probes (also known as TaqMan hydrolysis probes, etc.; U.S. Patent Nos. 5,210,015, 5,538,848, 5,487,972, and 5,804,375), molecular beacons (U.S. Patent No. 5,118,801), and FRET hybridization probes (International Publication Nos. 97 / 46707, 97 / 46712, and 97 / 46714), with TaqMan probes being preferred.

[0068] In one embodiment, the detection method of the present invention preferably uses at least one selected from the group consisting of the following probes (I) to (V): (I) HPV26 type detection probes having a sequence of bases including positions 97 to 126 in Sequence ID No. 1, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added in those sequences; (II) HPV53 type detection probes having a sequence of bases including positions 192-213 in Sequence ID No. 2, a complementary sequence thereof, or a sequence in which 1-3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added; (III) HPV70 type detection probes having a sequence of bases including positions 40 to 59 in Sequence ID No. 3, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added; (IV) HPV73 type detection probes having a consecutive nucleotide sequence including positions 386 to 407 in Sequence ID No. 4, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added; and (V) A probe for detecting HPV82 having a sequence of bases including positions 340 to 364 in Sequence ID No. 5, a complementary sequence thereof, or a sequence in which 1 to 3 bases (preferably 1 or 2, more preferably 1) are substituted, deleted, inserted, or added.

[0069] The length of the region to which each of the probes (I) to (V) binds (or the length of the probes (I) to (V)) is not particularly limited as long as it is a length suitable for specific detection, but for example it may be 15 to 31 base pairs, preferably 16 to 30 base pairs, more preferably 17 to 29 base pairs, and even more preferably 18 to 28 base pairs.

[0070] Probes (I) to (V) are not particularly limited as long as they can bind to specific regions of high-risk candidate HPV types. Furthermore, probes (I) to (V) may include degenerate sequences. Using degenerate probes may enable highly sensitive detection even when the target HPV type is a subtype.

[0071] In one preferred embodiment, probe (I) is preferably a probe (particularly a fluorescently labeled probe) consisting of the nucleotide sequences 97-126 of SEQ ID NO: 1, their complementary nucleotide sequences, or nucleotide sequences in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added. Using such a probe effectively suppresses cross-reactivity even from samples containing multiple HPV types, making it easier to more specifically detect HPV26, which is one of the high-risk candidate groups.

[0072] In one preferred embodiment, probe (II) is preferably a probe (particularly a fluorescently labeled probe) consisting of the nucleotide sequences 192-213 of SEQ ID NO: 2, their complementary nucleotide sequences, or nucleotide sequences in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added. Using such a probe effectively suppresses cross-reactivity even from samples containing multiple HPV types, making it easier to more specifically detect HPV type 53, which is one of the high-risk candidate groups.

[0073] In one preferred embodiment, probe (III) is preferably a probe (particularly a fluorescently labeled probe) consisting of the nucleotide sequences 40-59 of SEQ ID NO: 3, their complementary nucleotide sequences, or nucleotide sequences in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added. Using such a probe effectively suppresses cross-reactivity even from samples containing multiple HPV types, making it easier to more specifically detect HPV70, which is one of the high-risk candidate types.

[0074] In one preferred embodiment, probe (IV) is preferably a probe (particularly a fluorescently labeled probe) consisting of the nucleotide sequences 386-407 of SEQ ID NO: 4, their complementary nucleotide sequences, or nucleotide sequences in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added. Using such a probe effectively suppresses cross-reactivity even from samples containing multiple HPV types, making it easier to more specifically detect HPV73, which is one of the high-risk candidate types.

[0075] In one preferred embodiment, probe (V) is preferably a probe (particularly a fluorescently labeled probe) consisting of the nucleotide sequences 340-364 of SEQ ID NO: 5, their complementary nucleotide sequences, or nucleotide sequences in which 1-3 (preferably 1 or 2, more preferably 1) nucleotides are substituted, deleted, inserted, or added. Using such a probe effectively suppresses cross-reactivity even from samples containing multiple HPV types, making it easier to more specifically detect HPV82, which is one of the high-risk candidate types.

