Methods, uses, systems and kits for detecting human papilloma virus genotypes associated with onset of cervical cancer, oral cancer or anal genital cancer in subject and for determining one or more genotypes thereof

Abstract

The present invention relates to methods, uses, systems and kits for detecting human papilloma virus (HPV) genotypes associated with the onset of cervical cancer, oral cancer or anal genital cancer in a subject and for determining one or more genotypes of HPV thus detected. Furthermore, the invention relates to the use of a forward primer and / or a reverse primer or amplicon comprising a molecular tag produced by multiplex real-time PCR for identifying markers of genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68 of human papilloma virus.

Description

Technical Field

[0001] The present invention relates to the field of diagnostics, and in particular to the field of detecting an infection in a subject and determining the specific genotype causing the infection. Background Art

[0002] The problem of the present invention is to provide methods, uses, systems and kits that allow the detection of human papillomavirus (HPV) genotypes associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject, and when said human papillomavirus genotypes associated with the occurrence of cervical cancer, oral cancer or anogenital cancer are detected in a subject, the determination of said one or more genotypes of human papillomavirus infecting said subject, wherein the use of reagents and materials is minimized. In addition, the problem of the present invention is to provide methods, uses, systems and kits for these purposes that are accurate, precise and fully automated and can be based on samples taken by the subject from himself, without the intervention of a doctor or other medical staff, so that it is fully automated from subject to result. In addition, the problem of the present invention is to provide cheaper and faster methods, uses, systems and kits for these purposes. Summary of the invention

[0003] The present invention relates to a method for detecting a human papillomavirus (HPV) genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject and for determining one or more genotypes of the human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is detected in the subject, comprising:

[0004] (a) performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the DNA is extracted from the biological sample prior to performing the multiplex real-time PCR on the biological sample, wherein the multiplex real-time PCR amplifies:

[0005] (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and

[0006] (ii) a second sequence comprised in said DNA, wherein said second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker,

[0007] The composition comprises:

[0008] (iii) a set of probes comprising:

[0009] - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16;

[0010] - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and

[0011] - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and

[0012] (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene,

[0013] wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe,

[0014] Among them, when:

[0015] - when the fourth fluorophore of the probe bound to the second sequence is not detected and the first, second or third fluorophore of the probe bound to the first sequence is not detected, repeating step (a) for the subject;

[0016] - when the fourth fluorophore of the probe bound to the second sequence is detected and at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected;

[0017] - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected;

[0018] (b) subjecting each amplified first sequence and second sequence of the composition selected in step (a) to hybridization on a solid support, the solid support comprising probes attached thereto, wherein during hybridization, the composition selected in step (a) is heated to at least 60° C. and then cooled, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, and a forward primer and a reverse primer complementary to and specific for a locus of the marker are provided for the sequence of the marker in the multiplex real-time PCR in step (a),

[0019] wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence; and

[0020] (c) identifying each specific area of ​​the solid support to which a probe is attached and which hybridizes to the first sequence or the second sequence;

[0021] Among them, when:

[0022] (I) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the first fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 16 of human papillomavirus;

[0023] (II) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the second fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 18 of human papillomavirus;

[0024] (III) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in step (c), the genotype is determined by:

[0025] (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence;

[0026] (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for the probe sequence attached to each specific region identified in step (IIIa).

[0027] Furthermore, the present invention relates to a system for detecting a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer in a subject and for determining one or more genotypes of a human papillomavirus infecting the subject when the human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected in the subject, comprising:

[0028] (a) a device for performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the multiplex real-time PCR amplifies:

[0029] (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and

[0030] (ii) a second sequence comprised in said DNA, wherein said second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker,

[0031] The composition comprises:

[0032] (iii) a set of probes comprising:

[0033] - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16;

[0034] - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and

[0035] - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and

[0036] (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene,

[0037] wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe,

[0038] Among them, when:

[0039] - when the fourth fluorophore of the probe bound to the second sequence is not detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, repeating (a) for the subject;

[0040] - when the fourth fluorophore of the probe bound to the second sequence is detected and at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected;

[0041] - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected, and

[0042] wherein the apparatus for performing multiplex real-time PCR on at least one sequence contained in the DNA present in the biological sample comprises a multiplex real-time PCR instrument and multiplex real-time PCR reagents;

[0043] (b) means for hybridizing each amplified first sequence and second sequence of the composition selected in (a) on a solid support, the solid support comprising a probe attached thereto, means for heating the composition selected in (a) to at least 60° C. during hybridization, and means for cooling the composition selected in (a) during hybridization, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, forward primers and reverse primers complementary to and specific for a locus of the marker being provided for the sequence of the marker in the multiplex real-time PCR in (a),

[0044] wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence,

[0045] wherein the means for heating the composition selected in (a) comprises a heating device,

[0046] wherein the means for cooling the composition selected in (a) comprises a cooling device,

[0047] wherein the means for hybridizing each of the first and second sequences amplified in the composition selected in (a) on a solid support comprises a solid support and a hybridization reagent, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, a forward primer and a reverse primer complementary to and specific for a locus of the marker being provided for the sequence of the marker in the multiplex real-time PCR in (a); and

[0048] (c) means for identifying each specific area of ​​the solid support to which a probe is attached and which is hybridized to the first sequence or the second sequence, wherein the means comprises an image capture device;

[0049] Among them, when:

[0050] (I) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the first fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 16 of human papillomavirus;

[0051] (II) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the second fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 18 of human papillomavirus;

[0052] (III) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in (c), the genotype is determined by:

[0053] (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence;

[0054] (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for a probe sequence attached to each specific region identified in (IIIa).

[0055] The system may optionally further include a device for extracting DNA from a biological sample, such as a device for extracting DNA of human papillomavirus present in a biological sample obtained from a subject. The device for extracting DNA may be used prior to using a device for performing multiplex real-time PCR on at least one sequence contained in the DNA present in the biological sample obtained from the subject,

[0056] Furthermore, the present invention relates to a kit for detecting a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject and for determining the one or more genotypes of the human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is detected in the subject, comprising:

[0057] (a) a device for performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the multiplex real-time PCR amplifies:

[0058] (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and

[0059] (ii) second sequences comprised in said DNA, wherein each second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker,

[0060] The composition comprises:

[0061] (iii) a set of probes comprising:

[0062] - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16;

[0063] - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and

[0064] - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and

[0065] (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene,

[0066] wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe,

[0067] Among them, when:

[0068] - when the fourth fluorophore of the probe bound to the second sequence is not detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, repeating (a) for the subject;

[0069] - when the fourth fluorophore of the probe bound to the second sequence is detected and at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected;

[0070] - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected, and

[0071] wherein the apparatus for performing multiplex real-time PCR on at least one sequence contained in the DNA present in the biological sample comprises a multiplex real-time PCR instrument and multiplex real-time PCR reagents;

[0072] (b) means for hybridizing each amplified first sequence and second sequence of the composition selected in (a) on a solid support, the solid support comprising a probe attached thereto, means for heating the composition selected in (a) to at least 60° C. during hybridization, and means for cooling the composition selected in (a) during hybridization, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, and forward primers and reverse primers complementary to and specific for a locus of the marker are provided for the sequence of the marker in the multiplex real-time PCR in (a),

[0073] wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence,

[0074] wherein the means for heating the composition selected in (a) comprises a heating device,

[0075] wherein the means for cooling the composition selected in (a) comprises a cooling device,

[0076] wherein the means for hybridizing each of the first sequence and the second sequence amplified in the composition selected in (a) on a solid support comprises a solid support and a hybridization reagent, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for the sequence of the marker, and a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided for the sequence of the marker in the multiplex real-time PCR in (a); and

[0077] (c) means for identifying each specific area of ​​the solid support to which a probe is attached and which is hybridized to the first sequence or the second sequence, wherein the means comprises an image capture device;

[0078] Among them, when:

[0079] (I) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the first fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 16 of human papillomavirus;

[0080] (II) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the second fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 18 of human papillomavirus;

[0081] (III) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in (c), the genotype is determined by:

[0082] (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence;

[0083] (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for a probe sequence attached to each specific region identified in (IIIa).

[0084] The kit may optionally further include a device for extracting DNA from a biological sample, such as a device for extracting DNA of human papillomavirus present in a biological sample obtained from a subject. The device for extracting DNA may be used before using a device for performing multiplex real-time PCR on at least one sequence contained in the DNA present in the biological sample obtained from the subject,

[0085] The present invention also relates to a forward primer and / or a reverse primer

[0086] Use for obtaining amplified sequences comprising molecular tags by multiplex real-time PCR, wherein the multiplex real-time PCR amplifies sequences contained in DNA present in a biological sample obtained from a subject, wherein the DNA is extracted from the biological sample before subjecting the biological sample to multiplex real-time PCR.

[0087] The forward primer and / or reverse primer comprises

[0088] - sequences complementary to and specific for one or more loci of markers of genotype 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68 of human papillomavirus; and

[0089] - molecular tags; and

[0090] Use for identifying a specific area on a solid support attached to a probe, wherein the probe comprises a sequence complementary to and specific for a sequence of a marker, the forward primer and / or reverse primer are complementary to and specific for the sequence of the marker; and the amplified sequence comprising a molecular tag hybridizes with the sequence of the marker.

[0091] The present invention also relates to the use of amplified sequences comprising molecular tags, wherein said amplified sequences have been obtained in a multiplex real-time PCR, wherein said multiplex real-time PCR amplifies sequences comprised in DNA present in a biological sample obtained from a subject, wherein said DNA is extracted from said biological sample before subjecting said biological sample to the multiplex real-time PCR,

[0092] Amplification is performed using a forward primer and / or a reverse primer, wherein the forward primer and / or the reverse primer comprises

[0093] - sequences complementary to and specific for one or more loci of markers of genotype 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68 of human papillomavirus; and

[0094] - molecular tags; and

[0095] Used to identify a specific area on a solid support attached to a probe, wherein the probe comprises a sequence complementary to and specific for a sequence of a marker, the forward primer and / or reverse primer are complementary to and specific for the sequence of the marker; and the amplified sequence comprising the molecular tag hybridizes with the sequence of the marker. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 : According to the present invention, a flowchart of an example of a method for detecting a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer in a subject, and for determining the one or more genotypes of the human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer is detected in the subject.

[0097] Figure 2: Multiplex real-time PCR procedure according to (a) of the present invention, A. Amplification plot of positive control (PC) and B. Amplification plot of negative control (NTC) with water, where the expected count values ​​(ΔRn) of PC change over time (each multiplex PCR cycle): HPV 16 (ROX) 20±2; HPV 18 (Cy5) 20±2; HPV HR (genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of HPV, FAM) 20±2, Bglobin (human β-globin, JOE) 20±2. The experiment was performed in an Applied Biosystems QuantStudio™ 5 Real-Time PCR System. (O) β-globin; (◇) HPV HR, (Δ) HPV 16; (x) HPV 18.

[0098] Figure 3: A. Detection and determination of human papillomavirus genotype 56 in subjects infected therewith, based on a graph of β-globin and HPV HR amplification according to the multiplex real-time PCR procedure of the present invention (b), wherein the expected count values ​​(ΔRn) of PC: HPV HR (genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of HPV, FAM) change over time (per multiplex PCR cycle), B globin (human β-globin, JOE) 20 ± 2. The experiment was performed in an Applied Biosystems QuantStudio™ 5 Real-Time PCR System. (O) β-globin; (◇) HPV HR.

[0099] B. Visualization of the solid support after hybridization with the amplicons of the sample from the subject [obtained by multiplex real-time PCR according to (a) of the present invention], wherein specific regions 2H and 7C (specific regions are identified by numbers and letters, respectively, counting from the upper left specific region 1A of the solid support) show that hybridization has occurred, the specific regions corresponding to those regions of the solid support to which probes are attached, the probes comprising sequences complementary to and specific for the sequences of the markers of the HPV 56 genotype, as shown in C. Schematic diagram of the solid support. Additional hybridization has occurred: specific regions of the solid support to which probes are attached (1, A; 1, B; 2, I; 5, E and 8, A) used as hybridization controls (5 specific regions for correctly orienting the solid support), specific regions of the solid support to which probes are attached (1, C and 5, F) used as internal (human genomic) DNA controls, and specific regions of the solid support to which multiple probes are attached (1, D and 5, G) used as degenerate universal HPV controls. Specific regions 1, I; 5, C; 9, A; 9, F; 9; H and 9, I do not have probes attached thereto, while the remaining specific regions have probes attached thereto, wherein each probe is complementary to and specific for the sequence of a marker of one or more genotypes having one or more numbers shown in the schematic diagram, and two replicates are provided for each specific region of a given probe type. It is noted that specific regions 4, C; 4, F; 8, E and 8, H are specific regions to which probes are attached, and the probes are complementary to and specific for the sequences of markers of two genotypes.

[0100] Figure 4 : A further detailed flow chart of an example of a method for detecting a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject according to the present invention, and for determining the one or more genotypes of the human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is detected in the subject.

[0101] Figure 5: Detection and determination of human papillomavirus in subjects infected therewith, using different hybridization conditions. The same set of samples (liquid cytology samples with previous positive results for HR HPV genotype by real-time PCR) was assayed with different hybridization conditions. Hybridization condition 1 corresponds to real-time PCR with subsequent hybridization of the PCR products at a temperature of 41°C. Under these conditions, the undetermined HPV genotype in the sample is evaluated (marked "undetermined positive" in the figure) when the detected signal is provided only by the universal HPV probe. Hybridization condition 2 corresponds to amplification of the sample by end-point PCR without a fluorescent probe and subsequent hybridization of the PCR products at a temperature of 41°C. Hybridization condition 3 corresponds to the method according to the invention, real-time PCR, followed by hybridization at a temperature of 60°C and subsequent cooling to 41°C. DETAILED DESCRIPTION

[0102] The present invention relates to methods, systems, uses and kits for detecting human papillomavirus genotypes associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject and for determining the one or more genotypes of human papillomavirus infecting the subject when the human papillomavirus genotypes associated with the occurrence of cervical cancer, oral cancer or anogenital cancer are detected in the subject. Therefore, the methods, systems, uses and kits are for detecting human papillomavirus genotypes associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject and for determining one or more genotypes thereof when detected.

[0103] The human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject is a genotype of a human DNA virus from the family of Papillomaviridae. The human papillomavirus genotype is associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject, and is therefore a human papillomavirus genotype that can cause, increase, promote or otherwise lead to the occurrence of cervical cancer, oral cancer or anogenital cancer in the subject, respectively, when infected with the human papillomavirus genotype.

[0104] The cervical cancer is a cancer of the lower part of the uterus, preferably a cancer of the squamous-columnar junction of the cervix, more preferably a squamous cell carcinoma (epidermoid carcinoma) of the squamous-columnar junction of the cervix.

[0105] The oral cancer may be a cancer of the oral cavity and / or pharynx. It includes oral cancer, tonsil cancer and / or laryngeal cancer. The oral cancer may be oropharyngeal cancer.

