Methods, uses, systems, and kits for detecting and determining one or more human papillomavirus genotypes associated with the development of cervical, oral, or anogenital cancer in a subject

Abstract

The present invention relates to methods, uses, systems, and kits for detecting human papillomavirus (HPV) genotypes associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of HPV so detected in a subject. Furthermore, the present invention relates to the use of forward and / or reverse primers or amplicons generated by multiplex real-time PCR, comprising molecular tags for identifying markers of human papillomavirus genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66) and / or (68).

Description

[Technical Field]

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

[0002] The present invention aims to provide a method, use, system, and kit for detecting human papillomavirus (HPV) genotypes associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining the one or more genotypes of the human papillomavirus infecting the subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in the subject, wherein the method, use, system, and kit use a minimum amount of reagents and materials.Furthermore, the present invention aims to provide a method, use, system, and kit for these purposes that is accurate, precise, and fully automated, and can be based on a sample collected by the subject themselves without the need for intervention by a doctor or other medical professional, thus being fully automated from subject to result.Furthermore, the present invention aims to provide a method, use, system, and kit for these purposes that is cheaper and faster. Summary of the Invention

[0003] Short description The present invention provides a method for detecting a human papillomavirus (HPV) genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of the human papillomavirus infecting said subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in said subject, comprising: (a) subjecting at least one sequence contained in DNA present in a biological sample obtained from said subject to multiplex real-time PCR, wherein said DNA is extracted from said biological sample before subjecting said biological sample to multiplex real-time PCR, and said multiplex real-time PCR comprises: (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker for a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; of, (iii) a set of probes, - a probe comprising a first fluorophore and a sequence, said sequence being complementary to and specific for a sequence of a marker of 16 genotypes of human papillomavirus; - a probe comprising a second fluorophore and a sequence that is complementary to and specific for a sequence of a marker of 18 genotypes of human papillomavirus; and - a pool of probes, each probe of the pool of probes comprising a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus having a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes. and a set of probes comprising: (iv) a probe comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; amplifying in a composition comprising Each probe that binds to a sequence of the marker that is complementary to and specific thereto is detected; - repeating step (a) for said subject if the fourth fluorophore of the probe that binds to said second sequence is not detected and the first or second or third fluorophore of the probe that binds to the first sequence is not detected; - if the fourth fluorophore of the probe that binds to the second sequence is detected and at least one of the first or second fluorophore of the probe that binds to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected; - detecting a fourth fluorophore of the probe that binds to the second sequence and a third fluorophore of the probe of the pool of probes that binds to the first sequence, whereby a human papillomavirus genotype associated with the development of cervical, oral, 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 each second sequence of the compositions selected in step (a) to hybridization on a solid support comprising probes attached thereto, wherein during hybridization, the compositions selected in step (a) are subjected to heating to at least 60°C followed by cooling, and each probe comprises a forward primer and a reverse primer that are complementary to and specific for a marker locus and a sequence that is complementary to and specific for the marker sequence provided in the multiplex real-time PCR of step (a); each probe containing 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 containing the same sequence; (c) identifying specific regions of each of said solid supports to which probes are attached and to which the first sequence or the second sequence is hybridized, (I) in step (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the first fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the occurrence of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 16; (II) in step (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the second fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 18; (III) if in step (a) a fourth fluorophore of the probe that binds to the second sequence is detected and a third fluorophore of the probe that binds to the first sequence is detected, and in step (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified, the genotype is (IIIa) identifying specific regions of each of said solid supports to which probes are attached and to which first sequences are hybridized; (IIIb) determining which genotype of human papillomavirus is identified by a marker containing a sequence that is complementary and specific to the sequence of the probe attached to each specific region identified in step (IIIa); The steps and The present invention relates to a method comprising:

[0004] The present invention further provides a system for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in said subject, and for determining one or more genotypes of human papillomavirus infecting said subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in said subject, comprising: (a) a means for subjecting at least one sequence contained in DNA present in a biological sample obtained from said subject to multiplex real-time PCR, said multiplex real-time PCR comprising: (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker for a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; of, (iii) a set of probes, - a probe comprising a first fluorophore and a sequence, said sequence being complementary to and specific for a sequence of a marker of 16 genotypes of human papillomavirus; - a probe comprising a second fluorophore and a sequence that is complementary to and specific for a sequence of a marker of 18 genotypes of human papillomavirus; and - a pool of probes, each probe of the pool of probes comprising a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus having a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes. and a set of probes comprising: (iv) a probe comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; amplifying in a composition comprising Each probe that binds to a sequence of the marker that is complementary to and specific thereto is detected; - repeating (a) for said subject if the fourth fluorophore of the probe that binds to said second sequence is not detected and the first or second or third fluorophore of the probe that binds to the first sequence is not detected; - if the fourth fluorophore of the probe that binds to the second sequence is detected and at least one of the first or second fluorophore of the probe that binds to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected; - a composition comprising the amplified first and second sequences is selected, wherein a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected if the fourth fluorophore of the probe that binds to the second sequence is detected and the third fluorophore of the probe in the pool of probes that binds to the first sequence is detected; A means for subjecting at least one sequence contained in DNA present in the biological sample to multiplex real-time PCR, the means comprising a multiplex real-time PCR instrument and multiplex real-time PCR reagents; (b) means for subjecting each amplified first sequence and each second sequence of the compositions selected in (a) to hybridization on a solid support comprising probes attached thereto; means for heating the compositions selected in (a) to at least 60°C during hybridization; and means for cooling the compositions selected in (a) during hybridization, wherein each probe comprises a forward primer and a reverse primer that are complementary to and specific for a marker locus and that are complementary to and specific for the marker sequence provided in the multiplex real-time PCR of (a); Each probe containing 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 containing the same sequence; the means for heating the composition selected in (a) comprises a heating device; the means for cooling the composition selected in (a) comprises a cooling device; The means for subjecting each of the first and second sequences amplified in the composition selected in (a) to hybridization on a solid support comprises a solid support having probes attached thereto, each probe having a forward primer and a reverse primer complementary to and specific to a marker locus, the forward primer and the reverse primer comprising sequences complementary to and specific to the marker sequences provided in the multiplex real-time PCR in (a), and a hybridization reagent; (c) means for identifying specific regions of each of said solid supports having attached probes and hybridized with a first sequence or a second sequence, said means comprising an image capture device; (I) In (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the first fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the occurrence of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 16; (II) In (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the second fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 18; (III) if in (a) a fourth fluorophore of the probe that binds to the second sequence is detected and a third fluorophore of the probe that binds to the first sequence is detected, and in (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified, the genotype is (IIIa) identifying specific regions of each of said solid supports to which probes are attached and to which first sequences are hybridized; (IIIb) determining which genotype of human papillomavirus is identified by a marker containing a sequence that is complementary and specific to the sequence of the probe attached to each specific region identified in (IIIa); Determined by means and This relates to a system including:

[0005] The system may optionally further include a means for extracting DNA from a biological sample, such as a means for extracting human papillomavirus DNA present in the biological sample obtained from the subject. The means for extracting DNA can be used before using the means for subjecting at least one sequence contained in the DNA present in the biological sample obtained from the subject to multiplex real-time PCR.