[0076] The detection method of the present invention may use all of probes (I) to (V), or may select and use one, two, three, or four of them. For example, probe (I) may be used to detect HPV type 26, and probes that can bind to different regions than probes (II) to (V) (especially fluorescently labeled probes) may be used to detect HPV types 53, 70, 73, and / or 82. Also, in a particular embodiment of the present invention, for example, if it has already been determined that the suspected HPV type is HPV type 26, probe (I) may be used to detect HPV type 26 contained in the sample, and in this case, probes for detecting HPV types 53, 70, 73, and / or 82 may not be used. From the viewpoint of being able to detect infection of high-risk candidate HPV types with higher accuracy, it is preferable to use a combination of two or more, more preferably three or more, and even more preferably four or more probes from (I) to (V), and it is particularly preferable to use a combination of all of probes (I) to (V). By using all probes (I) to (V) in combination, even when performing multiplex PCR in cases where multiple HPV types may be present, cross-reactivity or nonspecific reactions can be suppressed, making it possible to detect high-risk candidate HPV types with greater specificity.

[0077] Any fluorescent compound known in the art can be used to label the probe, and can be selected, for example, to match the real-time PCR instrument being used. Specific examples of fluorescent compounds include, but are not limited to, rhodamine-based compounds such as rhodamine (ROX) or its derivatives (e.g., 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine, 5-carboxyrhodamine 6G (CR6G), tetramethylrhodamine (TAMRA)), or salts thereof; fluoroceine or its derivatives (e.g., FAM (carboxyfluorescein), JOE (6-carboxy-4',5'-dichloro2',7'-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (5'-hexachlorofluorescein-CE phosphoramidite), VIC®, BODIPY® series), Cy® dyes (e.g., Cy3, Cy5), or derivatives thereof, or salts thereof; and non-rhodamine-based compounds. Fluorescent compounds can use quenching agents that are appropriate for the fluorescent substance being used, as needed. Examples of quenching agents corresponding to the fluorescent substances mentioned above include, but are not limited to, TAMRA (tetramethyl-rhodamine), DABCYL (4-(4-dimethylaminophenylazo)benzoic acid), BHQ1 (BHQ: Black Hole Quencher®), BHQ2, BHQ3, etc.

[0078] When detecting HPV types 26, 53, 70, 73, and / or 82 using the aforementioned probes (especially fluorescently labeled probes), the concentration of the probes used (especially fluorescently labeled probes) is not particularly limited as long as the effects of the present invention are achieved. For example, it is preferable that the concentration of each probe relative to the entire reaction solution be 0.01 μM or more and 1.0 μM or less, more preferably 0.013 μM or more and 0.75 μM or less, and even more preferably 0.02 μM or more and 0.5 μM or less.

[0079] Examples of samples used in the present invention include, but are not limited to, liquid cytology preservation solutions, cervical swab samples, mouthwash samples, urine samples, and cervical swab samples. Any samples of biological origin can be used. The sample may be used for direct detection, or it may be suspended in water, saline, or a buffer solution to reduce the influence of contaminants on the reaction and obtain more stable test results. Examples of buffer solutions are not particularly limited, but include Hanks buffer, Tris buffer, phosphate buffer, glycine buffer, HEPES buffer, and tricine buffer. Furthermore, the sample used in the present invention may be a suspension in a preserve site solution (liquid cytology preservation solution), water, saline, or a buffer solution, or it may be a concentrate of such suspension. Even when using these samples, it has been confirmed in the examples described later that high-risk candidate HPV types can be detected with high sensitivity.

[0080] In certain preferred embodiments, the sample used in the present invention (for example, a sample derived from a living organism (including secretions from a living organism, etc.), or a sample derived from a living organism suspended in a preserve site solution (liquid cytological preservation solution), etc.) may be a sample that has not undergone prior nucleic acid isolation using a commercially available DNA purification kit, etc., and / or prior heat treatment to expose the DNA from the viral structure. In the method of the present invention, even with a sample that has not undergone prior nucleic acid isolation or heat treatment, heat treatment can be incorporated before and / or during the PCR reaction cycle to expose the DNA from the viral structure and subject it to the PCR reaction. By using a sample that has not undergone prior nucleic acid isolation or heat treatment in this way, not only can viral DNA testing be performed more simply and quickly, but the risk of sample loss and contamination of other samples due to carryover can also be reduced.

[0081] The PCR cycle in step (3) comprises two steps: 1. heat treatment and 2. PCR. Heat treatment steps to activate a hot-start enzyme may be included before and after each step. Heat treatment in step 1 includes a step to disrupt the virus and expose the nucleic acid within the virus, and / or activate a hot-start enzyme in the nucleic acid amplification reaction. Including these heat treatment steps makes it possible to expose (elute) DNA from the viral capsid structure. The temperature and time of heat treatment in step 1 should be 60°C or higher and 1 second or longer, preferably 70°C and 30 seconds or longer, more preferably 80°C and 30 seconds or longer, and particularly preferably 85°C and 30 seconds or longer. PCR in step 2 may include three steps: [1] DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), [2] annealing of primers to template single-stranded DNA, and [3] extension of the primers using DNA polymerase. Steps [2] and [3] may be performed at the same temperature to make it two steps. To perform rapid PCR, it is desirable to set the thermal cycler used for the PCR reaction to have a measurement program that combines the extension times of steps [2] and [3] to 15 seconds or less, more preferably 10 seconds or less. In this specification, "PCR extension time" refers to the time set on the thermal cycler.