[0106] The anogenital cancer includes cancer of the external sex organs and / or anal cancer. The cancer of the external sex organs is preferably selected from the group consisting of: cancer of the vulva, vagina, penis, scrotum and testicles and any anatomical part thereof. More preferably, the cancer of the external sex organs is selected from the group consisting of: cancer of the urethra, hymen, clitoral frenulum, clitoral glans, clitoral hood, labia majora, labia minora, frenulum of the labia minora, Skene's gland, vagina, foreskin, frenulum of penis, glans penis, scrotum and testicles and any anatomical part thereof. Even more preferably, the cancer of the external sex organs is selected from the group consisting of squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, small cell carcinoma, lymphoma, sarcoma and melanoma, and still more preferably, squamous cell carcinoma in any of the aforementioned external sex organ tissues. Therefore, the anogenital cancer can be anal cancer, vaginal cancer, vulvar cancer and / or penile cancer.

[0107] The anal cancer is preferably selected from the group consisting of: anal canal cancer, anal columns of Morgagni, squamous-columnar junction cancer, ischioanal fossa cancer, perianal space cancer, anal margin cancer, anal crypt cancer, anal gland cancer and anal sphincter cancer. More preferably, the anal cancer is selected from the group consisting of squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, small cell carcinoma, lymphoma, sarcoma and melanoma, even more preferably, squamous cell carcinoma in any of the aforementioned anal tissues, and still more preferably, squamous cell carcinoma of the squamous-columnar junction.

[0108] The subject is a human subject, preferably a male or female subject, more preferably a sexually active male subject or a sexually active female subject.

[0109] The human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject infected with a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is a genotype that places the subject infected with the genotype at a high risk of the occurrence of cervical cancer, oral cancer or anogenital cancer. Preferably, the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject infected with a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is selected from the group consisting of 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus, as also disclosed in other aspects of the invention described herein. The methods, uses, systems and kits of the present invention can detect one or more such genotypes in any given subject infected with the genotype.

[0110] Conversely, a human papillomavirus genotype that is not associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject infected with a human papillomavirus genotype that is not associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is a genotype that places the subject infected with the genotype at a low risk of developing cervical cancer, oral cancer or anogenital cancer. Preferably, the human papillomavirus genotype that is not associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject infected with a human papillomavirus genotype that is not associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is selected from the group consisting of 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82 and 84 genotypes of human papillomavirus. However, a human papillomavirus genotype that is not associated with the development of cervical cancer, oral cancer, or anogenital cancer in a subject infected with a human papillomavirus genotype that is not associated with the development of cervical cancer, oral cancer, or anogenital cancer can still infect a subject infected with one or more human papillomavirus genotypes associated with the development of cervical cancer, oral cancer, or anogenital cancer.

[0111] Therefore, in other words, the methods, uses, systems and kits of the present invention are used to detect one or more genotypes associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject infected with a genotype of human papillomavirus, and to determine one or more genotypes of human papillomavirus detected therein that are associated with the occurrence of cervical cancer, oral cancer or anogenital cancer, and optionally, one or more genotypes that are not associated with the occurrence of cervical cancer, oral cancer or anogenital cancer. In a particularly preferred embodiment, the methods, uses, systems and kits of the present invention are used to detect one or more genotypes associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in subjects infected with a genotype of human papillomavirus, and to determine one or more genotypes of human papillomavirus detected therein that are associated with the occurrence of cervical cancer, oral cancer or anogenital cancer, and optionally, one or more genotypes that are not associated with the occurrence of cervical cancer, oral cancer or anogenital cancer, wherein each genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is selected from the group consisting of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus.

[0112] The present invention includes two techniques, namely multiplex real-time PCR [described by step (a) in the method of the present invention and by means (a) in the system and kit of the present invention], and hybridization plus visualization [described by steps (b) plus (c) in the method of the present invention and by means (b) plus (c) in the system and kit of the present invention, respectively]. Multiplex PCR involves the simultaneous detection of multiple specific nucleotide sequences (i.e. genetic markers) in a single composition (i.e. PCR reaction well or container) when amplified by PCR using a different primer pair for each marker. Thus, multiplex real-time PCR involves said simultaneous detection in "real time" (i.e. at predetermined intervals), preferably after each amplification cycle of said PCR.

[0113] Therefore, in the method of the present invention, (a) comprises the step of subjecting at least one sequence contained in the DNA present in the biological sample obtained from the subject to multiplex real-time PCR ( Figure 1 100), and in the system and kit of the present invention, said (a) comprises a device for performing multiplex real-time PCR on at least one sequence contained in the DNA present in the biological sample obtained from the subject.

[0114] The PCR product added to the solid support contains residual probes from the real-time PCR that were not consumed during amplification, so there will be two complementary probes in the hybridization device, one in the liquid phase and the other in the solid phase, and both can compete for hybridization with the target amplicon. This is prevented by heating at a temperature of at least 60°C, which promotes the generated amplicons to preferentially hybridize with the probes in the solid phase (i.e., the probes in the array).

[0115] Thus, in the methods, systems and kits of the present invention, the composition selected in (a) is heated to at least 60°C and gradually cooled while hybridizing to the solid surface according to step (b).

[0116] Heating at a temperature of at least 60° C. promotes the generated amplicons to hybridize preferentially with the probes in the solid phase rather than with the remaining probes of the real-time PCR in the liquid phase, thereby improving the sensitivity and specificity of the method. If the heating temperature is lower than 60° C. (e.g., 50° C. or 41° C., as determined in Example 4), some of the remaining probes in the PCR may hybridize with the target amplicon and prevent the target amplicon from binding to the probes in the solid support, as shown in Example 4.

[0117] Example 4 shows that according to the method of the present invention, when real-time PCR is followed by hybridization at a temperature of 60° C., the genotypes present in the sample are correctly detected and identified, thereby improving the sensitivity and specificity of the method. When real-time PCR is followed by hybridization of the real-time PCR product at a temperature of 41° C., as determined in Example 4, some genotypes are not identified.

[0118] The biological sample is a sample containing biological fluid, free cells and / or tissue obtained from the subject. The sample can be obtained by a third party (e.g., a doctor, a nurse) or by the subject himself. In a preferred embodiment of the present invention, the biological sample obtained from the subject is obtained by the sample obtained by the subject himself. For example, a biological sample from the cervix obtained from the subject may have been obtained by the sample obtained by the subject himself. The sample is preferably obtained by a sample collection device such as a swab, a brush or a veil device applied to the cervix, oral or anal genital tissue (preferably cervical tissue) of the subject to obtain biological fluid and / or cells from the subject. Therefore, the sample collection device can be a swab, a brush or a vaginal veil device.

[0119] The biological sample comprises DNA. The DNA is a polynucleotide and therefore comprises at least one nucleotide sequence. If the subject is infected with a human papillomavirus associated with the occurrence of cervical cancer, oral cancer or anogenital cancer, the biological sample will comprise DNA from the human papillomavirus. If the subject is not infected with a human papillomavirus associated with the occurrence of cervical cancer, oral cancer or anogenital cancer, the biological sample will comprise DNA from other sources, i.e. DNA from the subject and / or DNA from a human papillomavirus that is not associated with the occurrence of cervical cancer, oral cancer or anogenital cancer.

[0120] DNA is usually extracted from a sample to perform the multiplex real-time PCR amplification. Thus, the amplification can be performed from a purified DNA sample.

[0121] The PCR (i.e. multiplex real-time PCR) amplification:

[0122] (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and

[0123] (ii) a second sequence contained in the DNA, wherein the second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of the second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of the marker ( Figure 1 of box 100).

[0124] Each amplification is performed by the PCR using a primer pair, wherein the primer pair comprises a locus-specific forward primer and a locus-specific reverse primer, which binds to different complementary sequences on the Watson and Crick chains adjacent to the nucleotide sequence, thereby identifying the 5' and 3' boundaries of the nucleotide sequence. In particular, the 3' end of the nucleotide sequence of the Watson chain begins at a nucleotide adjacent to the nucleotide at the 5' end of the sequence annealed with the forward primer. On the contrary, the 5' end of the nucleotide sequence of the Watson chain begins at a nucleotide complementary to the nucleotide adjacent to the nucleotide at the 5' end of the sequence annealed with the reverse primer. Similarly, the 3' end of the nucleotide sequence of the Crick chain begins at a nucleotide adjacent to the nucleotide at the 5' end of the sequence annealed with the reverse primer. On the contrary, the 5' end of the nucleotide sequence of the Crick chain begins at a nucleotide complementary to the nucleotide adjacent to the nucleotide at the 5' end of the sequence annealed with the forward primer. Therefore, DNA polymerase attaches to the 5' end of the above primers and replicates the nucleotide sequence multiple times.

[0125] The primers are locus-specific primers selected so as to be complementary to and thereby identify a specific nucleotide sequence (i.e., a genetic marker) that may be present in a biological sample, wherein the marker is:

[0126] - in the case of (i), is indicative of said human papillomavirus (i.e. the presence of human papillomavirus DNA in said sample, and therefore said subject is infected with said human papillomavirus), and

[0127] - in case of (ii), is indicative of said human housekeeping gene (ie the presence of human DNA from said subject in said sample).

[0128] Therefore, the methods, uses, systems and kits of the present invention rely on any genetic marker for human papillomavirus and any genetic marker for human housekeeping genes detectable using amplicon sequencing methods.

[0129] Since the multiplex real-time PCR amplifies at least one first sequence (a marker of the human papillomavirus genotype) and a second sequence (a marker of the human housekeeping gene genotype), and a forward primer and a reverse primer are used to amplify each sequence, each primer is complementary to and specific to the locus of any given marker, and the multiplex real-time PCR is performed using at least two pairs of forward primers and reverse primers. Preferably, the multiplex real-time PCR is performed using at least two pairs of forward primers and reverse primers for amplifying the first sequence and a forward primer and reverse primer pair for amplifying the second sequence, so that at least two different first sequences can be amplified by the multiplex real-time PCR, each first sequence is a marker of the human papillomavirus genotype, and so that the second sequence can be amplified by the multiplex real-time PCR, and the second sequence is a marker of the human housekeeping gene genotype. Any given forward primer and reverse primer pair used to amplify the first sequence can amplify markers common to one or more genotypes of human papillomavirus.

[0130] Each first sequence is a sequence of a human papillomavirus genotype marker, regardless of whether the human papillomavirus is associated with the occurrence of cervical cancer, oral cancer or anal genital cancer. In a preferred embodiment of the method, use, system and kit of the present invention, the multiplex real-time PCR amplification:

[0131] (i) at least one first sequence, wherein each first sequence is a sequence from a marker of a human papillomavirus genotype, which is present in a conserved region of the HPV L1 or HPV L2 gene, more preferably in a conserved region of the HPV L1 gene; and

[0132] (ii) the second sequence.

[0133] In another preferred embodiment of the methods, uses, systems and kits of the present invention, the multiplex real-time PCR amplification:

[0134] (i) at least one first sequence, wherein each first sequence is a sequence from a marker for a human papillomavirus genotype selected from the group consisting of genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, and 84 of human papillomavirus; and

[0135] (ii) the second sequence.

[0136] In another more preferred embodiment of the methods, uses, systems and kits of the present invention, the multiplex real-time PCR amplification:

[0137] (i) a first sequence, which is a sequence of a marker of genotype 16 of human papillomavirus;

[0138] - a first sequence which is a sequence of a marker of genotype 18 of human papillomavirus, and,

[0139] - at least one other first sequence, wherein each first sequence is a sequence from a marker of a human papillomavirus genotype selected from the group consisting of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus; and

[0140] (ii) the second sequence.

[0141] Each second sequence is a sequence of a marker of the genotype of a human housekeeping gene. Preferably, the human housekeeping gene is a constitutive gene that is continuously transcribed in the subject as required to maintain cell function. More preferably, the human housekeeping gene is a human beta globin gene (HBB gene) or a ribonuclease P gene (RNase P gene), even more preferably an HBB gene.

[0142] Preferably, the forward primer and reverse primer used to amplify each first sequence are selected from any sequence pairs of SEQ ID NO:1 to SEQ ID NO:2 disclosed in Table 1, and the forward primer and reverse primer used to amplify the second sequence are selected from any sequence pairs of SEQ ID NO:3 to SEQ ID NO:4 disclosed in Table 2.

[0143] Table 1: Forward and reverse primer pairs used to amplify each first sequence

[0144]

[0145] Table 2: Forward and reverse primer pairs used to amplify each second sequence

[0146]

[0147] *- indicates the 5' molecular tag of the primer, in this case the molecular tag is biotin.

[0148] The multiplex real-time PCR used in the present invention amplifies the DNA from the subject and / or the DNA from the human papillomavirus in the composition. In addition to the DNA, the composition also comprises a forward primer and a reverse primer and a plurality of probes, preferably a taqman probe. In particular, the plurality of probes contained in the composition comprises:

[0149] (iii) a set of probes comprising:

[0150] - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16;

[0151] - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and

[0152] - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and

[0153] (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a marker of the human housekeeping gene ( Figure 1 of box 100).

[0154] Each probe disclosed in (iii) and (iv) comprises a fluorophore and a sequence, wherein the sequence is complementary to and specific for the sequence of the marker of the gene. When the sequence binds / anneals with a complementary sequence (i.e., specific for) the marker of the gene amplified by the multiplex PCR, the fluorophore emits detectable light. In other words, each probe in which the sequence of the marker bound is detected is complementary to and specific for the probe.

[0155] In particular, each probe of the group of probes disclosed in (iii) comprises a fluorophore and a sequence, wherein the sequence is complementary to and specific for the sequence of markers of genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus. In this way, when the sequence of the probe is bound to the sequence of the marker, the sequence of the marker of any human papillomavirus genotype can be detected in the composition amplified by multiplex PCR by detecting the light emitted by the fluorophore of the probe in real time, wherein the probe comprises a sequence complementary to and specific for the sequence of the marker. Therefore, the methods, uses, systems and kits of the present invention allow human papillomavirus having any of the genotypes to be detected, and the genotype may cause the subject infected therewith to be at high risk of cervical cancer, oral cancer or anogenital cancer. Preferably, each probe of a given target (HPV genotype or HPV genotype group) comprises a sequence selected from any sequence shown in Table 3.

[0156] Table 3: Probe sequences used to detect each first sequence in multiplex real-time PCR

[0157]

[0158] Similarly, the probe disclosed in (iv) comprises a fluorophore and a sequence, wherein the sequence is complementary to and specific for the sequence of the human housekeeping gene amplified by the multiplex PCR. In this way, when the sequence of the probe binds to the sequence of the marker, the sequence of the marker of the human housekeeping gene can be detected in the composition amplified by multiplex PCR by detecting the light emitted by the fluorophore of the probe in real time, wherein the probe comprises a sequence that is complementary to and specific for the sequence of the marker. Therefore, the methods, uses, systems and kits of the present invention also allow for the detection of DNA from the subject. Preferably, each probe for a given human housekeeping gene comprises a sequence selected from any of the sequences shown in Table 4.