[0006] The present invention further provides a kit for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of human papillomavirus infecting said subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in said subject, comprising: (a) a means for subjecting at least one sequence contained in DNA present in a biological sample obtained from said subject to multiplex real-time PCR, said multiplex real-time PCR comprising: (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker for a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; of, (iii) a set of probes, - a probe comprising a first fluorophore and a sequence, said sequence being complementary to and specific for a sequence of a marker of 16 genotypes of human papillomavirus; - a probe comprising a second fluorophore and a sequence that is complementary to and specific for a sequence of a marker of 18 genotypes of human papillomavirus; and - a pool of probes, each probe of the pool of probes comprising a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus having a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes. and a set of probes comprising: (iv) a probe comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; amplifying in a composition comprising Each probe that binds to a sequence of the marker that is complementary to and specific thereto is detected; - repeating (a) for said subject if the fourth fluorophore of the probe that binds to said second sequence is not detected and the first or second or third fluorophore of the probe that binds to the first sequence is not detected; - if the fourth fluorophore of the probe that binds to the second sequence is detected and at least one of the first or second fluorophore of the probe that binds to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected; - a composition comprising the amplified first and second sequences is selected, wherein a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected if the fourth fluorophore of the probe that binds to the second sequence is detected and the third fluorophore of the probe in the pool of probes that binds to the first sequence is detected; A means for subjecting at least one sequence contained in DNA present in the biological sample to multiplex real-time PCR, the means comprising a multiplex real-time PCR instrument and multiplex real-time PCR reagents; (b) means for subjecting each amplified first sequence and each second sequence of the compositions selected in (a) to hybridization on a solid support comprising probes attached thereto; means for heating the compositions selected in (a) to at least 60°C during hybridization; and means for cooling the compositions selected in (a) during hybridization, wherein each probe comprises a forward primer and a reverse primer that are complementary to and specific for a marker locus and that are complementary to and specific for the marker sequence provided in the multiplex real-time PCR of (a); Each probe containing 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 containing the same sequence; the means for heating the composition selected in (a) comprises a heating device; the means for cooling the composition selected in (a) comprises a cooling device; The means for subjecting each of the first and second sequences amplified in the composition selected in (a) to hybridization on a solid support comprises a solid support having probes attached thereto, each probe having a forward primer and a reverse primer complementary to and specific to a marker locus, the forward primer and the reverse primer comprising sequences complementary to and specific to the marker sequences provided in the multiplex real-time PCR in (a), and a hybridization reagent; (c) means for identifying specific regions of each of said solid supports having attached probes and hybridized with a first sequence or a second sequence, said means comprising an image capture device; (I) In (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the first fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the occurrence of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 16; (II) In (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the second fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 18; (III) if in (a) a fourth fluorophore of the probe that binds to the second sequence is detected and a third fluorophore of the probe that binds to the first sequence is detected, and in (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified, the genotype is (IIIa) identifying specific regions of each of said solid supports to which probes are attached and to which first sequences are hybridized; (IIIb) determining which genotype of human papillomavirus is identified by a marker containing a sequence that is complementary and specific to the sequence of the probe attached to each specific region identified in (IIIa); Determined by means and The present invention relates to a kit comprising:

[0007] The kit may optionally further comprise a means for extracting DNA from a biological sample, such as a means for extracting human papillomavirus DNA present in the biological sample obtained from a subject. The means for extracting DNA can be used before using the means for subjecting at least one sequence contained in the DNA present in the biological sample obtained from the subject to multiplex real-time PCR.

[0008] The present invention also relates to the use of forward and / or reverse primers 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, and the DNA is extracted from the biological sample before being subjected to the multiplex real-time PCR; The forward and / or reverse primers are used to identify the specific region on the solid support to which the probe is attached. - a sequence complementary to and specific for one or more loci of markers of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68; and - Molecular tags and wherein the probe comprises a sequence that is complementary to and specific for the sequence of a marker to which the forward and / or reverse primer is complementary to and specific and to which the amplified sequence comprising a molecular tag is hybridized.

[0009] The present invention also relates to the use of amplified sequences comprising molecular tags, wherein the amplified sequences are obtained by multiplex real-time PCR, the multiplex real-time PCR amplifying sequences contained in DNA present in a biological sample obtained from a subject, and the DNA is extracted from the biological sample before being subjected to the multiplex real-time PCR; Amplification is performed using forward and / or reverse primers to identify the specific region on the solid support to which the probe is attached. - a sequence complementary to and specific for one or more loci of markers of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68; and - Molecular tags and wherein the probe comprises a sequence that is complementary to and specific for the sequence of a marker to which the forward and / or reverse primer is complementary to and specific and to which the amplified sequence comprising a molecular tag is hybridized. [Brief explanation of the drawings]

[0010] [Figure 1-1] 1 is a flow chart illustrating an example of a method according to the present invention for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of the human papillomavirus infecting the subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in the subject. [Figure 1-2] 1 is a flow chart illustrating an example of a method according to the present invention for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of the human papillomavirus infecting the subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in the subject. [Figure 2A] Graph showing the amplification of A. a positive control (PC) and B. a negative control (NTC) using water by the multiplex real-time PCR procedure (a) of the present invention. Expected counts over time (ΔRn) (per multiplex PCR cycle) for PC are as follows: HPV16 (ROX) 20±2, HPV18 (Cy5) 20±2, HPV HR (HPV genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, FAM) 20±2, and B globin (human beta globin, JOE) 20±2. Experiments were performed on an Applied Biosystems QuantStudio™ 5 real-time PCR system. (◯) beta globin, (◇) HPV HR, (△) HPV16, (×) HPV18. [Figure 2B]Graph showing the amplification of A. a positive control (PC) and B. a negative control (NTC) using water by the multiplex real-time PCR procedure (a) of the present invention. Expected counts over time (ΔRn) (per multiplex PCR cycle) for PC are as follows: HPV16 (ROX) 20±2, HPV18 (Cy5) 20±2, HPV HR (HPV genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, FAM) 20±2, and B globin (human beta globin, JOE) 20±2. Experiments were performed on an Applied Biosystems QuantStudio™ 5 real-time PCR system. (◯) beta globin, (◇) HPV HR, (△) HPV16, (×) HPV18. [Figure 3A] A. Detection and determination of human papillomavirus genotype 56 in an infected subject by the multiplex real-time PCR procedure of the present invention (b) based on a graph showing the amplification of beta globulin and HPV HR. The expected counts over time (ΔRn) (per multiplex PCR cycle) for PC are as follows: HPV HR (HPV genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, FAM) 20±2, B globin (human beta globin, JOE) 20±2. The experiment was performed on an Applied Biosystems QuantStudio™ 5 real-time PCR system. (○) Beta globin, (◇) HPV HR. [Figure 3B]B. A diagram showing a visualized image of the amplicon of the sample derived from the subject [obtained by multiplex real-time PCR according to (a) of the present invention] after hybridization with a solid support, in which specific regions 2H and 7C (specific regions are identified by numbers and letters, respectively, counting from specific region 1A at the top left of the solid support) indicate that hybridization has occurred, and the specific regions correspond to those on the solid support to which probes containing sequences complementary to and specific to the sequences of markers of the HPV56 genotype are attached, as shown in the schematic diagram of the solid support in C. Further hybridization occurs at specific regions (1A, 1B, 2I, 5E, and 8A) of the solid support to which probes are attached for use as hybridization controls (the five specific regions serve to properly orient the solid support), at specific regions (1C and 5F) of the solid support to which probes are attached for use as internal (human genomic) DNA controls, and at specific regions (1D and 5G) of the solid support to which multiple probes are attached for use as degenerate universal HPV controls. Specific regions 1I, 5C, 9A, 9F, 9H, and 9I have no probes attached to them, while the remaining specific regions have probes attached to them, each probe complementary to and specific for the sequence of the genotypic marker numbered as shown in the diagram, providing duplicates of each specific region of a given probe type. Note that specific regions 4C, 4F, 8E, and 8H are specific regions to which the probes attached are complementary to and specific for the sequences of two genotypic markers. [Figure 3C]B. A diagram showing a visualized image of the amplicon of the sample derived from the subject [obtained by multiplex real-time PCR according to (a) of the present invention] after hybridization with a solid support, in which specific regions 2H and 7C (specific regions are identified by numbers and letters, respectively, counting from specific region 1A at the top left of the solid support) indicate that hybridization has occurred, and the specific regions correspond to those on the solid support to which probes containing sequences complementary to and specific to the sequences of markers of the HPV56 genotype are attached, as shown in the schematic diagram of the solid support in C. Further hybridization occurs at specific regions (1A, 1B, 2I, 5E, and 8A) of the solid support to which probes are attached for use as hybridization controls (the five specific regions serve to properly orient the solid support), at specific regions (1C and 5F) of the solid support to which probes are attached for use as internal (human genomic) DNA controls, and at specific regions (1D and 5G) of the solid support to which multiple probes are attached for use as degenerate universal HPV controls. Specific regions 1I, 5C, 9A, 9F, 9H, and 9I have no probes attached to them, while the remaining specific regions have probes attached to them, each probe complementary to and specific for the sequence of the genotypic marker numbered as shown in the diagram, providing duplicates of each specific region of a given probe type. Note that specific regions 4C, 4F, 8E, and 8H are specific regions to which the probes attached are complementary to and specific for the sequences of two genotypic markers. [Figure 4]