[0082] The PCR solution added in step (2) is characterized by containing a heat-resistant DNA polymerase. Heat resistance means that the enzyme activity does not decrease by more than half even after heat treatment at 70°C for 1 minute or more. The origin is not particularly limited, but examples include Taq, Tth, Bst, Bca, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and their variants. The heat-resistant DNA polymerase contained in the PCR reaction solution added in step (2) is not limited as long as it is such a heat-resistant DNA polymerase. Particularly preferred are DNA polymerases belonging to family A, and preferably, heat-resistant DNA polymerases selected from the group consisting of Taq, Tth, Z05 and their variants. In one embodiment, from the viewpoint of easily obtaining high contamination resistance, it is preferable to use at least one selected from the group consisting of Tth polymerase, Hawk Z05 polymerase and their variants. The total amount of heat-stable DNA polymerase contained in the PCR reaction solution is, for example, at least 4.2 ng / μL, preferably 5.0 ng / μL or more, more preferably 5.8 ng / μL or more, and most preferably 8.3 ng / μL or more. There is no particular upper limit to the total amount of heat-stable DNA polymerase contained in the PCR reaction solution, but for example it can be 20 ng / μL or less, and even 16.7 ng / μL or less is sufficient to obtain the effects of the present invention. The amount of polymerase is a value quantified by the Bradford method or Nanodrop (Thermo Fisher), and may also be estimated from the Safety Data Sheet (SDS). If proteins such as BSA are included, it is desirable to calculate using the latter method.

[0083] In this specification, a variant of a heat-stable DNA polymerase refers to a variant that has, for example, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more of the amino acid sequence identity with respect to the wild-type DNA polymerase from which it is derived, and has the same DNA amplification activity as the wild-type DNA polymerase. Here, the method for calculating amino acid sequence identity can be any means known in the art. For example, it can be calculated using commercially available analysis tools or tools available via telecommunication lines (the Internet). As an example, it is possible to calculate amino acid sequence identity by using the default parameters of the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / . Furthermore, the mutants that can be used in the present invention are polypeptides consisting of amino acid sequences in which one or more amino acids are substituted, deleted, inserted, and / or added (hereinafter collectively referred to as "mutations") in the amino acid sequence of the wild-type DNA polymerase from which they are derived, and which may have the same activity as wild-type DNA polymerase in converting DNA to cDNA and in amplifying DNA. Here, one or more may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3, but are not particularly limited.

[0084] In step (2), the PCR reaction solution added is preferably used in combination with an anti-DNA polymerase antibody, or chemically modified to introduce a heat-unstable block group into the DNA polymerase, thereby suppressing the enzymatic activity of the DNA polymerase before the PCR reaction is performed, and enabling application to hot-start PCR.

[0085] The PCR reaction solution added in step (2) may contain, in addition to heat-resistant DNA polymerase, a buffer, a suitable salt (e.g., a magnesium salt), deoxynucleotide triphosphates (dNTPs), a primer pair corresponding to the target region of the viral DNA to be detected, and additional additives as needed.

[0086] The buffering agent included in the PCR solution added in step (2) is not particularly limited, but examples include Tris, Tricin, Bis-Tricine, and Bicine, and it is preferable that the pH is adjusted to 6-9, more preferably 7-9, with sulfuric acid, hydrochloric acid, acetic acid, or phosphoric acid. The concentration of the buffering agent to be added may be 10-200 mM, more preferably 20-150 mM. In this case, it is preferable to add a salt solution to create ionic conditions suitable for the reaction. Examples of salt solutions include, but are not limited to, potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, and ammonium acetate.

[0087] The dNTPs added to the PCR solution in step (2) preferably contain dATP, dCTP, dGTP, and dTTP in concentrations of 0.1 to 0.5 mM each, most commonly around 0.2 mM. Prophylactic measures against cross-contamination may be taken by using dUTP as a substitute for and / or part of dTTP. When prophylactic measures against cross-contamination are taken, it is preferable to include uracil-N-glycosylase (UNG).