[0159] Table 4: Probe sequences used to detect each secondary sequence in multiplex real-time PCR

[0160]

[0161] The multiplex real-time PCR thus:

[0162] - amplifying sequences of markers of a human papillomavirus genotype, whether or not the human papillomavirus is associated with the development of cervical cancer, oral cancer or anogenital cancer, and detecting any sequence amplified thereby comprising a sequence of a marker of any one of the human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68; and

[0163] - amplifying the sequence of a marker of a human housekeeping gene and detecting any sequence thereby amplified which comprises the sequence of the marker of said human housekeeping gene.

[0164] Thus, when the subject is infected with human papillomavirus (and the sample contains DNA from the subject and the human papillomavirus), the multiplex real-time PCR may amplify both (i) and (ii), but if the subject is not infected with human papillomavirus (and the sample contains DNA from the subject), the multiplex real-time PCR may amplify only (ii). On the other hand, if the sample contains DNA from the human papillomavirus but not from the subject, the multiplex real-time PCR will only amplify (i), indicating an invalid amplification procedure. If the sample does not contain DNA from the subject or from the human papillomavirus, the multiplex real-time PCR will not amplify any DNA. Thus, in the methods, uses, systems and kits of the present invention, when:

[0165] - the fourth fluorophore of the probe bound to the second sequence is not detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected ( Figure 1 102), repeat step (a) for the subject (“No” will Figure 1 The decision box 102 is connected to the box 100);

[0166] - detecting the fourth fluorophore of the probe bound to the second sequence (i.e. the sequence of the marker of the human housekeeping gene), and detecting at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence (i.e. the sequence of the marker of the 16 or 18 genotype of the human papillomavirus, respectively) Figure 1 104), then a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer is detected ( Figure 1 106);

[0167] - when the fourth fluorophore of the probe bound to the second sequence is detected, and the third fluorophore of the probe of the probe library bound to the first sequence (i.e., the sequence of the marker of any one of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus) is detected ( Figure 1 Decision box 108), a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is detected and a composition comprising the amplified first sequence and the second sequence is selected ( Figure 1 110 ), followed by step (b) when referring to the methods of the invention or followed by means (b) when referring to the systems and kits of the invention.

[0168] In preferred embodiments of the methods, systems and kits of the invention, said heating is from 60 to 70°C.

[0169] In preferred embodiments of the methods, systems and kits of the invention, said heating is from 60 to 65°C.

[0170] In preferred embodiments of the methods, systems and kits of the invention, the heating lasts for 1 to 20 minutes.

[0171] In preferred embodiments of the methods, systems and kits of the invention, the heating lasts for 1 to 10 minutes.

[0172] In preferred embodiments of the methods, systems and kits of the invention, the heating lasts for 3 to 20 minutes.

[0173] In a preferred embodiment of the methods, systems and kits of the present invention, the heating lasts for 1 minute.

[0174] In a preferred embodiment of the methods, uses, systems and kits of the invention, said cooling consists of cooling to 37 to 45°C.

[0175] In a preferred embodiment of the methods, uses, systems and kits of the invention, said cooling consists of cooling to 41 °C.

[0176] In a preferred embodiment of the methods, uses, systems and kits of the invention, said cooling lasts for at least 2 minutes.

[0177] In a preferred embodiment of the methods, uses, systems and kits of the invention, the cooling lasts for 2 to 10 minutes.

[0178] In a preferred embodiment of the methods, uses, systems and kits of the invention, said cooling lasts for 2 to 8 minutes.

[0179] In a preferred embodiment of the methods, uses, systems and kits of the invention, said cooling lasts for 2 to 6 minutes.

[0180] In a preferred embodiment of the methods, uses, systems and kits of the present invention, the cooling lasts for 4 minutes.

[0181] In a preferred embodiment of the methods, uses, systems and kits of the invention, the composition selected in (a) is heated to 95° C. for 10 minutes prior to the hybridization according to (b).

[0182] In a preferred embodiment of the methods, uses, systems and kits of the present invention, the composition selected in (a) is heated to at least 60°C for at least 10 minutes before cooling and hybridization according to (b). More preferably, the heating is maintained at 60 to 70°C for 1 to 20 minutes, even more preferably at 60 to 65°C for 3 to 10 minutes.

[0183] Preferred embodiments of the methods, uses, systems and kits of the present invention are additionally used to detect the absence of the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer in the subject, wherein when the fourth fluorophore of the probe bound to the second sequence is detected and the first fluorophore or the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer is not detected.

[0184] When no probe binding to the second sequence is detected and no probe binding to the first sequence is detected, the sample is considered to lack sufficient DNA. The probe binding to the second sequence may not be detected due to the presence of inhibitors in the PCR reaction. In a preferred embodiment of the method, use, system and kit of the present invention, when no probe binding to the second sequence is detected and no probe binding to the first sequence is detected, step (a) is repeated for the subject using a new sample obtained from the subject.

[0185] The first fluorophore, the second fluorophore, the third fluorophore and the fourth fluorophore of the corresponding probe (ie fluorescent probe) preferably each emit light at a different emission maximum wavelength. The first fluorophore, the second fluorophore, the third fluorophore and the fourth fluorophore are more preferably selected from hydroxycoumarin, methoxycoumarin, Alexa fluor 350, DY-415, aminocoumarin, Cy2, FAM, Alexa fluor 488, fluorescein FITC, Alexa fluor 430, JOE [6-JOE, SE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, succinimidyl ester)], VIC (2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein) fluorophore, Alexa fluor 532, HEX, Cy3, TRITC, Alexa fluor 546, Alexa fluor555, R-phycoerythrin (PE), rhodamine Red-X, Tamara, Cy3.5 581, Rox, Alexafluor 568, Red 613 (Red 613), Texas Red, Alexa fluor 594, Alexa fluor633, allophycocyanin, Alexa fluor 633, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor680 and Cy7.In one embodiment, one of the first, second, third and fourth fluorophores is a fluorophore selected from the group consisting of hydroxycoumarin, methoxycoumarin, Alexa fluor, aminocoumarin, Cy2 and FAM (blue channel); another of the first, second, third and fourth fluorophores is a fluorophore selected from the group consisting of Alexa fluor 488, fluorescein isothiocyanate (FITC), Alexafluor 430, JOE, VIC, Alexa fluor 532 and HEX (green channel); another of the first, second, third and fourth fluorophores is a fluorophore selected from the group consisting of Cy3, TRITC, Alexa fluor546, Alexa fluor 555, R-phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5 581, Rox, Alexafluor 568, Red 613, Texas Red and Alexa fluor 594 (yellow channel), and another of the first, second, third and fourth fluorophores is a fluorophore selected from the group consisting of Alexa fluor 633, allophycocyanin, Alexa fluor 633, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor 680 and Cy7 (red channel). Even more preferably, the first, second, third and fourth fluorophores are each different fluorophores selected from the group consisting of ROX, Cy5, FAM, HEX, JOE and VIC fluorophores. In an exemplary embodiment, the first fluorophore is ROX, the second fluorophore is Cy5, the third fluorophore is FAM, and the fourth fluorophore is HEX or JOE or VIC.

[0186] The light emitted from each fluorophore is detected simultaneously (when emitted) by a multiplex real-time PCR platform capable of detecting light at each of the emission maximum wavelengths of the first, second, third and fourth fluorophores. Preferably, the multiplex real-time PCR platform is selected from the group consisting of QuantStudio™5 real-time PCR system (Applied Biosystems), CFX96™ real-time PCR detection system (Bio-Rad) and VitroCycler (VitroS.A.) platforms, in which the methods, uses, systems and kits of the present invention have been validated. The detection limit and quantification limit can be determined as described in Forootan et al. (2017) Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR), BiomolDetect Quantif. 12: 1-6. For example, a threshold and a baseline can be established for each real-time PCR. The baseline of a real-time PCR reaction can, for example, refer to the signal level during the initial cycle of PCR, such as the average signal obtained during cycles 3 to 15 of PCR. The threshold can be used to distinguish the relevant amplified signal from the background.

[0187] The (b) in the method of the present invention comprises the step of hybridizing the amplified first sequence and the second sequence contained in the composition selected in step (a). Figure 1 The method of the present invention is to provide a method of hybridizing the amplified first sequence and the second sequence contained in the composition selected in (a) according to the present invention, wherein the amplified DNA is a DNA subjected to hybridization, the DNA being contained in the composition, in which the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe pool bound to the first sequence is detected.

[0188] The hybridization comprises combining the amplified first sequence and the amplified second sequence contained in the composition selected in (a) with at least one probe attached to a solid support. Each probe attached to the solid support comprises a sequence complementary to and specific for the sequence of the marker, and a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided for the marker in the multiplex real-time PCR in (a). Therefore, the hybridization comprises:

[0189] - binding / annealing of the amplified first sequence comprised in the composition selected in (a) and at least one sequence complementary to and specific for the sequence of the marker, providing the marker with a forward primer and a reverse primer complementary to and specific for the locus of the marker in the multiplex real-time PCR in (a), and

[0190] - binding / annealing of the amplified second sequence comprised in the composition selected in (a) and at least one sequence complementary to and specific for the sequence of the marker, for which forward primers and reverse primers complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in (a),

[0191] wherein the sequence of the marker is contained in a probe attached to the solid support.

[0192] The solid support is a three-dimensional porous environment, preferably selected from the group consisting of a hybridization membrane, a hybridization sheet and a loaded hybridization membrane. More preferably, the solid support is a nylon hybridization membrane, a charged nylon hybridization membrane, a nitrocellulose sheet or a loaded hybridization membrane comprising nitrocellulose loaded on a polymer membrane.

[0193] The solid support comprises probes attached thereto, wherein each probe comprises a sequence complementary to and specific to the sequence of the marker, and in the multiplex real-time PCR in step (a), a forward primer and a reverse primer complementary to and specific to the locus of the marker are provided for the marker, wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence. Therefore, the solid support comprises a plurality of non-overlapping specific regions, each of which comprises a plurality of probes, the probes comprising the same sequence, and which are different from the probe sequences attached to other specific regions of the solid support. Each specific region is preferably a circular, square or rectangular region of the solid support, more preferably a circular region (i.e., a dot or spot). The specific regions are preferably arranged in an array, a grid or an ordered pattern, more preferably in a square or rectangular array, and even more preferably in a square array of circular specific regions.

[0194] Each probe attached to a specific region of the solid support comprises a sequence complementary to and specific for a sequence of a marker for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR of step (a). Preferably, each probe attached to a specific region of the solid support comprises a sequence selected from any sequence shown in Table 5, which is complementary to and specific for the sequence of the corresponding human papillomavirus genotype or human housekeeping gene shown therein.

[0195] Table 5: Probe sequences used to detect each human papillomavirus genotype

[0196]

[0197]

[0198] Any given specific region of the solid support comprises probes, each probe having a sequence complementary to and specific for the same marker. The group of specific regions comprises at least two subgroups of specific regions, wherein:

[0199] - each specific region of a subset of said specific regions comprises probes, each probe having a sequence complementary to and specific for a sequence of the same marker, for which a forward primer and a reverse primer complementary to and specific for the locus of said marker of human papillomavirus are provided in said multiplex real-time PCR of step (a); and

[0200] - each specific region of a subset of said specific regions comprises probes, each probe having a sequence complementary to and specific for a sequence of the same marker, for which a forward primer and a reverse primer complementary to and specific for the locus of said marker of a human housekeeping gene are provided in said multiplex real-time PCR of step (a),

[0201] Each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence. When the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the third fluorophore of the probe bound to the first sequence is detected, at least one specific region of the solid support to which the probe is attached can hybridize with the first sequence. Therefore, when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the third fluorophore of the probe bound to the first sequence is detected, if only one genotype of human papillomavirus is infecting the subject and the genotype can be identified by a marker comprising a sequence complementary to and specific for the sequence of the probe attached to the solid support, wherein the sequence of the probe is complementary to and specific for the sequence of the marker, the first sequence will hybridize with the sequence of the probe in the multiplex real-time PCR in step (b) provided with a forward primer and a reverse primer complementary to and specific for the locus of the marker. Preferably, at least one specific region of the solid support to which the probe is attached can hybridize to a first sequence, and at least one specific region of the solid support to which the probe is attached can hybridize to a second sequence.

[0202] The (d) in the method of the present invention comprises the step of identifying each specific region of the solid support to which the probe is attached and to which the first sequence or the second sequence hybridizes ( Figure 1 114), while the (c) in the systems and kits of the present invention includes a device for identifying each specific area of ​​the solid support to which the probe is attached and to which the first sequence or the second sequence hybridizes.

[0203] Each specific area of ​​the solid support on which hybridization of the amplified sequence and / or the amplified first and second sequences (i.e., amplicons) of the composition selected in step (a) with the sequence of the probe attached to the solid support occurs can be detected and thus identified. Detection includes detecting light emitted from a fluorophore contained in a probe, the probe comprising a sequence that binds / anneals to any given amplicon, such as the amplicon binds / anneals to the sequence, or by using a molecular tag attached to the amplicon, wherein the molecular tag can be detected. Preferably, detection includes detection of light (i.e., visualization), wherein the molecular tag is a fluorophore or a reagent required to cause a colorimetric reaction. More preferably, and as exemplified, detection comprises visualization of a molecular tag attached to the amplicon, wherein the tag is biotin and is visualized as follows, by an immunoenzymatic colorimetric reaction with streptavidin-phosphatase and a chromogen (e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) (also referred to herein as NBT-BCIP)) and / or with streptavidin-horseradish peroxidase and 3,3-diaminobenzidine (DAB), producing an insoluble precipitate in the solid support in those specific areas where hybridization has occurred. Light irradiation through the solid support scatters to a greater extent in those specific areas where the insoluble precipitate is present (i.e., where hybridization has occurred), thereby allowing identification of those specific areas where hybridization has occurred. Typically, detection comprises detecting the concentration of the amplicon compared to the gray signal intensity and background noise. Suitable software can define threshold parameters to automatically perform the analysis of each point to distinguish the gray signal intensity and background noise from the amplicon signal.

[0204] Therefore, the molecular tag can be any suitable molecular tag. Suitable molecular tags are known to those skilled in the art. For example, the molecular tag can be biotin, digoxigenin or a fluorophore, as described elsewhere herein. Biotin can be visualized by immunoenzymatic colorimetric reaction with streptavidin-phosphatase and a suitable chromogen (e.g., NBT-BCIP) and / or with streptavidin-horseradish peroxidase and 3,3 diaminobenzidine (DAB). Leary et al. (1983) PNAS, Vol. 80, pp. 4045-4049, Genetics, in particular, describes how biotin contained in DNA / RNA can be visualized on a solid support. Visualization of digoxigenin-labeled amplicons can be performed in a similar manner, using anti-digoxigenin antibodies coupled to alkaline phosphatase (AP) or horseradish peroxidase (HRP) for colorimetric detection. Therefore, molecular tags (e.g., biotin or digoxigenin) can be identified by streptavidin containing a reporter. The reporter can be any suitable reporter. For example, the reporter can be horseradish peroxidase or alkaline phosphatase.