[0023] FIG. 1 is a further detailed flow chart illustrating an example of a method according to the present invention for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of human papillomavirus infecting the subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in the subject. [Figure 5-1]1 shows the detection and determination of human papillomavirus in subjects infected with the virus using various hybridization conditions. The same sample group (liquid cytological samples with previous positive results for HR HPV genotypes by real-time PCR) was assayed using various hybridization conditions. Hybridization condition 1 corresponds to real-time PCR followed by hybridization of the PCR product at a temperature of 41°C. Under these conditions, if the detected signal is provided only by the universal HPV probe, the sample is assessed as having an undetermined HPV genotype (marked "undetermined positive" in the figure). Hybridization condition 2 corresponds to amplification of the sample by end-point PCR without a fluorescent probe, followed by hybridization of the PCR product at a temperature of 41°C. Hybridization condition 3 corresponds to real-time PCR followed by hybridization at a temperature of 60°C and subsequent cooling to 41°C according to the method of the present invention. [Figure 5-2] 1 shows the detection and determination of human papillomavirus in subjects infected with the virus using various hybridization conditions. The same sample group (liquid cytological samples with previous positive results for HR HPV genotypes by real-time PCR) was assayed using various hybridization conditions. Hybridization condition 1 corresponds to real-time PCR followed by hybridization of the PCR product at a temperature of 41°C. Under these conditions, if the detected signal is provided only by the universal HPV probe, the sample is assessed as having an undetermined HPV genotype (marked "undetermined positive" in the figure). Hybridization condition 2 corresponds to amplification of the sample by end-point PCR without a fluorescent probe, followed by hybridization of the PCR product at a temperature of 41°C. Hybridization condition 3 corresponds to real-time PCR followed by hybridization at a temperature of 60°C and subsequent cooling to 41°C according to the method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to a method, system, use and kit for detecting human papillomavirus genotypes associated with the occurrence of cervical, oral or anogenital cancer in a subject, and for determining one or more genotypes of the human papillomavirus that infects the subject when the human papillomavirus genotypes associated with the occurrence of cervical, oral or anogenital cancer are detected in the subject.Therefore, the method, system, use and kit are for detecting human papillomavirus genotypes associated with the occurrence of cervical, oral or anogenital cancer in a subject, and for determining one or more genotypes when detected.

[0012] The human papillomavirus genotype associated with the occurrence of cervical, oral or anogenital cancer in subject is the genotype of human DNA virus from the Papillomaviridae family.The human papillomavirus genotype is associated with the occurrence of cervical, oral or anogenital cancer in subject, and therefore, when infected with it, may cause, increase, promote or otherwise lead to the occurrence of cervical, oral or anogenital cancer in subject.

[0013] The cervical cancer is cancer of the lower uterine region, preferably cancer of the squamous-columnar junction of the cervix, more preferably squamous cell carcinoma of the squamous-columnar junction of the cervix (epidermoid carcinoma).

[0014] The oral cancer may be cancer of the oral cavity and / or pharynx, including cancer of the oral cavity, tonsils, and / or pharynx. The oral cancer may be oropharyngeal cancer.

[0015] The anogenital cancer includes external genital cancer and / or anal cancer. The external genital cancer is preferably selected from the group consisting of cancers of the vulva, vagina, penis, scrotum, and testicles, and any anatomical part thereof. More preferably, the external genital cancer is selected from the group consisting of cancers of the urethra, hymen, clitoral frenulum, clitoral glans, clitoral hood, labia majora, labia minora, fourchette, Skene's glands, vagina, foreskin, penile frenulum, glans penis, scrotum, and testicles, and any anatomical part thereof. Even more preferably, the external genital cancer is selected from the group consisting of squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, small cell carcinoma, lymphoma, sarcoma, and melanoma, and even more preferably squamous cell carcinoma in any of the aforementioned external genital tissues. Thus, the anogenital cancer can be anal cancer, vaginal cancer, vulvar cancer, and / or penile cancer.

[0016] The anal cancer is preferably selected from the group consisting of cancers of the anal canal, anal pillars of Morgagni, squamocolumnar junction, ischio-anal fossa, perianal space, anal verge, anal crypts, anal glands, and anal sphincter. 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 of any of the aforementioned anal tissues, even more preferably squamous cell carcinoma of the squamocolumnar junction.

[0017] The subject is a human subject, preferably a male or female human subject, more preferably a sexually active male subject or a sexually active female human subject.

[0018] The human papillomavirus genotype associated with the development of cervical, oral or anogenital cancer in infected subjects is a genotype that causes the infected subject to have a high risk of developing cervical, oral or anogenital cancer.Preferably, the human papillomavirus genotype associated with the development of cervical, oral or anogenital cancer in infected subjects 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 embodiments of the present invention described herein.The method, use, system and kit of the present invention can detect one or more of these genotypes in any given infected subject.

[0019] In contrast, the human papillomavirus genotype that is not associated with the occurrence of cervical, oral or anogenital cancer in infected subjects is a genotype that causes the occurrence of cervical, oral or anogenital cancer in infected subjects to be at low risk.Preferably, the human papillomavirus genotype that is not associated with the occurrence of cervical, oral or anogenital cancer in infected subjects is selected from the group consisting of human papillomavirus 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82 and 84 genotypes.However, the human papillomavirus genotype that is not associated with the occurrence of cervical, oral or anogenital cancer in infected subjects can still infect subjects infected with one or more human papillomavirus genotypes that are associated with the occurrence of cervical, oral or anogenital cancer.

[0020] Therefore, in other words, the methods, uses, systems and kits of the present invention are for detecting one or more genotypes associated with the development of cervical, oral or anogenital cancer, and for determining one or more genotypes of human papillomavirus associated with the development of cervical, oral or anogenital cancer detected in a subject, and optionally one or more genotypes not associated with the development of cervical, oral or anogenital cancer in a subject infected therewith. In particularly preferred embodiments, the methods, uses, systems, and kits of the present invention are for detecting one or more genotypes associated with the development of cervical, oral, or anogenital cancer, and for determining one or more genotypes of human papillomavirus associated with the development of cervical, oral, or anogenital cancer, and optionally one or more genotypes not associated with the development of cervical, oral, or anogenital cancer, detected in a subject, wherein each genotype associated with the development of cervical, oral, or anogenital cancer is selected from the group consisting of 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus.

[0021] The present invention includes two techniques: multiplex real-time PCR (described by step (a) of the method of the present invention and step (a) of the system and kit of the present invention) and hybridization + visualization (described by steps (b) + (c) of the method of the present invention and step (b) + (c) of 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 vessel) during PCR amplification using a different primer pair for each marker. Thus, multiplex real-time PCR preferably involves said simultaneous detection in "real time" (i.e., at predetermined intervals) after each amplification cycle of said PCR.

[0022] Therefore, (a) of the method of the present invention includes a step of subjecting at least one sequence contained in DNA present in a biological sample obtained from the subject to multiplex real-time PCR (block 100 in Figure 1-1), while (a) of the system and kit of the present invention includes a means for subjecting at least one sequence contained in DNA present in a biological sample obtained from the subject to multiplex real-time PCR.

[0023] Because the PCR products applied to the solid support contained probes remaining from the real-time PCR that were not consumed during amplification, two complementary probes are present in the hybridization device, one in the liquid phase and the other in the solid phase, and they may compete to hybridize to the amplicon of interest. This is prevented by heating to a temperature of at least 60°C, which preferably promotes hybridization of the generated amplicons to the solid phase probes, i.e., those in the array.