[0088] Furthermore, it is preferable that the PCR solution added in step (2) contains divalent cations. Including divalent cations in this way provides greater stability, higher resistance to interference, and enables highly sensitive detection. The divalent cations are not particularly limited, but examples include magnesium ions, manganese ions, calcium ions, copper ions, iron ions, nickel ions, zinc ions, etc. Preferably, the divalent cations include magnesium ions and / or manganese ions. In certain embodiments, it is preferable that the reaction solution contains 1 mM or more of divalent cations, more preferably 1.5 mM or more, and even more preferably 2 mM or more. In the present invention, when magnesium ions are added to the PCR reaction solution, magnesium or a salt thereof may be added. Examples of magnesium or a salt thereof include magnesium, magnesium chloride, magnesium sulfate, magnesium acetate, etc. It is preferable that such magnesium or a salt thereof is added to the PCR reaction solution in a concentration of about 1 to 10 mM. In certain embodiments, the PCR reaction solution preferably contains 1 mM or more of magnesium or a salt thereof, more preferably 1.5 mM or more of magnesium or a salt thereof, and even more preferably 2 mM or more of magnesium or a salt thereof.

[0089] Furthermore, the PCR reaction solution may contain at least one additive selected from the group consisting of a quaternary ammonium salt having a structure in which three methyl groups are added to the amino group of an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), bovine serum albumin, sericin, BFP, glycerol, glycol, gelatin, and surfactant.

[0090] The concentration of bovine serum albumin in the PCR reaction solution is not particularly limited, but is preferably at least 0.5 mg / ml, more preferably at least 1 mg / ml, relative to the total PCR reaction solution. For samples with many impurities, a bovine serum albumin concentration of preferably 2 mg / ml or higher, and more preferably 3 mg / ml or higher, enables good detection. There is no particular upper limit, but as an example, it can be 10 mg / ml or less.

[0091] Examples of surfactants that can be included in the PCR reaction solution include Triton X-100, Triton X-114, Tween 20, Nonidet® P40, Briji 35, Briji 58, SDS, CHAPS, CHAPSO, and Emulgen 420, but are not particularly limited. The concentration of surfactant in the PCR reaction solution is also not particularly limited, but preferably 0.0001 w / v% or higher, more preferably 0.002 w / v% or higher, and even more preferably 0.005 w / v% or higher, which enables good detection. There is no particular upper limit, but as an example, it can be 0.1 w / v% or lower.

[0092] Examples of betaine-like quaternary ammonium compounds included in the PCR reaction solution include betaine (trimethylglycine) and carnitine, but are not particularly limited. The betaine structure is a compound with stable positive and negative charges within the molecule, exhibiting surfactant-like properties and is thought to cause destabilization of the viral structure. Furthermore, it is known to promote nucleic acid amplification by DNA polymerase. The preferred concentration of betaine-like quaternary ammonium is 0.1M to 2M, more preferably 0.2M to 1.2M.

[0093] Furthermore, it can be used in combination with substances known to promote PCR in the art. Useful promoting substances include, but are not limited to, glycerol, polyols, protease inhibitors, single-strand binding proteins (SSBs), T4 gene 32 protein, tRNA, sulfur or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, ectoin, trehalose, dextran, polyvinylpyrrolidone (PVP), tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), and polyethylene glycol. Chelating agents such as ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) can also be used to further reduce reaction inhibition.

[0094] Another aspect of the present invention is the above-described detection (or testing) kit, reagent, or composition comprising at least one of the primer sets (A) to (E). Preferably, the kit, reagent, or composition further comprises at least one of the probes (I) to (V). In certain preferred embodiments, the kit, reagent, or composition of the present invention may further contain a heat-stable DNA polymerase. In preferred embodiments, the kit, reagent, or composition of the present invention comprises a reaction solution capable of performing PCR (e.g., a real-time PCR reaction solution). Using this kit, reagent, or composition, it may be possible to test for high-risk candidate HPV types (particularly HPV types 26, 53, 70, 73, and / or 82) with high sensitivity and efficiency. The kits, reagents, or compositions of the present invention are preferable when used in combination with other commercially available kits that detect high-risk HPV types such as HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and combinations of two or more of these types, as this allows for more accurate and earlier detection of cervical cancer risk, leading to prevention and treatment.

[0095] The type and quantity of heat-resistant DNA polymerase, primers, or probes that may be used in the above embodiments may be the same as those detailed in the detection method described above. The kit of the present invention may include, for example, instructions explaining how to use (or how to detect) the kit. For example, the kit of the present invention may be provided in a package containing, for example, instructions for the kit, at least one of primer sets (A) to (E) for detecting high-risk candidate HPV types (particularly HPV types 26, 53, 70, 73, and / or 82), at least one of probes (I) to (V) which may be included as needed, and a heat-resistant DNA polymerase, either sealed in the same container or in separate containers. [Examples]

[0096] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to the following examples.