[0205] Therefore, the present invention also relates to a forward primer and / or a reverse primer

[0206] Use for obtaining amplified sequences comprising molecular tags by multiplex real-time PCR, wherein the multiplex real-time PCR amplifies sequences contained in DNA present in a biological sample obtained from a subject, wherein the DNA is extracted from the biological sample before subjecting the biological sample to the multiplex real-time PCR,

[0207] The forward primer and / or reverse primer comprises

[0208] - sequences complementary to and specific for one or more loci of markers of genotype 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68 of human papillomavirus; and

[0209] - molecular tags; and

[0210] Use for identifying a specific area on a solid support attached to a probe, wherein the probe comprises a sequence complementary to and specific for a sequence of a marker, the forward primer and / or reverse primer are complementary to and specific for the sequence of the marker; and an amplified sequence comprising a molecular tag hybridizes with the sequence of the marker.

[0211] Primers (forward and reverse) described herein are oligonucleotides. Such oligonucleotides can have a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or more nucleotides. For example, the oligonucleotide can have a length of 4 to 50 nucleotides, 5 to 30 nucleotides, 10 to 30 nucleotides or 15 to 30 nucleotides. The primers / oligonucleotides can have a sequence of any one of SEQ ID No. 1 to 4. Primers described herein are used to amplify one or more DNA sequences described herein.

[0212] The primers described herein may include a molecular tag. It is also contemplated that the primer may include 1, 2, 3, 4 or more molecular tags. The molecular tag may be attached to the primer by any suitable means. The molecular tag may be attached to the 5' end, the 3' end, or may be attached inside the primer sequence. Therefore, the forward primer and / or the reverse primer may include a molecular tag at its 5' end. Means for attaching the molecular tag to the primer are known to the skilled person and are described, for example, by Benktström et al. (1991) Biotinylation of Oligonucleotides and Their Use as Polymerase Chain Reaction Primers Nucleosides & Nucleotides 10 (1): 507-509. Different companies synthesize oligonucleotides containing molecular tags as needed, such as TriLink® BioTechnologies or Creative Biogene. For example, the molecular tag may be attached to the forward primer and / or the reverse primer by a suitable linker (preferably at its 5' end). Such suitable linkers are known in the art. For example, the linker can be 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (TFA-amino linker), 2-[2-(4-monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, 6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 6-(4,4'-dimethoxy-4"-methylsulfonyl-tritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 12-(4-monomethoxytritylamino)dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite acetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 10-(O-trifluoroacetylamino-N-ethyl)-triethylene glycol-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 3-(trifluoroacetylamino)propyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, S-trityl-6-mercaptohexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 10-carboxy-decyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, N-hydroxysuccinimide ester, 1-O-dimethoxytrityl-hexyl-disulfide, 1′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 6-(N 2 -Methyl-N 1-phthalimido)-hexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0213] As disclosed herein, the molecular tag can thus be attached to the amplified sequence (also referred to herein as an amplicon) obtained by multiplex real-time PCR. Thus, depending on the position of the molecular tag of the primers that generate the amplified sequence, the molecular tag can be attached to the 5' end of the amplified sequence (e.g., the first sequence and / or the second sequence) or attached within the amplified sequence. Preferably, the amplified sequence (e.g., the first amplified sequence and / or the second amplified sequence) comprises a molecular tag at its 5' end.

[0214] Therefore, the present invention also relates to the use of an amplified sequence comprising a molecular tag, wherein the amplified sequence has been obtained in a multiplex real-time PCR, wherein the multiplex real-time PCR amplifies a sequence comprised in a DNA present in a biological sample obtained from a subject, wherein the amplification is performed using a forward primer and / or a reverse primer, wherein the forward primer and / or the reverse primer comprises

[0215] - sequences complementary to and specific for one or more loci of markers of genotype 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68 of human papillomavirus; and

[0216] - molecular tags; and

[0217] Used to identify a specific area on a solid support attached to a probe, wherein the probe comprises a sequence complementary to and specific for a sequence of a marker, the forward primer and / or reverse primer are complementary to and specific for the sequence of the marker; and the amplified sequence comprising a molecular tag hybridizes with the sequence of the marker.

[0218] Furthermore, by knowing which fluorophores are detected in (a), and the genotype detectable at each specific region of the solid support (i.e., the probe attached to each specific region of the solid support contains a sequence complementary to and specific for the sequence of the marker of which genotype), hybridization of each specific region has been identified in (c), it is then possible to determine one or more genotypes of the human papillomavirus infecting the subject. Specifically, when:

[0219] (I) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the first fluorophore of the probe bound to the first sequence is detected ( Figure 1 In the decision block 116, the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer is determined to be genotype 16 of human papillomavirus ( Figure 1118);

[0220] (II) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the second fluorophore of the probe bound to the first sequence is detected ( Figure 1 In the decision block 120, the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer is determined to be genotype 18 of human papillomavirus ( Figure 1 122);

[0221] (III) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in step (c); Figure 1 Decision block 124), the genotype is determined by ( Figure 1 Box 130):

[0222] (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence ( Figure 1 126);

[0223] (IIIb) Determine which HPV genotype is identified by the marker ( Figure 1 ), the marker comprising a sequence complementary to and specific for the probe sequence identified in (IIIa) attached to each specific region.

[0224] It is noteworthy that when infection with genotype 16 or 18 of human papillomavirus is determined, the clinical management of the subject is generally different from the clinical management when infection with any one of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus is determined. Therefore, if only one or two of genotype 16 and genotype 18 of human papillomavirus are detected (i.e., the fourth fluorophore of the probe bound to the second sequence is detected in step (a), but the third fluorophore of the probe bound to the first sequence is not detected), then hybridization as defined in (b) does not need to be performed, which in itself saves reagents for hybridization and minimizes waste. However, in practice, genotype 16 and / or genotype 18 of human papillomavirus are usually (if not always) found in the presence of at least one of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus. However, in particularly preferred embodiments, wherein the methods, uses, systems and kits of the present invention are used to detect one or more genotypes associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject infected with a genotype of a human papillomavirus, and to determine one or more genotypes of a human papillomavirus detected in a subject that are associated with the occurrence of cervical cancer, oral cancer or anogenital cancer and, optionally, one or more genotypes that are not associated with the occurrence of cervical cancer, oral cancer or anogenital cancer, wherein each genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is selected from the group consisting of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus, the group of probes defined in (iii) preferably still comprises a probe comprising a first fluorophore and a probe comprising a second fluorophore so as to be able to detect and determine genotypes 16 and 18 of human papillomavirus in the subject.

[0225] However, another aspect of the present invention is a method for detecting a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in a subject, and for determining one or more genotypes of human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is detected in the subject, wherein each genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is selected from the group consisting of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus,

[0226] The method comprises:

[0227] (a) performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the DNA is extracted from the biological sample prior to performing the multiplex real-time PCR on the biological sample, wherein the multiplex real-time PCR amplifies:

[0228] (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and

[0229] (ii) second sequences comprised in said DNA, wherein each second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker,

[0230] The composition comprises:

[0231] (iii) a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus each have a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and

[0232] (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene,

[0233] wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe,

[0234] Among them, when:

[0235] - when the fourth fluorophore of the probe bound to the second sequence is not detected and the third fluorophore of the probe bound to the first sequence is not detected, repeating step (a) for the subject;

[0236] - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected;

[0237] (b) hybridizing each amplified first sequence and second sequence of the composition selected in step (a) on a solid support, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, the marker being provided with a forward primer and a reverse primer complementary to and specific for the locus of the marker in the multiplex real-time PCR in step (a),

[0238] wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence; and

[0239] (c) identifying each specific area of ​​the solid support to which a probe is attached and which hybridizes to the first sequence or the second sequence;

[0240] Among them, when

[0241] (III) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in step (c), the genotype is determined by:

[0242] (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence;

[0243] (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for the probe sequence attached to each specific region identified in step (IIIa).

[0244] Similarly, another aspect of the present invention is a system and a kit for detecting a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer, or anal genital cancer in a subject, and for determining the genotype of the human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer, or anal genital cancer is detected, wherein each genotype associated with the occurrence of cervical cancer, oral cancer, or anal genital cancer is selected from the group consisting of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus,

[0245] The system and kit include:

[0246] (a) a device for performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the multiplex real-time PCR amplifies:

[0247] (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and

[0248] (ii) a second sequence comprised in said DNA, wherein said second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker,

[0249] The composition comprises:

[0250] (iii) a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus each have a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and

[0251] (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene,

[0252] wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe,

[0253] Among them, when:

[0254] - when the fourth fluorophore of the probe bound to the second sequence is not detected and the third fluorophore of the probe bound to the first sequence is not detected, repeating step (a) for the subject;

[0255] - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected;

[0256] (b) means for hybridizing each amplified first sequence and second sequence of the composition selected in (a) on a solid support, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in (a),

[0257] wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence; and

[0258] (c) means for identifying each specific area of ​​the solid support to which a probe is attached and which hybridizes to the first sequence or the second sequence;

[0259] Among them, when:

[0260] (III) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in (c), the genotype is determined by:

[0261] (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence;

[0262] (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for a probe sequence attached to each specific region identified in (IIIa).

[0263] The present invention also encompasses methods and / or uses including extraction of DNA of human papillomavirus. The present invention may also include a system and / or a kit comprising a step (aa) device for extracting DNA of human papillomavirus present in a biological sample obtained from the subject. The device for extracting DNA may be used before using a device for subjecting at least one sequence contained in the DNA present in the biological sample obtained from the subject to multiplex real-time PCR, and step (aa) may therefore be performed before step (a) of the system and kit as described herein. Such a device may, for example, include a lysis buffer, a high concentration salt solution (e.g., for precipitating proteins), a solution containing isopropanol for precipitating DNA from a high salt solution, and / or a solution for eluting DNA (e.g., Tris-EDTA or nuclease-free water). Filter membranes and / or matrices (e.g., silica, cellulose, and ion exchange) may also be included by the device.

[0264] In another preferred embodiment of the methods, uses, systems and kits of the present invention, the DNA is extracted from the biological sample by lysis before subjecting the biological sample to multiplex real-time PCR, and:

[0265] - when performing multiplex real-time PCR on said at least one sequence comprised in the DNA present in the biological sample, as defined in step (a), a first control is also subjected to multiplex real-time PCR, wherein said first control is a blank in which said extraction is performed, and optionally

[0266] - when the at least one sequence contained in the DNA present in the biological sample is subjected to multiplex real-time PCR, as defined in step (a), the second control is also subjected to multiplex real-time PCR, wherein the second control comprises a DNA comprising a first sequence, the first sequence being a sequence of a marker from a human papillomavirus genotype selected from the group consisting of 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus; and optionally

[0267] - the first sequence and the second sequence amplified in step (a) and the solid support further comprise molecules attached thereto, wherein the molecules attached to the solid support are attached to specific regions of the solid support that are different from other specific regions of the solid support to which probes are attached, wherein the molecules attached to the first sequence and the second sequence amplified in step (a) and the solid support have the same chemical structure.

[0268] HPV DNA extraction methods are known to those skilled in the art. Corresponding kits are particularly available from MGI (HPV DNA extraction kit, number: 1000028433). In addition, methods for extracting HPV DNA are described, for example, in Example 1. By extracting DNA from a biological sample, at least 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1 μg, 1.5 μg, 2 μg or more DNA can be obtained. Typically, the extracted DNA is contained in an eluent (elution buffer). In multiplex real-time PCR, the DNA can be, for example, at about 100 pg / μL. -1 For example, for multiplex real-time PCR, the PCR master mix may include about 1 ng / μL -1 Up to 4 ng / μL -1 DNA.

[0269] In another preferred embodiment of the method, system and test kit of the present invention, each probe is located in at least two specific areas of the solid support. This makes it possible to obtain more than one result for each probe in one assay, thereby further improving the sensitivity of the method.

[0270] In (b), a hybridization control probe or hybridization control such as biotin (B), an endogenous amplification control probe (C) and / or a degenerate universal probe (U) can be attached to a specific region of the solid support.

[0271] In the case of the application of hybridization control probes (B), after the development of the solid support (i.e., hybridization) required for identification of specific areas where hybridization occurs, a strong signal (coloration / darkening / lightening) will appear in one or more specific areas to which the hybridization control probe is attached, thereby serving as a quality control for hybridization. This signal indicates that the hybridization reagents and development have worked properly. If the signal does not appear, it indicates that an error occurred during the hybridization process or that the reagents were not used correctly. Preferably, this signal allows the software to correctly orient the solid support (chip) so that subsequent analysis of the hybridization results and therefore genotyping is automated.

[0272] The endogenous amplification control probe (C) detects the sequence of the human housekeeping gene amplified in (a) (co-amplified with each first sequence) by multiplex real-time PCR. In the case of applying the endogenous amplification control probe, after the solid support (i.e., hybridization) required for identifying the specific area where hybridization occurs, the sample in which the sequence of the human housekeeping gene has been correctly amplified will hybridize (i.e., be able to be visualized and have a positive signal) with the specific area of ​​the solid support to which the endogenous amplification control probe is attached. This signal shows the quality / quantity of the DNA used in the amplification (and therefore, produced by it) by the multiplex real-time PCR. A positive signal shows that the amplification is correct and the quality and quantity of the starting DNA are optimal. The absence of a signal for this control means that there is an error during amplification, the quality / quantity of the DNA used in amplification is low, or human DNA is not present in the sample. This last case is possible when the number of human cells present in the sample is below the detection limit. In addition, if no positive signal for any HPV genotype is detected, the software (e.g., hybriSoft software) will preferably generate an automatic message that the sample is blank, there is inappropriate or insufficient material, and / or PCR is inhibited. On the other hand, when the sample tests positive at any specific area of ​​the solid support attached to the probe (the probe comprises a sequence complementary to and specific for the sequence of the marker of the HPV genotype), but the sample does not test positive at any specific area of ​​the solid support attached to the endogenous amplification control probe, the software will preferably generate an automatic message that insufficient material is present in the composition. In this case, the method, reagents and samples (e.g., quality) must be checked before verifying the hybridization results.

[0273] In another preferred embodiment of the methods, uses, systems and kits of the present invention, the specific region of the solid support different from other specific regions of the solid support to which probes are attached comprises a probe library (i.e. a universal probe library or a degenerate universal probe, U) attached thereto, wherein each probe in the probe library comprises a sequence complementary to and specific for a sequence of a conserved region of an HPV L1 or HPV L2 gene, wherein at least two probes in the probe library comprise sequences different from each other, wherein when:

[0274] - upon identifying a specific area of ​​the solid support to which the probe library is attached and which hybridizes to a sequence;

[0275] - upon identification of a specific area of ​​said solid support to which the probe is attached and which is hybridized to the second sequence; and

[0276] - when no specific area of ​​the solid support to which the probe is attached and which hybridizes to the first sequence is identified,

[0277] The genotype of human papillomavirus is determined to be different from each genotype identifiable by a marker for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in step (a).

[0278] In another preferred embodiment of the methods, uses, systems and kits of the present invention, the methods are additionally used to determine whether the subject is at risk of developing cervical cancer, oral cancer or anogenital cancer, wherein when any one of the 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus is determined, the subject is determined to be at risk of developing cervical cancer, oral cancer or anogenital cancer.