[0024] Thus, in the methods, systems, and kits of the present invention, the composition selected in (a) is subjected to heating to at least 60° C. and gradually cooled while hybridizing to the solid surface according to step (b).

[0025] Heating at a temperature of at least 60°C promotes hybridization of the generated amplicons to the probes in the solid phase and not to the probes remaining from the real-time PCR in the liquid phase, improving the sensitivity and specificity of the method. If the heating temperature is below 60°C (e.g., 50°C or 41°C as assayed in Example 4), some of the probes remaining from the PCR may hybridize to the amplicon of interest and prevent it from binding to the probe in the solid support, as shown in Example 4.

[0026] Example 4 shows that the method of the present invention correctly detects and identifies genotypes present in a sample when real-time PCR is followed by hybridization at a temperature of 60° C., thus improving the sensitivity and specificity of the method. As assayed in Example 4, 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.

[0027] The biological sample is a sample comprising biological fluid, free cells, and / or tissue obtained from the subject. The sample can be obtained by a third party (e.g., a doctor or nurse) or by the subject themselves. In a preferred embodiment of the present invention, the biological sample obtained from the subject is obtained by the subject obtaining the sample themselves. For example, a biological sample derived from the cervix of the subject may be obtained by the subject obtaining the sample themselves. The sample is preferably obtained by a sample collection device such as a swab, brush, or veil device applied to the subject's cervical, oral, or anogenital tissue, preferably cervical tissue, to obtain biological fluid and / or cells from the subject. Thus, the sample collection device can be a swab, brush, or vaginal veil device.

[0028] The biological sample contains DNA. The DNA is a polynucleotide, and therefore contains at least one sequence of nucleotides. If the subject is infected with a human papillomavirus associated with the development of cervical, oral, or anogenital cancer, the biological sample contains DNA derived from the human papillomavirus. If the subject is not infected with a human papillomavirus associated with the development of cervical, oral, or anogenital cancer, the biological sample contains DNA derived from other sources, i.e., DNA derived from the subject and / or DNA derived from a human papillomavirus not associated with the development of cervical, oral, or anogenital cancer.

[0029] To perform the multiplex real-time PCR amplification, DNA is usually extracted from the sample, and therefore the amplification can be performed from a purified DNA sample.

[0030] The PCR (i.e., multiplex real-time PCR) (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker of a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary and specific to the locus of the marker, respectively (block 100 in FIG. 1-1); Amplify.

[0031] Each amplification is performed by PCR using a pair of primers, including a locus-specific forward primer and a locus-specific reverse primer that bind to different complementary sequences on the Watson-Crick strand adjacent to the nucleotide sequence, thereby identifying the 5' and 3' limits of the nucleotide sequence. Specifically, the 3' end of the nucleotide sequence on the Watson strand begins with the nucleotide adjacent to the 5'-terminal nucleotide of the sequence annealed with the forward primer. Conversely, the 5' end of the nucleotide sequence on the Watson strand begins with the nucleotide complementary to the nucleotide adjacent to the 5'-terminal nucleotide of the sequence annealed with the reverse primer. Similarly, the 3' end of the nucleotide sequence on the Crick strand begins with the nucleotide adjacent to the 5'-terminal nucleotide of the sequence annealed with the reverse primer. Conversely, the 5' end of the nucleotide sequence on the Crick strand begins with the nucleotide complementary to the nucleotide adjacent to the 5'-terminal nucleotide of the sequence annealed with the forward primer. Thus, the DNA polymerase attaches to the 5' end of the aforementioned primer and replicates the nucleotide sequence multiple times.

[0032] The primers are locus-specific primers selected to be complementary to, and therefore identify, specific nucleotide sequences (i.e., genetic markers) that may be present in a biological sample, said markers being: - in case (i) is indicative of said human papillomavirus (i.e. the presence of human papillomavirus DNA in said sample and therefore infection by said human papillomavirus in said subject), - in case (ii), it is indicative of said human housekeeping gene (i.e. the presence of human DNA from said subject in said sample).

[0033] Thus, the methods, uses, systems and kits of the present invention rely on any genetic markers of human papillomavirus and human housekeeping genes that are detectable using amplicon sequencing techniques.

[0034] The multiplex real-time PCR amplifies at least one first sequence (of a marker for the genotype of human papillomavirus) and a second sequence (of a marker for the genotype of human housekeeping gene), and the amplification of each sequence is performed using a forward primer and a reverse primer, each of which is complementary and specific to the locus of any given marker, so that the multiplex real-time PCR is performed using at least two pairs of forward and reverse primers. Preferably, the multiplex real-time PCR is performed using at least two pairs of forward and reverse primers for amplifying the first sequence, and one pair of forward and reverse primers is used for amplifying the second sequence, so that at least two different first sequences can be amplified by the multiplex real-time PCR, and each first sequence is a marker for the genotype of human papillomavirus, and the second sequence may be amplified by the multiplex real-time PCR, and the second sequence is a marker for the genotype of human housekeeping gene. Any given pair of forward and reverse primers for amplification of a first sequence can amplify a marker common to one or more genotypes of human papillomavirus.

[0035] Each first sequence is a sequence of a marker for the genotype of a human papillomavirus, regardless of whether the human papillomavirus is associated with the development of cervical, oral, or anogenital cancer. In a preferred embodiment of the method, use, system, and kit of the present invention, the multiplex real-time PCR comprises: (i) at least one first sequence, each of which is a sequence of a marker derived from a human papillomavirus genotype present in a conserved region of the HPV L1 or HPV L2 gene, more preferably in a conserved region of the HPV L1 gene; (ii) the second sequence; and Amplify.

[0036] In another preferred embodiment of the method, use, system, and kit of the present invention, said multiplex real-time PCR comprises: (i) at least one first sequence, each first sequence being a sequence of a marker derived from a human papillomavirus genotype selected from 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 genotypes of human papillomavirus; (ii) the second sequence; and Amplify.

[0037] In another more preferred embodiment of the method, the use, the system, and the kit of the present invention, said multiplex real-time PCR comprises: (i) - a first sequence which is a sequence of markers derived from 16 genotypes of human papillomavirus; - a first sequence, which is a sequence of markers derived from 18 genotypes of human papillomavirus; and - at least one other first sequence, each of which is a sequence of a marker derived from a genotype of human papillomavirus selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus; and (ii) the second sequence; and Amplify.

[0038] Each second sequence is a marker sequence 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 needed to maintain cell function. More preferably, the human housekeeping gene is the human beta globin gene (HBB gene) or the ribonuclease P gene (RNase P gene), even more preferably the HBB gene.

[0039] Preferably, the forward and reverse primers for the amplification of each first sequence are selected from any of the pairs of sequences SEQ ID NO: 1 to SEQ ID NO: 2 disclosed in Table 1, while the forward and reverse primers for the amplification of the second sequence are selected from any of the pairs of sequences SEQ ID NO: 3 to SEQ ID NO: 4 disclosed in Table 2.

[0040] [Table 1]

[0041] [Table 2]

[0042] The multiplex real-time PCR used in the present invention amplifies DNA derived from a subject and / or DNA derived from human papillomavirus in a composition. In addition to the DNA, the composition contains a forward primer, a reverse primer, and multiple probes, preferably TaqMan probes. In particular, the multiple probes contained in the composition include: (iii) a set of probes, - a probe comprising a first fluorophore and a sequence, said sequence being complementary to and specific for a sequence of a marker of 16 genotypes of human papillomavirus; - a probe comprising a second fluorophore and a sequence that is complementary to and specific for a sequence of a marker of 18 genotypes of human papillomavirus; and - a pool of probes, each probe of the pool of probes comprising a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus having a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes. and a set of probes comprising: (iv) a probe (Block 100 in FIG. 1) comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; Includes.

[0043] Each of the probes disclosed in (iii) and (iv) comprises a fluorophore and a sequence, which is complementary and specific to the sequence of a genetic marker. When the sequence binds / anneals to the complementary sequence (i.e., specific for) of the genetic marker amplified by the multiplex PCR, the fluorophore emits detectable light. In other words, each probe that binds to the complementary and specific sequence of the marker is detected.