[0097] Example 1: Examination using primer sets for detecting each high-risk candidate HPV type. (1) Preparation of HPV solution Synthetic linear DNA of the E6 / E7 genes (Thermo Fisher Scientific) with sequence numbers 1-5 was used as a sample of HPV in the high-risk candidate group. HPV types 26, 53, 70, 73, and 82 were added in quantities of 1000 and 50 copies, respectively, per reaction. Measurements were performed for N=2 under each condition.

[0098] (2) Reaction solution The reaction solution with the composition shown below was used as the basic composition, and HPV in the reaction solution was detected by real-time PCR. For the primer set, each primer constituting the primer set was added at the concentrations described in (3) below. The reaction solution used was qPCR Mix (included with THUNDERBIRD® Probe qPCR Mix (Toyobo)) and 50X ROX reference dye (included with THUNDERBIRD® Probe qPCR Mix (Toyobo)). This reaction solution contained Mg, which was adjusted to have a final concentration of 1 mM or higher in the reaction solution. 2+ Includes. The reagents described above and the primer solutions described in (3) were mixed to prepare a real-time PCR reaction solution with a final volume of 19 μL. 1 μL of each type of HPV sample (DNA) solution was added to the real-time PCR reaction solution, and real-time PCR was performed using a 20 μL reaction system.

[0099] (3) Primer liquid The following primer solutions (including primers and probes) were added to the reaction mixture after being adjusted so that the final concentrations during real-time PCR matched those shown in Table 1 below.

[0100] [Table 1]

[0101] (4) Real-time PCR reaction conditions This was subjected to a real-time PCR reaction using a Thermo QuantStudio 5 Dx with the following temperature cycles: fluorescence values ​​were read during the 45-cycle extension step at 60°C. 95℃ for 1 minute (heat treatment) 95°C for 15 seconds - 60°C for 30 seconds, 45 cycles (PCR - fluorescence reading)

[0102] (5) Results The Ct value was calculated when the fluorescence signal of Cy5, the detection channel of the probe, was set to a threshold of 10000. The results are shown in Table 2 below. As shown in these results, using primer sets (A) to (E) enabled highly sensitive detection even when each HPV type in the high-risk candidate group had 50 copies / test.

[0103] [Table 2]

[0104] Test Example 2. Sensitivity Comparison Study in Simulated Samples (1) Specimen HPV-negative K-562 cells (ATCC) were suspended in a liquid cytology preservation solution (Hologic), and simulated samples were prepared by adding DNA templates (synthetic linear DNA of E6 / E7 genes (Thermo Fisher Scientific)) for each high-risk candidate group of HPV types (HPV types 26, 53, 70, 73, and 82). These simulated samples did not undergo nucleic acid purification and are equivalent to model samples containing biological contaminants. 10,000 and 1,000 copies of each HPV type DNA were added per reaction. Measurements were performed for N=2 under each condition.

[0105] (2) Reaction solution The reaction solution with the composition shown below was used as the basic composition, and high-risk candidate HPV types in the reaction solution were detected by real-time PCR. For the primer set, each primer solution was added at the concentrations described in (3). The reaction solution used was qPCR Mix (included with THUNDERBIRD® Probe qPCR Mix (Toyobo)) and 50X ROX reference dye (included with THUNDERBIRD® Probe qPCR Mix (Toyobo)). This reaction solution contained Mg, which was adjusted to have a final concentration of 1 mM or higher in the reaction solution. 2+ Includes. The reagents described above and the primer solutions described in (3) were mixed to prepare a real-time PCR reaction mixture with a final volume of 14 μL. HPV-negative cells were treated with 0.01 mg / mL ProK solution at 25°C for 5 minutes, then heat-treated at 95°C for 5 minutes, and 5 μL of these cells were added to the real-time PCR reaction mixture. 1 μL of each type of HPV solution was then added to the 20 μL reaction system, and real-time PCR was performed.

[0106] (3) Primer liquid Each HPV type detection primer solution (primer and probe set) shown in Table 1 of Test Example 1 was used at the concentrations indicated in Table 1.

[0107] (4) Real-time PCR reaction conditions This was subjected to a real-time PCR reaction using a Thermo QuantStudio 5 Dx with the following temperature cycles: fluorescence values ​​were read during the 45-cycle extension step at 60°C. 95℃ for 1 minute (heat treatment conditions) 95°C for 15 seconds - 60°C for 30 seconds, 45 cycles (PCR - fluorescence reading)

[0108] (5) Results The Ct values ​​were calculated when the fluorescence signal of Cy5, the detection channel for each HPV type probe, was set to a threshold of 10000. The Ct values ​​for each condition are shown in Table 3. These results demonstrate that high-risk candidate HPV types 26, 53, 70, 73, and 82 can be detected with high sensitivity in samples that have not been nucleic acid purified and contain biological contaminants.