[0279] In another preferred embodiment of the methods, uses, systems and kits of the present invention, the methods are additionally used to determine whether the subject is at risk of developing a benign lesion, wherein the subject is determined to be at risk of developing a benign lesion when any one of genotypes 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82 and 84 of human papillomavirus is determined. The benign lesion is not cervical cancer, oral cancer or anogenital cancer.

[0280] In preferred embodiments of the systems and kits of the invention:

[0281] - a device for subjecting the DNA present in the biological sample to multiplex real-time PCR comprising a multiplex real-time PCR instrument and multiplex real-time PCR reagents;

[0282] - a means for hybridizing each of the first and second sequences amplified in the composition selected in (a) on a solid support, comprising a solid support and a hybridization reagent, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in (a);

[0283] - The means for identifying each specific area of ​​said solid support to which a probe is attached and to which a sequence is hybridized comprises an image capture device.

[0284] Preferably, the multiplex real-time PCR instrument is a platform selected from the group consisting of the QuantStudio™ 5 Real-Time PCR System (Applied Biosystems), the CFX96™ Real-Time PCR Detection System (Bio-Rad), and the VitroCycler (Vitro SA) platform, also as described above, and the multiplex real-time PCR reagents are those in the human papillomavirus master mix necessary for performing multiplex real-time PCR, more preferably a hot-start polymerase, uracil DNA glycosylase, forward and reverse primer pairs, a fluorescent probe, a PCR reaction buffer, a deoxyuridine triphosphate (dUTP) solution, a deoxynucleoside triphosphate (dNTP) solution containing dATP, dCTP, dTTP, and dGTP, and molecularly labeled primers, each of the forward and reverse primer pairs being complementary to and specific for the locus of a marker of the genotype of human papillomavirus or a human housekeeping gene.

[0285] Preferably, the device for hybridizing each first sequence and second sequence amplified in the composition selected in (a) on a solid support comprises a solid support and a hybridization reagent, as defined herein, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for the sequence of the marker, and the marker is provided with a forward primer and a reverse primer complementary to and specific for the locus of the marker in the multiplex real-time PCR in (a). More preferably, the reagents include a hybridization solution (reagent A), a blocking solution (reagent B), streptavidin-phosphatase (streptavidin-alkaline phosphatase, reagent C), a wash buffer I (reagent D), an NBT-BCIP solution (reagent E) and a wash buffer II (reagent F). Even more preferably, the device for hybridizing each first sequence and second sequence amplified in the composition selected in (a) on a solid support further comprises a hybridization platform, such as an exemplary hybriSpot 12 or 24 platform, a thermal cycler, a thermal block or a thermostatic bath / heater and a cold plate.

[0286] Preferably, the means for identifying each specific area of ​​the solid support to which a probe is attached and hybridized to a sequence comprises an image capture device. The image capture device is preferably comprised in the above-mentioned hybridization platform, such as the exemplary hybriSpot 12 or 24 platform.

[0287] The multiplex real-time PCR instrument, hybridization platform, and image capture device may each comprise a computer program product.

[0288] The computer program product may include a computer-readable storage medium (or multiple media) having computer-readable program instructions thereon for causing a processor to execute aspects or steps of the present invention.

[0289] A computer-readable storage medium may be a tangible device that can retain and store instructions used by an instruction execution device. Computer-readable storage media may include, but are not limited to, electronic storage devices, magnetic storage devices, optical storage devices, electromagnetic storage devices, semiconductor storage devices, or any suitable combination of the foregoing. A non-exhaustive list of more specific examples of computer-readable storage media includes the following: portable computer diskettes, hard disks, random access memories (RAM), read-only memories (ROM), erasable programmable read-only memories (EPROM or flash memory), static random access memories (SRAM), portable compact disk read-only memories (CD-ROMs), digital versatile disks (DVDs), memory sticks, floppy disks, mechanical encoding devices (such as punch cards with instructions recorded thereon or raised structures in grooves), and any suitable combination of the foregoing.

[0290] The computer-readable program instructions described herein may be downloaded from a computer-readable storage medium to a corresponding computing / processing device, or downloaded to an external computer or external storage device via a network (e.g., the Internet, a local area network, a wide area network, and / or a wireless network). The network may include copper transmission cables, optical transmission fibers, wireless transmissions, routers, firewalls, switches, gateway computers, and / or edge servers. The network adapter or network interface in each computing / processing device receives the computer-readable program instructions from the network and forwards the computer-readable program instructions to be stored in a computer-readable storage medium within the corresponding computing / processing device.

[0291] The computer-readable program instructions for performing the operations of the present invention may be assembly instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, state setting data, or source code or object code written in any combination of one or more programming languages, including object-oriented programming languages ​​(such as Smalltalk, C++, etc.) and conventional procedural programming languages ​​(such as "C" programming language or similar programming languages). The computer-readable program instructions may be executed entirely on the user's computer, partially on the user's computer, as a stand-alone software package, partially on the user's computer and partially on a remote computer, or entirely on a remote computer or server. In the latter case, the remote computer may be connected to the user's computer via any type of network, or may be connected to an external computer (e.g., via the Internet using an Internet service provider), including a local area network (LAN) or a wide area network (WAN). In some embodiments, electronic circuits including, for example, programmable logic circuits, field programmable gate arrays (FPGAs), or programmable logic arrays (PLAs) may execute computer-readable program instructions by utilizing the state information of the computer-readable program instructions to personalize the electronic circuits so as to perform aspects or steps of the present invention.

[0292] Aspects or steps of the invention are described herein with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), uses and kits according to embodiments and / or steps of the invention (see e.g. Figure 1 and 4 ). It should be understood that each square or diamond box in the flowchart and / or block diagram and the combination of boxes in the flowchart and / or block diagram can be implemented by biotechnology or computer-readable program instructions or a combination thereof.

[0293] These computer-readable program instructions may be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing device to produce a machine, such that the instructions executed via the processor of the computer or other programmable data processing device create a device for implementing the functions / actions specified in the flowchart and / or one or more block diagram blocks. These computer-readable program instructions may also be stored in a computer-readable storage medium, which may direct a computer, a programmable data processing device, and / or other device to operate in a specific manner, such that the computer-readable storage medium having the instructions stored therein contains an article of manufacture, which includes instructions for implementing aspects of the functions / actions specified in the flowchart and / or one or more block diagram blocks.

[0294] Computer-readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device, so that a series of operational steps are performed on the computer, other programmable apparatus, or other device to produce a computer-implemented process, so that the instructions executed on the computer, other programmable apparatus, or other device implement the functions / actions specified in the flowchart and / or one or more block diagram blocks.

[0295] The flow chart and block diagram in the accompanying drawings show the possible architecture, function and operation of the system, method and test kit according to various embodiments of the present invention.In this respect, each block in the flow chart or block diagram can represent a module, segment or part of an instruction, which includes one or more executable instructions for realizing one or more specified logical functions.In some alternative embodiments, the function marked in the block may not occur in the order marked in the accompanying drawings.For example, depending on the function involved, the two blocks shown in succession can actually be performed substantially simultaneously, or these blocks can sometimes be performed in reverse order.It should also be noted that the combination of each block of the block diagram and / or the flow chart diagram and the block diagram and / or the flow chart diagram can be realized by a system based on dedicated hardware that performs a specified function or action or performs a combination of dedicated hardware and computer instructions.

[0296] All means or steps in the following claims plus corresponding structures, materials, acts, and equivalents of functional elements are intended to include any structure, material, or act for performing a function in combination with elements of other claims such as specific claims. The description of the present invention has been presented for the purpose of illustration and description, but the description of the present invention is not intended to be exhaustive or limited to the embodiments of the present invention in a disclosed form. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the embodiments of the present invention. The embodiments are selected and described in order to best explain the principles and practical applications of the embodiments of the present invention, and to enable other persons of ordinary skill in the art to understand the embodiments of the present invention, as well as various embodiments for various modifications suitable for the intended specific use.

[0297] The present invention also relates to the following items:

[0298] Item 1. A method for detecting a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer in a subject and for determining the one or more genotypes of a human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer is detected in the subject, comprising:

[0299] (a) performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the DNA is extracted from the biological sample prior to performing the multiplex real-time PCR on the biological sample, wherein the multiplex real-time PCR amplifies:

[0300] (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and

[0301] (ii) a second sequence comprised in said DNA, wherein said second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker,

[0302] The composition comprises:

[0303] (iii) a set of probes comprising:

[0304] - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16;

[0305] - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and

[0306] - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and

[0307] (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene,

[0308] wherein each probe is detected that binds to a sequence of a marker to which each probe is complementary and specific,

[0309] Among them, when:

[0310] - when the fourth fluorophore of the probe bound to the second sequence is not detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, repeating step (a) for the subject;

[0311] - when the fourth fluorophore of the probe bound to the second sequence is detected and at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected;

[0312] - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected;

[0313] (b) hybridizing each amplified first sequence and second sequence of the composition selected in step (a) on a solid support, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, the marker being provided with a forward primer and a reverse primer complementary to and specific for the locus of the marker in the multiplex real-time PCR in step (a),

[0314] wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence; and

[0315] (c) identifying each specific area of ​​the solid support to which a probe is attached and which hybridizes to the first sequence or the second sequence;

[0316] Among them, when:

[0317] (I) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the first fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 16 of human papillomavirus;

[0318] (II) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the second fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 18 of human papillomavirus;

[0319] (III) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in step (c), the genotype is determined by:

[0320] (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence;

[0321] (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for the probe sequence attached to each specific region identified in step (IIIa).

[0322] Item 2. The method according to Item 1, wherein the multiplex real-time PCR amplification:

[0323] (i) at least one first sequence, wherein each first sequence is a sequence from a marker of a human papillomavirus genotype that is present in a conserved region of the HPV L1 or HPV L2 gene; and

[0324] (ii) the second sequence.

[0325] Item 3. The method according to any one of Items 1 and 2, wherein the multiplex real-time PCR amplification:

[0326] (i) at least one first sequence, wherein each first sequence is a sequence from a marker for a human papillomavirus genotype selected from the group consisting of genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, and 84 of human papillomavirus; and

[0327] (ii) the second sequence.

[0328] Item 4. The method according to Item 3, wherein the multiplex real-time PCR amplification:

[0329] (i) a first sequence, which is a sequence of a marker of genotype 16 of human papillomavirus;

[0330] - a first sequence which is a sequence of a marker of genotype 18 of human papillomavirus, and,

[0331] - at least one other first sequence, wherein each first sequence is a sequence from a marker of a human papillomavirus genotype selected from the group consisting of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus; and

[0332] (ii) the second sequence.

[0333] Item 5. The method according to any one of items 1 to 4, wherein the DNA is extracted from the biological sample by lysis before the biological sample is subjected to multiplex real-time PCR, and:

[0334] - when performing multiplex real-time PCR on said at least one sequence comprised in the DNA present in the biological sample, as defined in step (a), a first control is also subjected to multiplex real-time PCR, wherein said first control is a blank in which said extraction is performed, and optionally

[0335] - when the at least one sequence contained in the DNA present in the biological sample is subjected to multiplex real-time PCR, as defined in step (a), the second control is also subjected to multiplex real-time PCR, wherein the second control comprises a DNA comprising a first sequence, the first sequence being a sequence of a marker from a human papillomavirus genotype selected from the group consisting of 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus; and optionally

[0336] - the first sequence and the second sequence amplified in step (a) and the solid support further comprise molecules attached thereto, wherein the molecules attached to the solid support are attached to specific regions of the solid support that are different from other specific regions of the solid support to which probes are attached, wherein the molecules attached to the first sequence and the second sequence amplified in step (a) and the solid support have the same chemical structure.

[0337] Item 6. The method according to any one of Items 1 to 5, wherein when no probe bound to the second sequence is detected and no probe bound to the first sequence is detected, step (a) is repeated on the subject using a new sample obtained from the subject.

[0338] Item 7. A method according to any one of Items 1 to 6, wherein the method is additionally used to detect the absence of the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in the subject,

[0339] When the fourth fluorophore of the probe bound to the second sequence is detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is not detected.

[0340] Item 8. A method according to any one of Items 1 to 7, wherein the specific area of ​​the solid support different from other specific areas of the solid support to which probes are attached comprises a probe library attached thereto, wherein each probe in the probe library comprises a sequence complementary to and specific for a sequence of a conserved region of an HPV L1 or HPV L2 gene, wherein at least two probes in the probe library comprise sequences different from each other, wherein when:

[0341] - upon identifying a specific area of ​​the solid support to which the probe library is attached and which hybridizes to a sequence;

[0342] - upon identification of a specific area of ​​said solid support to which the probe is attached and which is hybridized to the second sequence; and

[0343] - when no specific area of ​​the solid support to which the probe is attached and which hybridizes to the first sequence is identified,

[0344] The genotype of human papillomavirus is determined to be different from each genotype identifiable by a marker for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in step (a).

[0345] Item 9. A method according to any one of Items 1 to 8, wherein the method is additionally used to determine whether the subject is at risk of developing cervical cancer or anogenital cancer, wherein when any one of the 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus is determined, the subject is determined to be at risk of developing cervical cancer or anogenital cancer.

[0346] Item 10. A method according to any one of Items 1 to 8, wherein the method is additionally used to determine whether the subject is at risk of developing benign lesions, wherein when any one of 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82 and 84 genotypes of human papillomavirus is determined, the subject is determined to be at risk of developing benign lesions.

[0347] Item 11. The method according to any one of Items 1 to 10, wherein the composition selected in step (a) is heated to 60° C. for at least 10 minutes before cooling and hybridizing according to step (b).

[0348] Item 12. The method according to any one of Items 1 to 11, wherein the biological sample obtained from the subject is obtained by obtaining the sample from the subject himself.

[0349] Item 13. A system for detecting a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer, or anal genital cancer in a subject and for determining the one or more genotypes of a human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer, or anal genital cancer is detected in the subject, comprising:

[0350] (a) a device for performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the multiplex real-time PCR amplifies:

[0351] (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and

[0352] (ii) a second sequence comprised in said DNA, wherein said second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker,

[0353] The composition comprises:

[0354] (iii) a set of probes comprising:

[0355] - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16;

[0356] - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and

[0357] - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and

[0358] (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene,

[0359] wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe,

[0360] Among them, when:

[0361] - when the fourth fluorophore of the probe bound to the second sequence is not detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, repeating (a) for the subject;

[0362] - when the fourth fluorophore of the probe bound to the second sequence is detected and at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected;

[0363] - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected;

[0364] (b) means for hybridizing each amplified first sequence and second sequence of the composition selected in (a) on a solid support, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in (a),

[0365] wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence; and

[0366] (c) means for identifying each specific area of ​​the solid support to which a probe is attached and which hybridizes to the first sequence or the second sequence;

[0367] Among them, when:

[0368] (I) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the first fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 16 of human papillomavirus;

[0369] (II) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the second fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 18 of human papillomavirus;

[0370] (III) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in (c), the genotype is determined by:

[0371] (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence;

[0372] (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for a probe sequence attached to each specific region identified in (IIIa).