[0044] In particular, each probe in the set of probes disclosed in (iii) comprises a fluorophore and a sequence that is complementary to and specific to the sequence of a marker for the 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus. In this way, the sequence of a marker for any of the human papillomavirus genotypes can be detected in a composition subjected to multiplex PCR by real-time detection of light emitted from the fluorophore of a probe containing a sequence that is complementary to and specific to the sequence of the marker when the sequence of the probe binds to the sequence of the marker during amplification. Thus, the methods, uses, systems, and kits of the present invention enable the detection of human papillomaviruses having any of the genotypes that may cause a high risk of developing cervical, oral, or anogenital cancer in infected subjects. Preferably, each probe for a given target (HPV genotype or set of HPV genotypes) comprises a sequence selected from any of the sequences shown in Table 3.

[0045] [Table 3]

[0046] Similarly, the probe disclosed in (iv) comprises a fluorophore and a sequence that is complementary and specific to the sequence of the human housekeeping gene amplified by the multiplex PCR. In this way, the sequence of the marker for the human housekeeping gene can be detected in a composition subjected to multiplex PCR by real-time detection of light emitted from the fluorophore of the probe, which comprises a sequence that is complementary and specific to the sequence of the marker, when the sequence of the probe binds to the sequence of the marker during amplification. Thus, the method, use, system, and kit of the present invention also enable DNA from the subject to be detected. Preferably, each probe for a given human housekeeping gene comprises a sequence selected from any of the sequences shown in Table 4.

[0047] [Table 4]

[0048] Therefore, the multiplex real-time PCR is - amplifying sequences of markers for human papillomavirus genotypes, regardless of whether said human papillomavirus is associated with the development of cervical, oral, or anogenital cancer, and detecting any sequences so amplified, including sequences of markers for any of the 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus; and - amplifying a sequence of a marker of a human housekeeping gene and detecting any sequence so amplified which comprises the sequence of said marker of a human housekeeping gene.

[0049] Therefore, if the subject is infected with human papillomavirus (and the sample contains DNA from the subject and the human papillomavirus), the multiplex real-time PCR can amplify (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 can only amplify (ii). On the other hand, if the sample contains DNA from the human papillomavirus but not DNA from the subject, the multiplex real-time PCR will only amplify (i), which is an indication of an ineffective 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. Therefore, in the methods, uses, systems, and kits of the present invention, - if the fourth fluorophore of the probe binding to said second sequence is not detected and the first or second or third fluorophore of the probe binding to the first sequence is not detected (decision box 102 in FIG. 1-1), repeat step (a) for said subject ("No" connecting decision box 102 in FIG. 1-1 with block 100); - if the fourth fluorophore of the probe that binds to the second sequence (i.e., the sequence of the marker for the human housekeeping gene) is detected and at least one of the first or second fluorophores of the probe that binds to the first sequence (i.e., the sequence of the marker for 16 or 18 genotypes of human papillomavirus, respectively) is detected (decision box 104 in Figure 1-1), a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected (block 106 in Figure 1-1); - If the fourth fluorophore of the probe that binds to the second sequence is detected and the third fluorophore of the probe of the probe pool that binds to the first sequence (i.e., a marker sequence of any of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus) is detected (decision box 108 in Figure 1-1), a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected, and a composition containing the amplified first sequence and second sequence is selected for subsequent step (b), which refers to the method of the present invention (block 110 in Figure 1-1) or means (b) when referring to the system and kit of the present invention.

[0050] In a preferred embodiment of the method, system, and kit of the present invention, the heating is at 60 to 70°C.

[0051] In a preferred embodiment of the method, system, and kit of the present invention, the heating is at 60 to 65°C.

[0052] In a preferred embodiment of the method, system, and kit of the present invention, the heating is for 1 to 20 minutes.

[0053] In a preferred embodiment of the method, system, and kit of the present invention, the heating is for 1 to 10 minutes.

[0054] In a preferred embodiment of the method, system, and kit of the present invention, the heating is for 3 to 20 minutes.

[0055] In a preferred embodiment of the methods, systems, and kits of the present invention, the heating is for 1 minute.

[0056] In a preferred embodiment of the methods, uses, systems and kits of the present invention, said cooling comprises cooling to 37-45°C.

[0057] In a preferred embodiment of the methods, uses, systems and kits of the present invention, said cooling comprises cooling to 41°C.

[0058] In preferred embodiments of the methods, uses, systems and kits of the present invention, said cooling is for at least 2 minutes.

[0059] In a preferred embodiment of the method, use, system, and kit of the present invention, the cooling is for 2 to 10 minutes.

[0060] In a preferred embodiment of the method, use, system and kit of the present invention, said cooling is for 2 to 8 minutes.

[0061] In a preferred embodiment of the method, use, system, and kit of the present invention, the cooling is for 2 to 6 minutes.

[0062] In a preferred embodiment of the methods, uses, systems and kits of the present invention, said cooling is for 4 minutes.

[0063] In a preferred embodiment of the methods, uses, systems and kits of the present invention, the composition selected in (a) is heated to 95° C. for 10 minutes prior to hybridization in (b).

[0064] In a preferred embodiment of the methods, uses, systems, and kits of the invention, the composition selected in (a) is subjected to heating to at least 60°C for at least 10 minutes before being cooled and subjected to hybridization in step (b). More preferably, the heating is to 60-70°C for 1-20 minutes, and even more preferably to 60-65°C for 3-10 minutes.

[0065] A preferred embodiment of the method, use, system, and kit of the present invention is further for detecting the absence of a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in the subject, wherein if the fourth fluorophore of the probe that binds to the second sequence is detected and the first, second, or third fluorophore of the probe that binds to the first sequence is not detected, then the human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is not detected.

[0066] If the probe binding to the second sequence is not detected and the probe binding to the first sequence is not 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 during the PCR reaction. In a preferred embodiment of the method, use, system, and kit of the present invention, if the probe binding to the second sequence is not detected and the probe binding to the first sequence is not detected, step (a) is repeated for the subject using a new sample obtained therefrom.

[0067] The first, second, third and fourth fluorophores of each probe (ie, fluorescent probe) preferably each emit light at a different emission maximum wavelength. The first, second, third, and fourth fluorophores are more preferably 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-carboxy-fluorescein) fluorophore, Alexa fluor 532, HEX, Cy3, TRITC, Alexa fluor 546, Alexa fluor 555, R-phycoerythrin (PE), rhodamine red-X, Tamara, Cy3.5 581, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, allophycocyanin, Alexa fluor 633, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor 680, 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), Alexa fluor 430, JOE, VIC, Alexa fluor 532, and HEX (green channel); and another of the first, second, third, and fourth fluorophores is a fluorophore selected from the group consisting of Cy3, TRITC, Alexa fluor 546, Alexa fluor 555, R-phycoerythrin (PE), rhodamine red-X, Tamara, Cy3.5 581, Rox, Alexa fluor 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 a different fluorophore 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, JOE, or VIC.

[0068] The light emitted from each fluorophore is simultaneously detected (if emitted) by a multiplex real-time PCR platform capable of detecting light at the emission maxima wavelengths of the first, second, third, and fourth fluorophores. Preferably, the multiplex real-time PCR platform is 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, on 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 by Forootan et al. (2017), "Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR)," Biomol Detect Quantif., 12:1-6. For example, a threshold and baseline can be established for each real-time PCR. The baseline of a real-time PCR reaction can refer to the signal level during the initial cycles of PCR, such as the average signal obtained during cycles 3-15 of PCR. A threshold value can be used to distinguish relevant amplification signals from background.

[0069] While step (b) of the method of the present invention includes a step of subjecting the amplified first and second sequences contained in the composition selected in step (a) to hybridization (block 112 in Figures 1-2), step (b) of the system and kit of the present invention includes a means for subjecting the amplified first and second sequences contained in the composition selected in step (a) to hybridization. Thus, the DNA amplified by multiplex real-time PCR contained in the composition in which the fourth fluorophore of the probe that binds to the second sequence is detected and the third fluorophore of the probe of the probe pool that binds to the first sequence is detected is subjected to hybridization.