[0109] [Table 3]

[0110] Test Example 3. Examination of Cross-Reactivity (1) Specimen For the high-risk candidate group of HPV, HPV templates of types 26, 53, 70, 73, and 82 (synthetic linear DNA of the E6 / E7 gene (Thermo Fisher Scientific)) were added in quantities equivalent to 50 or 1000 copies. Measurements were performed for N=2 under each condition.

[0111] (2) Reaction solution The reaction solution with the composition shown below was used as the basic composition, and HPV in the reaction solution was detected by real-time PCR. The reaction solution used was qPCR Mix (included with THUNDERBIRD® Probe qPCR Mix (Toyobo)) and 50X ROX reference dye (included with THUNDERBIRD® Probe qPCR Mix (Toyobo)). This reaction solution contained Mg, which was adjusted to have a final concentration of 1 mM or higher in the reaction solution. 2+ Includes. The reagents described above and the primer solutions described in (3) were mixed to prepare a real-time PCR reaction mixture with a final volume of 19 μL. 1 μL of sample solution containing the HPV types of the high-risk candidate group was added to the real-time PCR reaction mixture, and real-time PCR was performed using a 20 μL reaction system.

[0112] (3) Primer liquid Each HPV type detection primer solution (primer and probe set) shown in Table 1 of Test Example 1 was used at the concentrations indicated in Table 1.

[0113] (4) Real-time PCR reaction conditions This was subjected to a real-time PCR reaction using a Thermo QuantStudio 5 Dx with the following temperature cycles: fluorescence values ​​were read during the 45-cycle extension step at 60°C. 95℃ for 1 minute (heat treatment conditions) 95°C for 15 seconds - 60°C for 30 seconds, 45 cycles (PCR - fluorescence reading)

[0114] (5) Results The Ct values ​​were calculated when the fluorescence signal of Cy5, the detection channel for each probe, was set to a threshold of 10000. The Ct values ​​for each condition are shown in Table 4. As shown in the results in Table 4, when using primer sets (A) to (E), no reaction occurred with molds other than the respective mold. Since there was no cross-reaction among them, it became clear that accurate mold identification is possible by using the primer sets of the present invention.

[0115] [Table 4] - indicates not detected.

[0116] Example 4. Investigation of multi-detection of high-risk candidate HPV types. (1) Preparation of HPV solution Synthetic linear DNA of the E6 / E7 genes (Thermo Fisher Scientific) with sequence numbers 1-5 was used as a sample of HPV in the high-risk candidate group. HPV types 26, 53, 70, 73, and 82 were added in quantities of 1000 and 50 copies, respectively, per reaction. Measurements were performed for N=2 under each condition.

[0117] (2) Reaction solution The reaction solution with the composition shown below was used as the basic composition, and HPV in the reaction solution was detected by real-time PCR. For the primer set, each primer constituting the primer set was added at the concentrations described in (3) below. The reaction solution used was qPCR Mix (included with THUNDERBIRD® Probe qPCR Mix (Toyobo)) and 50X ROX reference dye (included with THUNDERBIRD® Probe qPCR Mix (Toyobo)). This reaction solution contained Mg, which was adjusted to have a final concentration of 1 mM or higher in the reaction solution. 2+ Includes. The reagents described above and the primer solutions described in (3) were mixed to prepare a real-time PCR reaction mixture with a final volume of 19 μL. 1 μL of each type of HPV sample (DNA) solution was added to the real-time PCR reaction mixture, and real-time PCR was performed using a 20 μL reaction system.

[0118] (3) Primer liquid The primers listed in Table 5 were added to the reaction mixture so that the final concentrations of the primer solutions during real-time PCR were as shown below.

[0119] [Table 5]

[0120] (4) Real-time PCR reaction conditions This was subjected to a real-time PCR reaction using a Thermo QuantStudio 5 Dx with the following temperature cycles: fluorescence values ​​were read during the 45-cycle extension step at 60°C. 95℃ for 1 minute (heat treatment) 95°C for 15 seconds - 60°C for 30 seconds, 45 cycles (PCR - fluorescence reading)

[0121] (5) Results The Ct value was calculated when the fluorescence signal of Cy5, the detection channel of the probe, was set to a threshold of 10000. The results are shown in Table 6 below. As shown in these results, even when primer sets (A) to (E) were used in multiplex PCR, each HPV type in the high-risk candidate group was detected reliably and without omission. Furthermore, highly sensitive detection was possible with multiplex PCR even when the copy number in the sample was as low as 50 copies / test.