[0373] Item 14. The system according to Item 13, wherein the multiplex real-time PCR amplification:

[0374] (i) at least one first sequence, wherein each first sequence is a sequence from a marker of a human papillomavirus genotype that is present in a conserved region of the HPV L1 or HPV L2 gene; and

[0375] (ii) the second sequence.

[0376] Item 15. A system according to any one of Items 13 and 14, wherein the multiplex real-time PCR amplification:

[0377] (i) at least one first sequence, wherein each first sequence is a sequence from a marker for a human papillomavirus genotype selected from the group consisting of genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, and 84 of human papillomavirus; and

[0378] (ii) the second sequence.

[0379] Item 16. The system according to Item 15, wherein the multiplex real-time PCR amplification:

[0380] (i) a first sequence, which is a sequence of a marker of genotype 16 of human papillomavirus;

[0381] - a first sequence which is a sequence of a marker of genotype 18 of human papillomavirus, and,

[0382] - at least one other first sequence, wherein each first sequence is a sequence from a marker for a human papillomavirus genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus; and

[0383] (ii) the second sequence.

[0384] Item 17. A system according to any one of Items 13 to 16, wherein the DNA is extracted from the biological sample by lysis before the biological sample is subjected to multiplex real-time PCR, and:

[0385] - when multiplex real-time PCR is performed on said at least one sequence comprised in the DNA present in the biological sample, as defined in (a), a first control is also subjected to multiplex real-time PCR, wherein said first control is a blank in which said extraction is performed, and optionally

[0386] - when the at least one sequence contained in the DNA present in the biological sample is subjected to multiplex real-time PCR, as defined in (a), the second control is also subjected to multiplex real-time PCR, wherein the second control comprises DNA containing a first sequence, the first sequence being a sequence of a marker from a human papillomavirus genotype selected from the group consisting of 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus; and optionally

[0387] - the first sequence and the second sequence amplified in (a) and the solid support further comprise molecules attached thereto, wherein the molecules attached to the solid support are attached to specific regions of the solid support that are different from other specific regions of the solid support to which probes are attached, wherein the molecules attached to the first sequence and the second sequence amplified in (a) and the solid support have the same chemical structure.

[0388] Item 18. A system according to any one of Items 13 to 17, wherein when no probe binding to the second sequence is detected and no probe binding to the first sequence is detected, step (a) is repeated on the subject using a new sample obtained from the subject.

[0389] Item 19. A system according to any one of Items 13 to 18, wherein the system is additionally used to detect the absence of the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in the subject,

[0390] When the fourth fluorophore of the probe bound to the second sequence is detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is not detected.

[0391] Item 20. A system according to any one of Items 13 to 19, wherein the specific area of ​​the solid support different from other specific areas of the solid support to which probes are attached comprises a library of probes attached thereto, wherein each probe in the library comprises a sequence complementary to and specific for a sequence of a conserved region of an HPV L1 or HPV L2 gene, wherein at least two probes in the library comprise sequences different from each other, wherein when:

[0392] - upon identifying a specific area of ​​the solid support to which the probe library is attached and which hybridizes to a sequence;

[0393] - upon identification of a specific area of ​​said solid support to which the probe is attached and which is hybridized to the second sequence; and

[0394] - when no specific area of ​​the solid support to which the probe is attached and which hybridizes to the first sequence is identified,

[0395] The genotype of human papillomavirus is determined to be different from each genotype identifiable by a marker for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in (a).

[0396] Item 21. A system according to any one of Items 13 to 20, wherein the system is additionally used to determine whether the subject is at risk of developing cervical cancer or anogenital cancer, wherein when any one of the 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of the human papillomavirus is determined, the subject is determined to be at risk of developing cervical cancer or anogenital cancer.

[0397] Item 22. A system according to any one of Items 13 to 20, wherein the system is further used to determine whether the subject is at risk of developing a benign lesion, wherein when any one of 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82 and 84 genotypes of human papillomavirus is determined, the subject is determined to be at risk of developing a benign lesion.

[0398] Item 23. The system according to any one of Items 13 to 22, wherein the composition selected in (a) is heated to 60° C. for at least 10 minutes prior to cooling and hybridizing according to (b).

[0399] Item 24. A system according to any one of Items 13 to 23, wherein the biological sample obtained from the subject is obtained by obtaining the sample from the subject himself.

[0400] Item 25. A system according to any one of Items 13 to 24, wherein:

[0401] - a device for subjecting the DNA present in the biological sample to multiplex real-time PCR comprising a multiplex real-time PCR instrument and multiplex real-time PCR reagents;

[0402] - a means for hybridizing each of the first and second sequences amplified in the composition selected in (a) on a solid support, comprising a solid support and a hybridization reagent, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of the marker, and a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided for the marker in the multiplex real-time PCR in (a);

[0403] - The means for identifying each specific area of ​​said solid support to which a probe is attached and to which a sequence is hybridized comprises an image capture device.

[0404] Item 26. A kit for detecting a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer in a subject and for determining the one or more genotypes of the human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer is detected in the subject, comprising:

[0405] (a) a device for performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the multiplex real-time PCR amplifies:

[0406] (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and

[0407] (ii) a second sequence comprised in said DNA, wherein said second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker,

[0408] The composition comprises:

[0409] (iii) a set of probes comprising:

[0410] - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16;

[0411] - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and

[0412] - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and

[0413] (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene,

[0414] wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe,

[0415] Among them, when:

[0416] - when the fourth fluorophore of the probe bound to the second sequence is not detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, repeating (a) for the subject;

[0417] - when the fourth fluorophore of the probe bound to the second sequence is detected and at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected;

[0418] - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected;

[0419] (b) means for hybridizing each amplified first sequence and second sequence of the composition selected in (a) on a solid support, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in (a),

[0420] wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence; and

[0421] (c) means for identifying each specific area of ​​the solid support to which a probe is attached and which hybridizes to the first sequence or the second sequence;

[0422] Among them, when:

[0423] (I) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the first fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 16 of human papillomavirus;

[0424] (II) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the second fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 18 of human papillomavirus;

[0425] (III) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in (c), the genotype is determined by:

[0426] (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence;

[0427] (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for a probe sequence attached to each specific region identified in (IIIa).

[0428] Item 27. The kit according to Item 26, wherein the multiplex real-time PCR amplification:

[0429] (i) at least one first sequence, wherein each first sequence is a sequence from a marker of a human papillomavirus genotype that is present in a conserved region of the HPV L1 or HPV L2 gene; and

[0430] (ii) the second sequence.

[0431] Item 28. A kit according to any one of Items 26 and 27, wherein the multiplex real-time PCR amplification:

[0432] (i) at least one first sequence, wherein each first sequence is a sequence from a marker for a human papillomavirus genotype selected from the group consisting of genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, and 84 of human papillomavirus; and

[0433] (ii) the second sequence.

[0434] Item 29. The kit according to Item 28, wherein the multiplex real-time PCR amplification:

[0435] (i) a first sequence, which is a sequence of a marker of genotype 16 of human papillomavirus;

[0436] - a first sequence which is a sequence of a marker of genotype 18 of human papillomavirus, and,

[0437] - at least one other first sequence, wherein each first sequence is a sequence from a marker of a human papillomavirus genotype selected from the group consisting of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus; and

[0438] (ii) the second sequence.

[0439] Item 30. The kit according to any one of Items 26 to 29, wherein the DNA is extracted from the biological sample by lysis before the biological sample is subjected to multiplex real-time PCR, and:

[0440] - when multiplex real-time PCR is performed on said at least one sequence comprised in the DNA present in the biological sample, as defined in (a), a first control is also subjected to multiplex real-time PCR, wherein said first control is a blank in which said extraction is performed, and optionally

[0441] - when the at least one sequence contained in the DNA present in the biological sample is subjected to multiplex real-time PCR, as defined in (a), the second control is also subjected to multiplex real-time PCR, wherein the second control comprises DNA containing a first sequence, the first sequence being a sequence of a marker from a human papillomavirus genotype selected from the group consisting of 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus; and optionally

[0442] - the first sequence and the second sequence amplified in (a) and the solid support further comprise molecules attached thereto, wherein the molecules attached to the solid support are attached to specific regions of the solid support that are different from other specific regions of the solid support to which probes are attached, wherein the molecules attached to the first sequence and the second sequence amplified in (a) and the solid support have the same chemical structure.

[0443] Item 31. A kit according to any one of Items 26 to 30, wherein when no binding to the probe of the second sequence is detected and no binding to the probe of the first sequence is detected, step (a) is repeated on the subject using a new sample obtained from the subject.

[0444] Item 32. A kit according to any one of Items 26 to 31, wherein the kit is additionally used to detect the absence of the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer in the subject,

[0445] When the fourth fluorophore of the probe bound to the second sequence is detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is not detected.

[0446] Item 33. A kit according to any one of Items 26 to 32, wherein the specific area of ​​the solid support different from other specific areas of the solid support to which probes are attached comprises a probe library attached thereto, wherein each probe in the probe library comprises a sequence complementary to and specific for a sequence of a conserved region of an HPV L1 or HPV L2 gene, wherein at least two probes in the probe library comprise sequences different from each other, wherein when:

[0447] - upon identifying a specific area of ​​the solid support to which the probe library is attached and which hybridizes to a sequence;

[0448] - upon identification of a specific area of ​​said solid support to which the probe is attached and which is hybridized to the second sequence; and

[0449] - when no specific area of ​​the solid support to which the probe is attached and which hybridizes to the first sequence is identified,

[0450] The genotype of human papillomavirus is determined to be different from each genotype identifiable by a marker for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in (a).

[0451] Item 34. A kit according to any one of Items 26 to 33, wherein the kit is additionally used to determine whether the subject is at risk of developing cervical cancer or anogenital cancer, wherein when any one of the 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus is determined, the subject is determined to be at risk of developing cervical cancer or anogenital cancer.

[0452] Item 35. A kit according to any one of Items 26 to 33, wherein the kit is additionally used to determine whether the subject is at risk of developing a benign lesion, wherein when any one of 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82 and 84 genotypes of human papillomavirus is determined, the subject is determined to be at risk of developing a benign lesion.

[0453] Item 36. The kit according to any one of Items 26 to 35, wherein the composition selected in (a) is heated to 60° C. for at least 10 minutes prior to cooling and hybridization according to (b).

[0454] Item 37. The kit according to any one of Items 26 to 36, wherein the biological sample obtained from the subject is obtained by obtaining the sample from the subject himself.

[0455] Item 38. A kit according to any one of Items 26 to 37, wherein:

[0456] - a device for subjecting the DNA present in the biological sample to multiplex real-time PCR comprising a multiplex PCR instrument and multiplex PCR reagents;

[0457] - a means for hybridizing each of the first and second sequences amplified in the composition selected in (a) on a solid support, comprising a solid support and a hybridization reagent, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for the sequence of the marker, and a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided for the marker in the multiplex PCR in (a);

[0458] - The means for identifying each specific area of ​​said solid support to which a probe is attached and to which a sequence is hybridized comprises an image capture device.

[0459] Item 39. The method of any one of items 1-18, the system of any one of items 13-25, or the kit of any one of items 26-38, wherein the forward primer and / or the reverse primer comprises a molecular tag.

[0460] Item 40. Forward primer and / or reverse primer

[0461] Use for obtaining amplified sequences comprising molecular tags by multiplex real-time PCR, wherein the multiplex real-time PCR amplifies sequences contained in DNA present in a biological sample obtained from a subject, wherein the DNA is extracted from the biological sample before subjecting the biological sample to the multiplex real-time PCR, and

[0462] The forward primer and / or reverse primer comprises

[0463] - sequences complementary to and specific for one or more loci of markers of genotype 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68 of human papillomavirus; and

[0464] - molecular tags; and

[0465] Use for identifying a specific area on a solid support attached to a probe, wherein the probe comprises a sequence complementary to and specific for a sequence of a marker, the forward primer and / or reverse primer are complementary to and specific for the sequence of the marker; and the amplified sequence comprising a molecular tag hybridizes with the sequence of the marker.

[0466] Item 41. Forward primer and / or reverse primer

[0467] Use for obtaining amplified sequences comprising molecular tags by multiplex real-time PCR, as defined in step (a) of the method of any one of items 1 to 18 or 39, the system of any one of items 13 to 25 or 39, or the kit of any one of items 26 to 39; and

[0468] Use for identifying a specific area on a solid support to which a probe is attached, wherein the probe comprises a sequence complementary to and specific for the sequence of a marker, the forward primer and / or reverse primer are complementary to and specific for the sequence of the marker; and the amplified sequence comprising a molecular tag hybridizes with the sequence of the marker, as defined in step (b) and identified as defined in step (c) of the method described in any one of items 1-18 or 39, the system described in any one of items 13-25 or 39, or the kit described in any one of items 26-39.

[0469] Item 42. Use of an amplified sequence comprising a molecular tag, wherein the amplified sequence has been obtained in a multiplex real-time PCR, wherein the multiplex real-time PCR amplifies a sequence contained in DNA present in a biological sample obtained from a subject, wherein the DNA is extracted from the biological sample before subjecting the biological sample to the multiplex real-time PCR, and wherein amplification is performed using a forward primer and / or a reverse primer, wherein the forward primer and / or the reverse primer comprises

[0470] - sequences complementary to and specific for one or more loci of markers of genotype 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68 of human papillomavirus; and

[0471] - molecular tags; and

[0472] Used to identify a specific area on a solid support attached to a probe, wherein the probe comprises a sequence complementary to and specific for a sequence of a marker, the forward primer and / or reverse primer are complementary to and specific for the sequence of the marker; and the amplified sequence comprising a molecular tag hybridizes with the sequence of the marker.

[0473] Item 43. Use of an amplified first sequence and / or second sequence, wherein the amplified first sequence and / or second sequence has been obtained in step (a) of the method described in any one of items 1-18 or 39, in the system described in any one of items 13-25 or 39, or in the kit described in any one of items 26-39,

[0474] For identifying a specific area on a solid support to which a probe is attached, as defined in step (c) of the method described in any one of items 1-18 or 39, in the system described in any one of items 13-25 or 39, or in the kit described in any one of items 26-39, wherein the amplified first and / or second sequence comprises a molecular tag and is hybridized on the solid support, as defined in step (b) of the method described in any one of items 1-18 or 39, in the system described in any one of items 13-25 or 39, or in the kit described in any one of items 26-39.

[0475] Item 44. The method of any one of items 1-18 or 39, the system of any one of items 13-25 or 39, or the kit of any one of items 26-39, or the use of any one of items 40-43, wherein the molecular tag is biotin.

[0476] Item 45. The method of any one of items 1-18, 39 or 44, the system of any one of items 13-25, 39 or 44, the kit of any one of items 26-39 or 44, or the use of any one of items 40-44, wherein the molecular tag biotin is identified by streptavidin comprising a reporter.

[0477] Item 46. The method described in any one of items 1-18, 39, 44 or 45, the system described in any one of items 13-25, 39, 44 or 45, the kit described in any one of items 26-39, 44 or 45, or the use described in any one of items 40-45, wherein the reporter is horseradish peroxidase or alkaline phosphatase.