[0070] The hybridization comprises binding of the amplified first sequence and the amplified second sequence contained in the composition selected in (a) to 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 marker sequence, for which a forward primer and a reverse primer complementary to and specific for the marker locus are provided in the multiplex real-time PCR of (a). Thus, the hybridization comprises: - the amplified first sequence contained in the composition selected in (a) and at least one sequence complementary to and specific for the sequence of the marker, for which forward and reverse primers complementary to and specific for the locus of the marker are provided in the multiplex real-time PCR of (a); and - an amplified second sequence contained in the composition selected in (a), and at least one sequence complementary to and specific for the marker locus, wherein a forward primer and a reverse primer complementary to and specific for the marker locus are provided in the multiplex real-time PCR of (a); wherein the sequence of said marker is contained in a probe attached to said solid support.

[0071] The solid support is a three-dimensional porous environment preferably selected from the group consisting of a hybridization membrane, a hybridization sheet, and a supported hybridization membrane. More preferably, the solid support is a nylon hybridization membrane, a charged nylon hybridization membrane, a nitrocellulose sheet, or a supported hybridization membrane comprising nitrocellulose supported on a polymer membrane.

[0072] The solid support comprises probes attached thereto, each probe comprising a sequence complementary to and specific for the marker locus provided in the multiplex real-time PCR of step (a) as a forward primer and a reverse primer, each complementary to and specific for the marker locus. 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. Thus, the solid support comprises multiple non-overlapping specific regions, each of which comprises multiple probes comprising the same sequence and whose sequences are different from those of the probes 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, grid, or regular pattern, more preferably a square or rectangular array, and even more preferably a square array of circular specific regions.

[0073] Each probe attached to the specific region of the solid support comprises a sequence complementary to and specific for the marker sequence, for which a forward primer and a reverse primer complementary to and specific for the marker locus are provided in the multiplex real-time PCR of step (a). Preferably, each probe attached to the specific region of the solid support comprises a sequence selected from any of the sequences shown in Table 5, which is complementary to and specific for the corresponding human papillomavirus genotype or human housekeeping gene shown therein.

[0074] [Table 5]

[0075] Any given specific region of the solid support contains probes each having a sequence complementary to and specific for the same marker sequence. The set of specific regions includes at least two specific region subsets, - each specific region of one subset of said specific regions comprises probes, each having a sequence complementary to and specific for the locus of said marker of human papillomavirus, 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); - each specific region of one subset of said specific regions comprises probes, each having a sequence complementary to and specific for the locus of said marker of a human housekeeping gene, for which a forward primer and a reverse primer are provided in said multiplex real-time PCR of step (a); Each probe containing 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 containing the same sequence. In step (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the third fluorophore of the probe that binds to the first sequence is detected, then at least one specific region of the solid support to which a probe is attached can hybridize to the first sequence. Therefore, in step (a), if the fourth fluorophore of the probe binding to the second sequence is detected and the third fluorophore of the probe binding to the first sequence is detected, the subject is infected with only one genotype of human papillomavirus, and the genotype can be identified by a marker comprising a sequence that is complementary and specific to the sequence of the probe attached to the solid support, and the sequence of the probe is complementary and specific to the sequence of the marker, for which forward and reverse primers complementary and specific to the marker locus are provided in step (b) in the multiplex real-time PCR, the first sequence hybridizes with the sequence of the probe. Preferably, at least one specific region of the solid support to which the probe is attached can hybridize with the first sequence, and at least one specific region of the solid support to which the probe is attached can hybridize with the second sequence.

[0076] While the step (d) of the method of the present invention includes a step of identifying specific regions of the solid support to which the probe is attached and to which the first sequence or the second sequence is hybridized (block 114 in Figure 1-2), the step (c) of the system and kit of the present invention includes a means for identifying specific regions of the solid support to which the probe is attached and to which the first sequence or the second sequence is hybridized.

[0077] The specific regions of each solid support where hybridization of the amplified sequences and / or the amplified first and second sequences (i.e., amplicons) of the composition selected in step (a) to the sequences of the probes attached to the solid support occurs can be detected, and therefore identified. Detection involves detection of light emitted from a fluorophore contained in a probe containing the sequence to which any given amplicon binds / anneals (as the amplicon binds / anneals to it), or by the use of a detectable molecular tag attached to the amplicon. Preferably, detection involves detection of light (i.e., visualization), and the molecular tag is a fluorophore or reagent necessary to cause a colorimetric reaction. More preferably, as exemplified, detection involves visualization of a molecular tag attached to the amplicon, the tag being biotin, which is visualized by an immunoenzymatic colorimetric reaction using a chromogen such as streptavidin-phosphatase and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) (also referred to herein as NBT-BCIP), and / or streptavidin-horseradish peroxidase and 3,3-diaminobenzidine (DAB), which precipitates in the solid support in specific areas where hybridization has occurred. Light shone through the solid support is scattered to a higher degree in specific areas where the insoluble precipitate is present (i.e., where hybridization has occurred), thereby allowing the specific areas where hybridization has occurred to be identified. Typically, detection involves detecting the concentration of the amplicon relative to the gray signal intensity and background noise. Suitable software can define grey intensity and threshold parameters to distinguish background noise from amplicon signal in order to automate the analysis of all dots.

[0078] Thus, 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 reactions using streptavidin-phosphatase and an appropriate chromogen (e.g., NBT-BCIP) and / or streptavidin-horseradish peroxidase and 3,3-diaminobenzidine (DAB). Methods for visualizing biotin contained in DNA / RNA on a solid support are described, inter alia, by Leary et al. (1983), PNAS, vol. 80, pp. 4045-4049, Genetics. Visualization of digoxigenin-labeled amplicons can be achieved in a similar manner using anti-digoxigenin antibodies coupled with either alkaline phosphatase (AP) or horseradish peroxidase (HRP) for colorimetric detection. Thus, molecular tags such as 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.

[0079] Therefore, the present invention also provides the use of forward and / or reverse primers 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, and the DNA is extracted from the biological sample before being subjected to the multiplex real-time PCR; The forward and / or reverse primers are used to identify the specific region on the solid support to which the probe is attached. - a sequence complementary to and specific for one or more loci of markers of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68; and - Molecular tags and wherein the probe comprises a sequence that is complementary to and specific for the sequence of a marker to which the forward and / or reverse primer is complementary to and specific and to which the amplified sequence comprising a molecular tag is hybridized.

[0080] The primers (forward and reverse) described herein are oligonucleotides. Such oligonucleotides can be 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 in length. For example, the oligonucleotides can be 4 to 50 nucleotides, 5 to 30 nucleotides, 10 to 30 nucleotides, or 15 to 30 nucleotides in length. The primers / oligonucleotides can have the sequence of any one of SEQ ID NOs: 1 to 4. The primers described herein are used to amplify the DNA sequences described herein.

[0081] The primers described herein may contain molecular tags. It is also contemplated that a primer may contain one, two, three, four, or more molecular tags. Molecular tags may be attached to a primer by any suitable means. Molecular tags may be attached to the 5' end, the 3' end, or may be internally attached within the primer sequence. Thus, the forward and / or reverse primer may contain a molecular tag at its 5' end. Means for attaching molecular tags to primers are known to those skilled in the art and are described, for example, in Benktstrom et al. (1991), Biotinylation of Oligonucleotides and Their Use as Polymerase Chain Reaction Primers. Nucleosides & Nucleotides, 10(1):507-509. Various companies, such as TriLink® BioTechnologies or Creative Biogene, synthesize oligonucleotides containing molecular tags on demand. For example, molecular tags may be attached to the forward and / or reverse primer, preferably at its 5' end, via a suitable linker. Such suitable linkers are known in the art.For example, the linker may 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 1-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 10-(O-trifluoroacetamido-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 -phthalimidyl)-hexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0082] Therefore, as disclosed herein, molecular tags can be attached to the amplified sequences (also referred to herein as amplicons) obtained by multiplex real-time PCR. Thus, molecular tags can be attached to the 5'-end of the amplified sequences (e.g., the first and / or second sequences) or within the amplified sequences, depending on the location of the molecular tags on the primers that generated the amplified sequences. Preferably, the amplified sequences (e.g., the first and / or second amplified sequences) contain molecular tags at their 5'-ends.