[0122] [Table 6] [Industrial applicability]

[0123] The present invention is suitably used in tests for the purpose of clinical testing, molecular biological research, and other applications where it is desirable to detect high-risk candidates for HPV.

Claims

1. A method for detecting human papillomavirus (HPV) types 26, 53, 70, 73, and / or 82 that may be present in a sample, (A) A primer set for detecting HPV type 26 that can amplify the region containing the nucleotide sequences 97 to 126 in the nucleotide sequence shown in Sequence ID No. 1; (B) A primer set for detecting HPV type 53 that can amplify the region containing the nucleotide sequences 192 to 213 in the nucleotide sequence shown in Sequence ID No. 2; (C) A primer set for detecting HPV type 70 that can amplify the region containing the nucleotide sequences 40 to 59 in the nucleotide sequence shown in Sequence ID No. 3; (D) A primer set for detecting HPV type 73 that can amplify the region containing the nucleotide sequences 386 to 407 in the nucleotide sequence shown in Sequence ID No. 4; and (E) A primer set for detecting HPV82 that can amplify the region containing the nucleotide sequences 340 to 364 in the nucleotide sequence shown in Sequence ID No.

5. A method comprising the step of carrying out a nucleic acid amplification reaction using a reaction solution containing at least one selected from the group consisting of the following.

2. The primer set (A) is a primer set comprising a primer having a consecutive nucleotide sequence including positions 19 to 38 in SEQ ID NO: 1, 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, and a primer having a consecutive nucleotide sequence including positions 169 to 188 in SEQ ID NO: 1, 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; The primer set (B) is a primer set comprising a primer having a consecutive nucleotide sequence including positions 109 to 128 in SEQ ID NO: 2, 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, and a primer having a consecutive nucleotide sequence including positions 253 to 272 in SEQ ID NO: 2, 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; The primer set (C) is a primer set comprising a primer having a consecutive nucleotide sequence including positions 3 to 22 in SEQ ID NO: 3, 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, and a primer having a consecutive nucleotide sequence including positions 169 to 188 in SEQ ID NO: 3, 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; The primer set (D) is a primer set comprising a primer having a consecutive nucleotide sequence including positions 307 to 326 in SEQ ID NO: 4, 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, and a primer having a consecutive nucleotide sequence including positions 419 to 438 in SEQ ID NO: 4, 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; and / or The primer set (E) is a primer set comprising a primer having a consecutive nucleotide sequence including positions 315 to 334 in SEQ ID NO: 5, 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, and a primer having a consecutive nucleotide sequence including positions 398 to 417 in SEQ ID NO: 5, 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. 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 set comprising a primer consisting of the nucleotide sequences 19 to 38 of SEQ ID NO: 1 or their complementary nucleotide sequences, and a primer consisting of the nucleotide sequences 169 to 188 of SEQ ID NO: 1 or their complementary nucleotide sequences; The primer set (B) is a primer set comprising a primer consisting of the nucleotide sequences 109 to 128 of SEQ ID NO: 2 or their complementary nucleotide sequences, and a primer consisting of the nucleotide sequences 253 to 272 of SEQ ID NO: 2 or their complementary nucleotide sequences; The primer set (C) is a primer set comprising a primer consisting of the nucleotide sequences 3 to 22 of SEQ ID NO: 3 or their complementary nucleotide sequences, and a primer consisting of the nucleotide sequences 169 to 188 of SEQ ID NO: 3 or their complementary nucleotide sequences; The primer set (D) is a primer set comprising a primer consisting of the nucleotide sequences 307 to 326 of SEQ ID NO: 4 or their complementary nucleotide sequences, and a primer consisting of the nucleotide sequences 419 to 438 of SEQ ID NO: 4 or their complementary nucleotide sequences; and / or The primer set (E) is a primer set comprising a primer consisting of the nucleotide sequences 315 to 334 of SEQ ID NO: 5 or their complementary nucleotide sequences, and a primer consisting of the nucleotide sequences 398 to 417 of SEQ ID NO: 5 or their complementary 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.