[0478] Item 47. The method described in any one of items 1-18, 39, 44, 45, 46; the system described in any one of items 13-25, 39, 44, 45, 46; the kit described in any one of items 26-39, 44, 45, 46; or the use described in any one of items 40-46, wherein the sample is obtained via a sample collection device.

[0479] Item 48. The method described in any one of items 1-18, 39, 44, 45, 46, 47; the system described in any one of items 13-25, 39, 44, 45, 46, 47; the kit described in any one of items 26-39, 44, 45, 46, 47; or the use described in any one of items 40-47, wherein the sample collection device is a swab, a brush or a covering device.

[0480] Item 49. The system of any one of Items 13-25, 39, 44, 45, 46, 47 or 48; or the kit of any one of Items 26-39, 44, 45, 46, 47 or 48, wherein the system and / or kit further comprises a step (aa) device for extracting DNA of human papillomavirus present in a biological sample obtained from a subject.

[0481] Item 50. The system of any one of Items 13-25, 39, 44, 45, 46, 47, 48 or 49; or the kit of any one of Items 26-39, 44, 45, 46, 47, 48 or 49, wherein step (aa) precedes step (a).

[0482] Example

[0483] The following examples illustrate the invention and should not be considered limiting, but rather as illustrating the invention.

[0484] The following examples are based on the amplification of a fragment in the viral region L1 of papillomavirus by PCR followed by hybridization to a membrane with a DNA-specific probe by using a technique suitable for analysis in both automated and manual hybriSpot platforms.

[0485] Example 1: Multiplex real-time PCR of samples obtained from subjects infected with human papillomavirus.

[0486] Example 1 is a specific embodiment of (a) of the method, use, system and kit of the present invention.

[0487] Reagents:

[0488] Samples were obtained from 24 subjects infected with human papillomavirus (HPV) by using swabs of the anogenital area (self-swab).

[0489] Use DNase / RNase-free double distilled water to treat clinical samples:

[0490] A lyophilized multiplex real-time PCR mixture was prepared for analysis of 24 clinical samples on the hybriSpot 24 platform. The lyophilized multiplex real-time PCR mixture contained PCR buffer, dNTPs (U / T), DNase / RNase-free water, biotinylated primers, DNA polymerase and uracil-DNA glycosylase (Cod-UNG), and fluorescent probes. The biotinylated primers included a pair of primers for detecting at least 35 HPV genotypes, wherein each primer was specific for amplification of a fragment of the HPV L1 region. In addition, the remaining biotinylated primers included a pair of primers for amplifying a human housekeeping gene (β-globin gene) and were used as internal controls for the multiplex real-time PCR. The fluorescent probes used were ROX (for HPV 16 detection), Cy5 (for HPV 18 detection), FAM (for detection of HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68=HPV HR genotype), and JOE (used as an internal control).

[0491] Sequences of markers of human papillomavirus genotypes selected from genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus were used as positive controls (PCs), and DNase / RNase-free DEPC-treated water was used as negative control (NTC).

[0492] The reagents were transported and stored in the dark at 2-8°C in an area not contaminated by DNA or PCR products.

[0493] method:

[0494] Purified genetic material from different types of biological samples have been used, such as liquid-based cytology and vaginal and rectal swabs. DNA was extracted using the DNA / RNA Purification Kit and Extraction Device*, starting with 200 μl of biological sample and eluting in 100 μl of elution buffer (for purification with Opentrons, it started with 92 μl of clinical sample and eluted in 60 μl of elution solution):

[0495]

[0496] Alternatively, DNA was extracted using the RNA / DNA Pathogen Extraction Kit (for Robot Nextractor® NX-48S) Ref. MAD-003955M-EX.

[0497] 8 μL of eluted DNA was added to 12 μL of PCR master mix. Thus, the PCR reaction was performed in each tube containing the multiplex real-time PCR reaction mixture in a final volume of 20 μL to obtain template DNA for the PCR reaction. A negative control was made by adding 8 μL of water to 12 μL of PCR master mix. Additionally, a positive control was made by adding 8 μL of the positive DNA control HPV Screening to 12 μL of PCR master mix.

[0498] Place each tube in a thermal cycler and use the following PCR amplification steps indicated in Table 6:

[0499] Table 6: PCR amplification steps used

[0500]

[0501] *Fluorescence data should be collected during the extension phase (*) for ROX (HPV 16), Cy5 (HPV 18), FAM (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), and HEX, JOE, or VIC (internal control) channels.

[0502] Table 7 below is used to explain the fluorescence data obtained for the positive and negative controls (PC and NTC, respectively), whereby the detection of the signal (amplification signal) is determined by a rapid and steady increase in the fluorescence data values ​​rather than by peak phenomena or a gradual increase in the background signal (irregular background or increased background noise). Figure 2A and 2B ).

[0503] Table 7: Interpretation of multiplex real-time PCR fluorescence data obtained for positive (PC) and negative (NTC) controls

[0504]

[0505] *Amplification signal must be determined by a rapid and steady increase in fluorescence value, not by a peak phenomenon or a gradual increase in background signal (irregular background or increased background noise).

[0506] The PCR run was considered valid when the detection count (Ct) values ​​obtained for the above standards for all reaction controls and in the positive controls for the different targets were within the expected range of 20 ± 2 for HPV 16 (ROX), 20 ± 2 for HPV 18 (Cy5), 20 ± 2 for HPV HR (FAM), and 20 ± 2 for β-globin (JOE).

[0507] The results obtained for each clinical sample are interpreted according to Table 8 below:

[0508] Table 8: Interpretation of multiplex real-time PCR fluorescence data obtained for each clinical sample

[0509]

[0510] *Negative or below the limit of detection.

[0511] ‡Problems with extraction and / or amplification

[0512] The default threshold line was automatically established by the VitroCycler (Vitro SA) instrument or adjusted manually if necessary until it was within the exponential phase of the fluorescence curve and above any background signal.

[0513] A sample was considered positive if a Ct value of ≤40 was obtained, even if the internal control showed no amplification (sometimes the internal control was not amplified correctly due to the presence of a high initial copy number of the target bacterial nucleic acid, which could result in preferential amplification of the latter).

[0514] If no amplification above the threshold is detected, and if the internal control does show amplification, the sample is considered negative. Inhibition of the PCR reaction can be ruled out by amplification of the internal control.

[0515] It has been demonstrated that the method of the present invention has a detection limit of 10 copies / reaction for HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 59, HPV 66 and HPV 68 genotypes.

[0516] Example 2: Hybridization of amplicons from Example 1.

[0517] Example 2 is a specific embodiment of (c) of the method, use, system and kit of the present invention.

[0518] Reagents:

[0519] The biotinylated amplicons generated during PCR from Example 1 were used for hybridization.

[0520] Prepare the following reagents for analysis of 24 clinical samples obtained from subjects infected with human papillomavirus (HPV) on the hybriSpot 24 platform. The reagents include hybridization solution (reagent A), blocking solution (reagent B), streptavidin-phosphatase (streptavidin-alkaline phosphatase, reagent C), wash buffer I (reagent D), substrate + chromogen (reagent E), and wash buffer II (reagent F). Immediately before use, preheat reagent A to 41°C in a thermostatic bath or heater, while use the remaining reagents B to F at room temperature (20-25°C).

[0521] The solid support used is a hybriSpot chip (membrane) containing probes attached to their specific areas. Each chip includes four subsets of specific areas, where:

[0522] - Each specific region in a subset of the specific regions comprises a probe, each probe having a sequence selected from SEQ ID NO: 20 to SEQ ID NO: 54, the sequence being complementary to and specific for a sequence of a marker of human papillomavirus, wherein each probe comprising the same sequence is attached to a specific region of the solid support;

[0523] - Each specific region in a subset of the specific regions comprises a probe having a sequence of SEQ ID NO: 55, which is complementary to and specific for a sequence of a marker of the human HBB gene (endogenous amplification control, C), wherein each probe comprising the same sequence is attached to a specific region of the solid support.

[0524] - Biotin protein used as hybridization control.

[0525] - Each specific region in a subset of said specific regions comprises a degenerate universal probe having a sequence selected from SEQ ID NO:56.

[0526] The reagents were transported and stored at 2-8°C in an area not contaminated by DNA or PCR products.

[0527] method:

[0528] The biotinylated amplicons generated during the PCR in Example 1 were hybridized to an array containing specific probes for each HPV genotype associated with the development of cervical cancer, oral cancer, and / or anogenital cancer, as well as a membrane containing amplification and hybridization control probes. The hybridization process was performed semi-automatically on the hybriSpot (HS12) platform following instructions provided by the wizard of the platform. Specifically, reagents were added manually, and the incubation time, temperature, and discharge of the reagents were controlled by firmware. However, the hybridization was also fully automated in parallel on the hybriSpot HS12a or HS24 platform, achieving the same results (not shown here).

[0529] Compared to hybridization on conventional surfaces, this technique allows for very rapid binding of PCR products and their specific probes in the three-dimensional porous environment of the membrane. Once binding between specific amplicons and their corresponding probes occurs, the signal is visualized by an immunoenzymatic colorimetric reaction with streptavidin-phosphatase and chromogen (NBT-BCIP), producing an insoluble precipitate in the membrane in those specific areas where hybridization occurred.

[0530] Management of samples, capture of images, and analysis and reporting of results are automatically performed by hybriSoft™ software.

[0531] Before starting the hybridization process, perform the following steps:

[0532] (i) denaturing the biotinylated amplicon produced during PCR in Example 1 by heating at 95° C. for 10 minutes in a thermal cycler and rapidly cooling in ice for at least 2 minutes;

[0533] (ii) preheating reagent A to 41°C; and

[0534] (iii) Place each HPV chip (one chip for each valid clinical sample that tests positive for HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68 in Example 1) at the indicated position in the platform (HS12).

[0535] It is worth noting that the PCR products added to the array contain the remnants of the Taqman probes from the real-time PCR that were not consumed during amplification, so there will be two complementary probes in the hybridization device, one in the liquid phase and the other in the solid phase, and both can compete for hybridization with the target amplicon. To prevent this from happening and ensure that the generated amplicon is fully hybridized with the solid phase probe (i.e. the probe in the array), we use a temperature switch for the first hybridization step. The temperature is first set to 60°C. In this way, the Taqman probe remnants are separated from the liquid phase amplicons and their hybridization is avoided, thereby increasing the sensitivity of the technique. This is because the temperature is above the melting temperature of all probes (including the array's probes and the real-time probes). If the initial temperature increase does not occur, some Taqman probe fragments can hybridize with the target amplicon and prevent it from binding to the chip probe.

[0536] Once all samples were added to the array, they were incubated at 60° C. for 1 minute, and then the temperature was lowered to 41° C. Specifically, the samples were incubated at 41° C. for another 6 minutes.

[0537] By setting the temperature at 41°C, PCR products (amplicons, amplified DNA) bind to the array probes according to the melting temperatures of these probes.

[0538] After incubation at 41°C for 6 min, the hybridization protocol was continued as described below.

[0539] The manual hybridization protocol consists of the following steps:

[0540] (a) Set the temperature of the device to 60 °C and add 300 μL of reagent A (hybridization solution) preheated at 41 °C to each chip and incubate until the temperature reaches 60 °C;

[0541] (b) Once the temperature reaches 60°C, remove reagent A (hybridization solution) by activating the vacuum pump.

[0542] (c) Mix 20 μL of each PCR sample (previously denatured and kept on ice) with 270 μL of reagent A (hybridization solution at 41 °C) and dispense the mixture onto the corresponding HVP chip;

[0543] (d) Incubate at 60°C for 1 min;

[0544] (e) setting the temperature of the apparatus to 41°C and once reached incubating for 6 minutes;

[0545] (f) activating the pump for at least 30 seconds to remove the liquid containing the PCR products (including amplicons, amplified DNA obtained by multiplex real-time PCR);

[0546] (g) Wash three times with 300 μL of reagent A (hybridization solution at 41°C);

[0547] (h) setting the temperature to 29°C;

[0548] (i) Add 300 μL of Reagent B (Blocking Solution) and incubate for 5 min;

[0549] (j) starting the pump to remove reagent B;

[0550] (k) Once the temperature reaches 29°C, add 300 μL of reagent C (streptavidin-alkaline phosphatase) to each chip.

[0551] (l) Incubate at 29°C for 5 min;

[0552] (m) activating the pump to remove the reagent;

[0553] (n) Setting the temperature to 36°C;

[0554] (o) Wash the membrane four times with 300 μL of reagent D (wash buffer I);

[0555] (p) Once the temperature reached 36°C, 300 μL of Reagent E was added to each chip and incubated at 36°C for 10 min.

[0556] (q) starting the pump to remove the reagent E containing the substrate and the chromogen to perform the enzymatic colorimetric reaction;

[0557] (r) Wash the membrane twice with 300 μL of Reagent F (Wash Buffer II).

[0558] (s) starting the pump to remove the reagent F (buffer having a very low salt concentration) to stop the enzymatic colorimetric reaction; and

[0559] (t) Follow the instructions in the HS12 User Manual to perform image capture, analysis and reporting of results.

[0560] Example 3: Determining the 56 genotype of the human papillomavirus infecting the subject.

[0561] Example 3 is a specific embodiment of (c) of the methods, uses, systems and kits of the present invention. One of the 24 samples obtained from the subject was tested positive for HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and / or 68 after multiplex real-time PCR according to Example 1, as shown in the third row of Table 8. Hybridization was then performed on a solid support according to Example 2, resulting in hybridization being visualized as occurring in a specific area, which is represented by:

[0562] (i) Figure 3B specific areas 1, A; 1, B; 2, I; 5, E and 8, A (specific areas are identified by numbers and letters, respectively, counting from the upper left specific area 1A of the solid support), according to Figure 3C Schematic diagram of the region corresponding to those specific regions to which the hybridization control probe (B) is attached;

[0563] (ii) Figure 3B The specific areas 1, C and 5, F, according to Figure 3C , which corresponds to those specific regions to which the endogenous amplification control probe (C) is attached; and

[0564] (iii) Figure 3B The specific areas 1, D and 5, G, according to Figure 3C Schematic diagram of a PCR product that corresponds to those specific regions to which the HPV degenerate universal probe (U) is attached, indicating that hybridization is effective. In addition, hybridization is visualized as occurring in specific regions, which are represented by the following:

[0565] (iv) Figure 3B The specific areas 2, H and 7, C, according to Figure 3C Schematic diagram of the specific regions to which probes for the HPV 56 genotype are attached.

[0566] A degenerate HPV universal probe (U) is attached.

[0567] Thus, the subject from which the sample was obtained is determined to be infected with an HPV 56 genotype associated with the development of cervical cancer, oral cancer, or anogenital cancer in the subject.

[0568] Example 4: Comparison of different hybridization conditions.

[0569] The same set of samples (liquid cytology samples with previous positive results for HR HPV genotypes by real-time PCR) were assayed using different assay conditions. Results are shown in Figure 5 middle.

[0570] Twelve clinical samples that had previous positive results for HPV by real-time PCR screening without hybridization were used. Previous real-time PCR results without hybridization are shown in Figure 5 middle.