[0083] Therefore, the present invention also relates to the use of amplified sequences comprising molecular tags, wherein the amplified sequences are obtained 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, and the amplification is carried out using forward and / or reverse primers, wherein the forward and / or reverse primers are capable of amplifying specific regions on a solid support to which probes are attached, - a sequence complementary to and specific for one or more loci of markers of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68; and - Molecular tags and wherein the probe comprises a sequence that is complementary to and specific for the sequence of a marker to which the forward and / or reverse primer is complementary to and specific and to which the amplified sequence comprising a molecular tag is hybridized.

[0084] Furthermore, by identifying which fluorophore was detected in (a) and the respective specific regions where hybridization occurred in (c), and knowing the genotype detectable in each specific region of the solid support (i.e., which genotype marker sequence contains the sequence to which the probe attached to each specific region of the solid support is complementary and specific), it is subsequently possible to determine the genotype(s) of the human papillomavirus infecting the subject. In particular, (I) In step (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the first fluorophore of the probe that binds to the first sequence is detected (decision box 116 in Figure 1-2), the detected human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is determined to be human papillomavirus 16 genotype (block 118 in Figure 1-2). (II) in step (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the second fluorophore of the probe that binds to the first sequence is detected (decision box 120 in Figure 1-2), the detected human papillomavirus genotype associated with the occurrence of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 18 (block 122 in Figure 1-2); (III) if in step (a) a fourth fluorophore of the probe that binds to the second sequence is detected and a third fluorophore of the probe that binds to the first sequence is detected, and in step (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified (decision box 124 of Figure 1-2), the genotype is (IIIa) identifying specific regions of each of the solid supports to which probes are attached and to which first sequences are hybridized (Block 126 of FIG. 1-2); (IIIb) determining which genotype of human papillomavirus is identified by a marker containing a sequence that is complementary and specific to the sequence of the probe attached to each specific region identified in step (IIIa) (block 128 of Figure 1-2). (block 130 in FIG. 1-2).

[0085] It should be noted that the clinical management of a subject determined to be infected with human papillomavirus genotype 16 or 18 generally differs from that determined to be infected with any of human papillomavirus genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Thus, if one or both of human papillomavirus genotypes 16 and 18 are exclusively detected [i.e., in step (a), the fourth fluorophore of the probe that binds to the second sequence is detected, but the third fluorophore of the probe that binds to the first sequence is not detected], then it is not necessary to perform the hybridization defined in (b), which in itself conserves hybridization reagents and minimizes waste. However, in practice, human papillomavirus genotypes 16 and / or 18 are frequently, if not always, found in the presence of at least one of human papillomavirus genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Nevertheless, the methods, uses, systems and kits of the present invention are for detecting one or more genotypes associated with the development of cervical, oral or anogenital cancer, and for determining one or more genotypes of human papillomavirus associated with the development of cervical, oral or anogenital cancer detected in a subject, and optionally one or more genotypes not associated with the development of cervical, oral or anogenital cancer in a subject infected therewith, wherein each genotype associated with the development of cervical, oral or anogenital cancer is selected from the group consisting of 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 genotypes of human papillomavirus. In a particularly preferred embodiment, the set of probes defined in (iii) still preferably comprises a probe comprising a first fluorophore and a probe comprising a second fluorophore, in order to enable the detection and determination of 16 and 18 genotypes of human papillomavirus in said subject.

[0086] However, in another aspect of the present invention, the method is for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of human papillomavirus infecting said subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in said subject, wherein each genotype associated with the development of cervical, oral, or anogenital cancer is selected from the group consisting of 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus; (a) subjecting at least one sequence contained in DNA present in a biological sample obtained from said subject to multiplex real-time PCR, wherein said DNA is extracted from said biological sample before subjecting said biological sample to multiplex real-time PCR, and said multiplex real-time PCR comprises: (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker for a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; of, (iii) a pool of probes, each probe of the pool of probes comprising a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus having a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes; (iv) a probe comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; amplifying in a composition comprising Each probe that binds to a sequence of the marker that is complementary to and specific thereto is detected; - repeating step (a) for said subject if the fourth fluorophore of the probe that binds to said second sequence is not detected and the third fluorophore of the probe that binds to said first sequence is not detected; - detecting a fourth fluorophore of the probe that binds to the second sequence and a third fluorophore of the probe of the pool of probes that binds to the first sequence, whereby a human papillomavirus genotype associated with the development of cervical, oral, 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 each second sequence of the compositions selected in step (a) to hybridization on a solid support comprising a probe attached thereto, wherein each probe comprises a forward primer and a reverse primer that are complementary to and specific for a marker locus and a sequence that is complementary to and specific for the marker sequence provided in the multiplex real-time PCR of step (a); each probe containing 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 containing the same sequence; (c) identifying specific regions of each of said solid supports to which probes are attached and to which the first sequence or the second sequence is hybridized, (III) if in step (a) a fourth fluorophore of the probe that binds to the second sequence is detected and a third fluorophore of the probe that binds to the first sequence is detected, and in step (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified, the genotype is (IIIa) identifying specific regions of each of said solid supports to which probes are attached and to which first sequences are hybridized; (IIIb) determining which genotype of human papillomavirus is identified by a marker containing a sequence that is complementary and specific to the sequence of the probe attached to each specific region identified in step (IIIa); The steps and Includes.

[0087] Similarly, in another aspect of the present invention, the system and kit are for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of human papillomavirus infecting the subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in the subject, wherein each genotype associated with the development of cervical, oral, or anogenital cancer is selected from the group consisting of 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus; (a) a means for subjecting at least one sequence contained in DNA present in a biological sample obtained from said subject to multiplex real-time PCR, said multiplex real-time PCR comprising: (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker for a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; of, (iii) a pool of probes, each probe of the pool of probes comprising a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus having a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes; (iv) a probe comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; amplifying in a composition comprising Each probe that binds to a sequence of the marker that is complementary to and specific thereto is detected; - repeating step (a) for said subject if the fourth fluorophore of the probe that binds to said second sequence is not detected and the third fluorophore of the probe that binds to said first sequence is not detected; - detecting a fourth fluorophore of the probe that binds to the second sequence and a third fluorophore of the probe of the pool that binds to the first sequence, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected, and selecting a composition comprising the amplified first sequence and the second sequence; (b) means for subjecting each amplified first sequence and each second sequence of the compositions selected in (a) to hybridization on a solid support comprising probes attached thereto, wherein each probe comprises a forward primer and a reverse primer complementary to and specific for a marker locus, the forward primer and the reverse primer comprising sequences complementary to and specific for the marker sequences provided in the multiplex real-time PCR of (a); each probe containing 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 containing the same sequence; (c) means for identifying the specific regions of each of said solid supports to which probes are attached and to which the first sequence or the second sequence is hybridized, said means comprising: (III) if in (a) a fourth fluorophore of the probe that binds to the second sequence is detected and a third fluorophore of the probe that binds to the first sequence is detected, and in (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified, the genotype is (IIIa) identifying specific regions of each of said solid supports to which probes are attached and to which first sequences are hybridized; (IIIb) determining which genotype of human papillomavirus is identified by a marker containing a sequence that is complementary and specific to the sequence of the probe attached to each specific region identified in (IIIa); Determined by means and Includes.