4. The method according to claim 1, wherein the reaction solution comprises all of the primer sets (A) to (E).

5. The reaction solution is (I) HPV26 detection probes having a continuous nucleotide sequence including positions 97 to 126 in Sequence ID No. 1, 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; (II) HPV53 detection probes having a continuous nucleotide sequence including positions 192 to 213 in Sequence ID No. 2, 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; (III) HPV 70 detection probes having a continuous nucleotide sequence including positions 40 to 59 in Sequence ID No. 3, 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; (IV) HPV73 detection probes having a continuous nucleotide sequence including positions 386 to 407 in Sequence ID No. 4, 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 (V) HPV82 detection probe having a continuous nucleotide sequence including positions 340 to 364 in Sequence ID No. 5, 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 at least one selected from the group consisting of the following.

6. The probe (I) is a probe consisting of the nucleotide sequence 97 to 126 of Sequence ID No. 1 or a complementary nucleotide sequence thereof; The probe (II) is a probe consisting of the nucleotide sequence from position 192 to 213 of Sequence ID No. 2 or a complementary nucleotide sequence thereof; The probe (III) is a probe consisting of the 40th to 59th base sequence of Sequence ID No. 3 or a complementary base sequence thereof; The probe (IV) is a probe consisting of the nucleotide sequence 386-407 of Sequence ID No. 4 or a complementary nucleotide sequence thereof; and / or The method according to claim 5, wherein the probe (V) is a probe consisting of the nucleotide sequence 340 to 364 of sequence number 5 or a nucleotide sequence complementary thereto.

7. The method according to claim 5, wherein the reaction solution comprises all of the probes (I) to (V).

8. The method according to claim 5, wherein the probes (I) to (V) are fluorescently labeled probes.

9. The method according to claim 8, wherein the fluorescently labeled probe is a TaqMan probe.

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

11. The method according to claim 10, wherein the heat-stable DNA polymerase belonging to Family A is at least one selected from the group consisting of Taq polymerase, Tth polymerase, Hawk Z05 polymerase, and their variants.

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

13. The method according to claim 1, wherein the nucleic acid amplification reaction is a PCR reaction.

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

15. 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.

16. The method according to claim 1, wherein the sample is a suspension or concentrate thereof suspended in a liquid cytological preservation solution, water, physiological saline, or buffer solution.

17. A reagent or kit for use in the method according to any one of claims 1 to 16, comprising at least one selected from the group consisting of the primer sets (A) to (E).

18. The reagent or kit according to claim 17, comprising at least one selected from the group consisting of probes (I) to (V) as described in claim 5.

19. At least one primer set selected from the following groups: (A) A primer set for detecting HPV type 26, comprising a primer having a consecutive nucleotide sequence including positions 19 to 38 in SEQ ID NO: 1, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; and a primer having a consecutive nucleotide sequence including positions 169 to 188 in SEQ ID NO: 1, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (B) A primer set for detecting HPV type 53, comprising a primer having a consecutive nucleotide sequence including positions 109 to 128 in SEQ ID NO: 2, 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; and a primer having a consecutive nucleotide sequence including positions 253 to 272 in SEQ ID NO: 2, 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; (C) A primer set for detecting HPV type 70, comprising a primer having a consecutive nucleotide sequence including positions 3 to 22 in Sequence ID No. 3, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; and a primer having a consecutive nucleotide sequence including positions 169 to 188 in Sequence ID No. 3, a complementary nucleotide sequence thereto, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences; (D) A primer set for detecting HPV type 73, comprising a primer having a consecutive nucleotide sequence including positions 307 to 326 in SEQ ID NO: 4, 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; and a primer having a consecutive nucleotide sequence including positions 419 to 438 in SEQ ID NO: 4, 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; and (E) A primer set for detecting HPV type 82, comprising a primer having a consecutive nucleotide sequence including positions 315 to 334 in SEQ ID NO: 5, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences, and a primer having a consecutive nucleotide sequence including positions 398 to 417 in SEQ ID NO: 5, its complementary nucleotide sequence, or a nucleotide sequence in which 1 to 3 nucleotides are substituted, deleted, inserted, or added in those sequences. (Here, in each primer set, one primer is complementary to the extension product of the other primer.)

20. At least one probe selected from the following group: (I) HPV26 detection probes having a continuous nucleotide sequence including positions 97 to 126 in Sequence ID No. 1, 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; (II) HPV53 detection probes having a continuous nucleotide sequence including positions 192 to 213 in Sequence ID No. 2, 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; (III) HPV 70 detection probes having a continuous nucleotide sequence including positions 40 to 59 in Sequence ID No. 3, 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; (IV) HPV73 detection probes having a continuous nucleotide sequence including positions 386 to 407 in Sequence ID No. 4, 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 (V) A probe for detecting HPV type 82 having a continuous nucleotide sequence including positions 340 to 364 in Sequence ID No. 5, 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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