[0571] Genetic material was extracted from the samples using the RNA / DNA Pathogen Extraction Assay (VITRO SA, Granada, Spain) and a total volume of 60 μl was obtained. Using these samples, three hybridization conditions were tested:

[0572] Hybridization condition 1: Real-time PCR followed by hybridization of PCR products at 41°C. 8 μl of each sample was mixed with 12 μl of the master mix necessary for multiplex real-time PCR with in vitro HPV screening assay. The products of this real-time PCR were denatured by heating at 95°C for 10 minutes and hybridized to a membrane with HPV-specific probes using DNAFlow technology on a hybriSpot platform at 41°C.

[0573] Hybridization condition 2: Amplification of samples by end-point PCR without fluorescent probes, followed by hybridization of PCR products at a temperature of 41°C. This is considered the gold standard method. 30 μl of each sample was subjected to PCR amplification with a mixture (lyophilized PCR mix) without any fluorescent probes. Under these conditions, interference with the probes on the solid surface should not occur. The products of this real-time PCR were denatured by heating at 95°C for 10 minutes and hybridized to a membrane with HPV-specific probes using DNAFlow technology on the hybriSpot platform at a temperature of 41°C.

[0574] Hybridization condition 3: According to the method of the present invention, real-time PCR, followed by hybridization of the PCR products at a temperature of 60°C, and then cooling to 41°C. 8 μl of each sample was mixed with 12 μl of a master mix necessary for multiplex real-time PCR with an in vitro HPV screening assay. The products of this real-time PCR were denatured by heating at 95°C for 10 minutes and hybridized to a membrane with HPV-specific probes using DNAFlow technology on the hybriSpot platform, with a temperature gradient starting at 60°C and ending at 41°C. The product was kept at 60°C for 1 minute, then the temperature began to gradually decrease to 41°C over 4 minutes, and finally the product was kept at 41°C for 6 minutes.

[0575] For all conditions, the entire hybridization process is performed automatically and the TM The software performs the management of samples, the capture of images, and the analysis and reporting of results. The same batch of reagents is used for all conditions.

[0576] When real-time PCR was followed by hybridization of the PCR products at a temperature of 41°C (not according to the invention), some genotypes were not identified (see Figure 5Hybridization condition 1): genotype 18 was not identified in sample M1, genotype 16 was not identified in sample M2, genotypes 18, 53 and 67 were not identified in sample M4, genotypes 16 and 58 were not identified in sample M5, genotype 16 was not identified in sample M6, genotype 16 was not identified in sample M7, genotype 16 was not identified in sample M8, genotype 18 was not identified in sample M9, and genotype 18 was not identified in sample M11. When the PCR products are hybridized at a temperature of 60°C after real-time PCR according to the method of the present invention and then cooled to 41°C (see Figure 5 3) and the gold standard method (see Figure 5 All of these genotypes were correctly identified under the hybridization conditions in 2).

[0577] The PCR products added to the array contain remaining probes from the real-time PCR that were not consumed during amplification, so there will be two complementary probes in the hybridization device, one in the liquid phase and the other in the solid phase, and both can compete for hybridization with the target amplicon. This is prevented by hybridizing the PCR products at a temperature of 60°C, which promotes the generated amplicons to hybridize preferentially with the solid phase probes (i.e., the probes in the array) rather than with the probes remaining from the PCR in the liquid phase, thereby improving the sensitivity and specificity of the method. This is because the temperature is above the melting temperature of all probes (including those of the array and those of the real-time PCR). If the heating temperature is lower than 60°C, some of the probes remaining from the PCR can hybridize with the target amplicon and prevent it from binding to the chip probes.

[0578] This example shows that the genotypes present in the sample are properly detected and identified when, according to the method of the present invention, real-time PCR is followed by hybridization of the PCR products at a temperature of 60° C. and then cooled to 41° C. When real-time PCR is followed by hybridization of the real-time PCR products at a temperature of 41° C., some genotypes are not identified.

Claims

1. A method for detecting a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer in a subject and for determining the one or more genotypes of the human papillomavirus infecting the subject when the human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected in the subject, comprising: (a) performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the DNA is extracted from the biological sample prior to performing the multiplex real-time PCR on the biological sample, wherein the multiplex real-time PCR amplifies: (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and (ii) a second sequence comprised in said DNA, wherein said second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker, The composition comprises: (iii) a set of probes comprising: - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16; - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene, wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe, Among them, when: - when the fourth fluorophore of the probe bound to the second sequence is not detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, repeating step (a) for the subject; - when the fourth fluorophore of the probe bound to the second sequence is detected and at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected; - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected; (b) subjecting each amplified first sequence and second sequence of the composition selected in step (a) to hybridization on a solid support, the solid support comprising probes attached thereto, wherein the composition selected in step (a) is heated to at least 60° C. during hybridization and subsequently, cooled, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker for which a forward primer and a reverse primer complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR in step (a), wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence; and (c) identifying each specific area of ​​the solid support to which a probe is attached and which hybridizes to the first sequence or the second sequence; Among them, when: (I) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the first fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 16 of human papillomavirus; (II) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the second fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 18 of human papillomavirus; (III) when the fourth fluorophore of the probe bound to the second sequence is detected in step (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in step (c), the genotype is determined by: (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence; (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for the probe sequence attached to each specific region identified in step (IIIa).

2. A system for detecting a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer in a subject and for determining the one or more genotypes of a human papillomavirus infecting the subject when the human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected in the subject, comprising: (a) a device for performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the multiplex real-time PCR amplifies: (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and (ii) a second sequence comprised in said DNA, wherein said second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker, The composition comprises: (iii) a set of probes comprising: - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16; - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene, wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe, Among them, when: - when the fourth fluorophore of the probe bound to the second sequence is not detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, repeating (a) for the subject; - when the fourth fluorophore of the probe bound to the second sequence is detected and at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected; - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected, and wherein the apparatus for performing multiplex real-time PCR on at least one sequence contained in the DNA present in the biological sample comprises a multiplex real-time PCR instrument and multiplex real-time PCR reagents; (b) means for hybridizing each amplified first sequence and second sequence of the composition selected in (a) on a solid support, the solid support comprising a probe attached thereto, means for heating the composition selected in (a) to at least 60° C. during hybridization, and means for cooling the composition selected in (a) during hybridization, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, and forward primers and reverse primers complementary to and specific for the locus of the marker are provided for the marker in the multiplex real-time PCR in (a), wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence, wherein the means for heating the composition selected in (a) comprises a heating device, wherein the means for cooling the composition selected in (a) comprises a cooling device, wherein the means for hybridizing each first sequence and second sequence amplified in the composition selected in (a) on a solid support comprises a solid support and a hybridization reagent, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of the marker, and a forward primer and a reverse primer complementary to and specific for a locus of the marker are provided for the marker in the multiplex real-time PCR in (a); and (c) means for identifying each specific area of ​​the solid support to which a probe is attached and which is hybridized to the first sequence or the second sequence, wherein the means comprises an image capture device; Among them, when: (I) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the first fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 16 of human papillomavirus; (II) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the second fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 18 of human papillomavirus; (III) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in (c), the genotype is determined by: (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence; (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for a probe sequence attached to each specific region identified in (IIIa).

3. A kit for detecting a human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer in a subject and for determining the one or more genotypes of the human papillomavirus infecting the subject when the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anal genital cancer is detected in the subject, comprising: (a) a device for performing multiplex real-time PCR on at least one sequence contained in DNA present in a biological sample obtained from the subject, wherein the multiplex real-time PCR amplifies: (i) at least one first sequence contained in said DNA, wherein each first sequence is a sequence of a marker of a human papillomavirus genotype, wherein amplification of each sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker; and (ii) second sequences comprised in said DNA, wherein each second sequence is a sequence of a marker of a human housekeeping gene, wherein amplification of said second sequence is performed using a forward primer and a reverse primer, each primer being complementary to and specific for the locus of said marker, The composition comprises: (iii) a set of probes comprising: - a probe comprising a first fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus genotype 16; - a probe comprising a second fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of the human papillomavirus 18 genotype; and - a library of probes, wherein each probe of the library of probes comprises a third fluorophore and a sequence complementary to and specific for a sequence of a marker of a genotype selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus, wherein each of genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus has a marker comprising a sequence complementary to and specific for at least one sequence of a probe of the library of probes; and (iv) a probe comprising a fourth fluorophore and a sequence, wherein the sequence is complementary to and specific for a sequence of a marker of a human housekeeping gene, wherein each probe is detected that binds to a sequence of a marker that is complementary to and specific for each probe, Among them, when: - when the fourth fluorophore of the probe bound to the second sequence is not detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, repeating (a) for the subject; - when the fourth fluorophore of the probe bound to the second sequence is detected and at least one of the first fluorophore or the second fluorophore of the probe bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected; - when the fourth fluorophore of the probe bound to the second sequence is detected and the third fluorophore of the probe of the probe library bound to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer is detected, and a composition comprising the amplified first sequence and the second sequence is selected, and wherein the apparatus for performing multiplex real-time PCR on at least one sequence contained in the DNA present in the biological sample comprises a multiplex real-time PCR instrument and multiplex real-time PCR reagents; (b) means for hybridizing each amplified first sequence and second sequence of the composition selected in (a) on a solid support, the solid support comprising a probe attached thereto, means for heating the composition selected in (a) to at least 60° C. during hybridization, and means for cooling the composition selected in (a) during hybridization, wherein each probe comprises a sequence complementary to and specific for a sequence of a marker, and forward primers and reverse primers complementary to and specific for the locus of the marker are provided for the marker in the multiplex real-time PCR in (a), wherein each probe comprising the same sequence is attached to a specific region of the solid support, and each specific region of the solid support is attached to a probe comprising the same sequence, wherein the means for heating the composition selected in (a) comprises a heating device, wherein the means for cooling the composition selected in (a) comprises a cooling device, wherein the means for hybridizing each first sequence and second sequence amplified in the composition selected in (a) on a solid support comprises a solid support and a hybridization reagent, the solid support comprising probes attached thereto, wherein each probe comprises a sequence complementary to and specific for a sequence of the marker, and a forward primer and a reverse primer complementary to and specific for a locus of the marker are provided for the marker in the multiplex real-time PCR in (a); and (c) means for identifying each specific area of ​​the solid support to which a probe is attached and which is hybridized to the first sequence or the second sequence, wherein the means comprises an image capture device; Among them, when: (I) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the first fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 16 of human papillomavirus; (II) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the second fluorophore of the probe bound to the first sequence is detected, determining the detected human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer as genotype 18 of human papillomavirus; (III) when the fourth fluorophore of the probe bound to the second sequence is detected in (a) and the third fluorophore of the probe bound to the first sequence is detected, and at least one specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence is identified in (c), the genotype is determined by: (IIIa) identifying each specific area of ​​the solid support to which the probe is attached and hybridized to the first sequence; (IIIb) determining which genotype of the human papillomavirus is identified by a marker comprising a sequence complementary to and specific for a probe sequence attached to each specific region identified in (IIIa).

4. The method of claim 1, the system of claim 2, or the kit of claim 3, wherein the multiplex real-time PCR amplification: (i) at least one first sequence, wherein each first sequence is a sequence from a marker of a human papillomavirus genotype that is present in a conserved region of the HPV L1 or HPV L2 gene; and (ii) the second sequence.

5. The method of any one of claims 1 and 4, the system of any one of claims 2 and 4, or the kit of claims 3 or 4, wherein the multiplex real-time PCR amplification: (i) at least one first sequence, wherein each first sequence is a sequence from a marker for a human papillomavirus genotype selected from the group consisting of genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, and 84 of human papillomavirus; and (ii) the second sequence.

6. The method of any one of claims 1, 4 or 5, the system of any one of claims 2, 4 or 5, or the kit of any one of claims 3 to 5, wherein the method is additionally used to detect the absence of the human papillomavirus genotype associated with the development of cervical cancer, oral cancer or anogenital cancer in the subject, When the fourth fluorophore of the probe bound to the second sequence is detected and the first fluorophore, the second fluorophore or the third fluorophore of the probe bound to the first sequence is not detected, the human papillomavirus genotype associated with the occurrence of cervical cancer, oral cancer or anogenital cancer is not detected.

7. The method of any one of claims 1 or 4 to 6, the system of any one of claims 2 or 4 to 6, or the kit of any one of claims 3 to 6, wherein the method, system or kit is additionally used to determine whether the subject is at risk of developing cervical cancer, oral cancer or anogenital cancer, wherein when any one of the 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus is determined, the subject is determined to be at risk of developing cervical cancer, oral cancer or anogenital cancer.

8. The method of any one of claims 1 or 4 to 7, the system of any one of claims 2 or 4 to 7, or the kit of any one of claims 3 to 7, wherein the method, system or kit is additionally used to determine whether the subject is at risk of developing a benign lesion, wherein when any one of 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82 and 84 genotypes of human papillomavirus is determined, the subject is determined to be at risk of developing a benign lesion.

9. The method of any one of claims 1 or 4 to 8, the system of any one of claims 2 or 4 to 8, or the kit of any one of claims 3 to 8, wherein the heating is from 60 to 70°C.

10. The method of any one of claims 1 or 4 to 9, the system of any one of claims 2 or 4 to 9, or the kit of any one of claims 3 to 9, wherein the heating is from 60 to 65°C.

11. The method according to any one of claims 1 or 4 to 10, the system according to any one of claims 2 or 4 to 10, or the kit according to any one of claims 3 to 10, wherein the heating is for 1 to 20 minutes.

12. The method of any one of claims 1 or 4 to 11, the system of any one of claims 2 or 4 to 11, or the kit of any one of claims 3 to 11, wherein the heating is for 3 to 20 minutes.

13. The method of any one of claims 1 or 4 to 11, the system of any one of claims 2 or 4 to 11, or the kit of any one of claims 3 to 11, wherein the heating is for at least 10 minutes.

14. The method of any one of claims 1 or 4 to 13, the system of any one of claims 2 or 4 to 13, or the kit of any one of claims 3 to 13, wherein the cooling consists of cooling to 41°C.

15. The method according to any one of claims 1 or 4 to 14, the system according to any one of claims 2 or 4 to 14, or the kit according to any one of claims 3 to 14, wherein the forward primer and / or the reverse primer comprises a molecular tag.

16. The method, system or kit of claim 15, wherein the molecular tag is biotin.

17. The method of any one of claims 1 or 4 to 16, the system of any one of claims 2 or 4 to 16, or the kit of any one of claims 3 to 16, wherein the sample is obtained by a sample collection device.

18. The method, system or kit of claim 17, wherein the sample collection device is a swab, a brush or a covering device.

19. The method according to any one of claims 1 or 4 to 18; the system according to any one of claims 2 or 4 to 18, or the kit according to any one of claims 3 to 18, wherein Prior to hybridization according to (b), the composition selected in (a) was heated to 95°C for 10 minutes.

20. The system according to any one of claims 2 or 4 to 19 or the kit according to any one of claims 3 to 19, wherein the system and / or kit further comprises a (aa) device for extracting DNA of human papillomavirus present in a biological sample obtained from a subject.

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