[0088] The present invention also contemplates that the method and / or use may include extracting human papillomavirus DNA. The present invention may also include a system and / or kit comprising step (aa) a means for extracting human papillomavirus DNA present in a biological sample obtained from the subject. The means for extracting DNA may be used before using the means for subjecting at least one sequence contained in the DNA present in the biological sample obtained from the subject to multiplex real-time PCR; thus, step (aa) may precede step (a) of the systems and kits described herein. Such means may include, for example, a lysis buffer, a high salt solution (e.g., for precipitating proteins), a solution containing isopropanol for precipitated DNA from a high salt solution, and / or a solution for eluting DNA, such as Tris-EDTA or nuclease-free water. Filters and / or matrices, such as silica, cellulose, and ion exchange, may also be included by the means.

[0089] In another preferred embodiment of the method, use, system and kit of the invention, said DNA is extracted from said biological sample by lysis before subjecting it to multiplex real-time PCR, - a first control is also subjected to multiplex real-time PCR as defined in step (a), when at least one sequence comprised in DNA present in said biological sample is subjected thereto, said first control being a blank subjected to said extraction, and optionally - when at least one sequence comprised in DNA present in said biological sample is subjected thereto, a second control is also subjected to multiplex real-time PCR as defined in step (a), said second control comprising DNA comprising a first sequence which is a sequence of a marker derived from a genotype of human papillomavirus selected from genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus, and optionally - the first and second sequences amplified in step (a) and the solid support further comprise molecules attached thereto, the molecules attached to the solid support being attached to specific regions of the solid support that are different from other specific regions of the solid support to which probes are attached, and the first and second sequences amplified in step (a) and the molecules attached to the solid support have the same chemical structure.

[0090] Methods for HPV DNA extraction are known to those skilled in the art. Respective kits are available, inter alia, from MGI (HPV DNA Extraction Kit, item number 1000028433). Further methods for HPV DNA extraction are described, for example, in Example 1. DNA extraction from a biological sample can yield 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 of DNA. Typically, the extracted DNA is contained in an elution buffer. In multiplex real-time PCR, DNA can be, for example, about 100 pg / μL. -1 It can be added at a concentration of ~100ng / μl. For example, in multiplex real-time PCR, approximately 1ng / μL of DNA -1~ 4ng / μL -1 can be included in the PCR master mix.

[0091] In another preferred embodiment of the methods, systems, and kits of the present invention, each probe is located in at least two specific regions of the solid support, which allows for multiple results to be obtained for each probe in one assay, thus further improving the sensitivity of the method.

[0092] In (b), hybridization controls (B), such as hybridization control probes or biotin proteins, endogenous amplification control probes (C), and / or degenerate universal probes (U) can be attached to specific regions of the solid support.

[0093] When using a hybridization control probe (B), after the development of the solid support (i.e., hybridization), which is necessary to identify the specific region where hybridization has occurred, a strong signal (coloring / darkening / lightening) appears in the specific region where the hybridization control probe is attached, thereby serving as a quality control for hybridization. This signal indicates that the hybridization reagent and development worked correctly. If no signal appears, this indicates that an error occurred during the hybridization process or that the reagent was not used correctly. Preferably, this signal allows software to correctly orient the solid support (chip) so that the subsequent analysis of the hybridization results, and therefore genotyping, can be automated.

[0094] The endogenous amplification control probe (C) detects the human housekeeping gene sequence amplified by multiplex real-time PCR in (a) (co-amplified with each first sequence). When using an endogenous amplification control probe, after the development of the solid support (i.e., hybridization) required to identify the specific region where hybridization occurred, samples in which the human housekeeping gene sequence was correctly amplified hybridize (i.e., are visualized and have a positive signal) with the specific region of the solid support to which the endogenous amplification control probe is attached. This signal indicates the quality / quantity of the DNA used in (and therefore resulting from) the multiplex real-time PCR amplification. A positive signal indicates that the amplification worked correctly and that the quality and quantity of the starting DNA were optimal. The absence of a signal for this control indicates an error during amplification, low quality / quantity of DNA used in the amplification, or the absence of human DNA in the sample. This last case is possible when the number of human cells present in the sample is below the detection limit. Furthermore, if no positive signal is detected for any HPV genotype, the software (e.g., hybriSoft software) preferably generates an automatic message that the sample is blank, that the material is inappropriate or insufficient, and / or that PCR is inhibited. On the other hand, if the sample tests positive at any specific region of the solid support to which a probe containing a sequence complementary and specific to the sequence of an HPV genotype marker is attached, but the sample does not test positive at any specific region of the solid support to which an endogenous amplification control probe is attached, the software preferably generates an automatic message that the material present in the composition is insufficient. Under such circumstances, the method, reagents, and sample must be inspected (e.g., for quality) before the hybridization results can be confirmed.

[0095] In another preferred embodiment of the methods, uses, systems, and kits of the present invention, the specific region of the solid support that is different from the other specific regions of the solid support to which probes are attached has attached thereto a pool of probes (i.e., a universal probe pool, or degenerate universal probes, U), each probe of the probe pool comprises a sequence that is complementary to and specific for a sequence of a conserved region of an HPV L1 or HPV L2 gene, and at least two probes in the probe pool comprise sequences that are different from each other; - identifying specific regions of the solid support to which the pool of probes is attached and to which sequences hybridize; - identifying specific regions of the solid support to which probes are attached and to which second sequences are hybridized; - the specific region of the solid support to which the probe is attached and to which the first sequence hybridizes is not identified; In this case, the genotype of the human papillomavirus is determined as each genotype that can be identified by the markers provided in the multiplex real-time PCR of step (a) using forward and reverse primers that are complementary and specific to the loci of the markers.

[0096] In another preferred embodiment of the methods, uses, systems, and kits of the present invention, the method further comprises determining whether the subject is at risk of developing cervical, oral, or anogenital cancer, and wherein the subject is determined to be at risk of developing cervical, oral, or anogenital cancer if any one of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 is determined.

[0097] In another preferred embodiment of the method, use, system, and kit of the present invention, the method further comprises determining whether the subject is at risk of developing a benign lesion, and if the subject is determined to have any one of human papillomavirus genotypes 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82, and 84, the subject is determined to be at risk of developing a benign lesion. The benign lesion is not cervical, oral, or anogenital cancer.

[0098] In a preferred embodiment of the system and kit of the present invention, - the means for subjecting DNA present in said biological sample to multiplex real-time PCR comprises a multiplex real-time PCR instrument and multiplex real-time PCR reagents; - the means for subjecting each of the first and second sequences amplified in the composition selected in (a) to hybridization on a solid support comprises a solid support having probes attached thereto, each probe having a forward primer and a reverse primer complementary to and specific for a marker locus, the forward primer and the reverse primer comprising sequences complementary to and specific for the marker sequence provided in the multiplex real-time PCR of (a), and a hybridization reagent; - the means for identifying each specific area of ​​said solid support to which a probe is attached and a sequence is hybridized comprises an image capture device.

[0099] 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, all of which are described above, while the multiplex real-time PCR reagents are those of a human papillomavirus master mix required for performing multiplex real-time PCR, more preferably a hot-start polymerase, uracil DNA glycosylase, a pair of forward and reverse primers, each complementary to and specific for a locus of a human papillomavirus genotypic marker or a human housekeeping gene, a fluorescent probe, a PCR reaction buffer, a deoxyuridine triphosphate (dUTP) solution, a deoxynucleotide triphosphate (dNTP) solution containing dATP, dCTP, dTTP, and dGTP, and molecularly tagged primers.

[0100] Preferably, the means for subjecting each of the first and second sequences amplified in the composition selected in (a) to hybridization on a solid support comprises a solid support as defined herein having attached thereto probes, each probe comprising a forward primer and a reverse primer complementary to and specific to the locus of the marker, as defined herein, and a sequence complementary to and specific to the sequence of the marker provided in the multiplex real-time PCR of (a), and hybridization reagents. More preferably, the reagents comprise a hybridization solution (reagent A), a blocking solution (reagent B), streptavidin-phosphatase (streptavidin-alkaline phosphatase, reagent C), wash buffer I (reagent D), an NBT-BCIP solution (reagent E), and wash buffer II (reagent F). Even more preferably, the means for subjecting each of the first and second sequences amplified in the composition selected in (a) to hybridization on a solid support also includes hybridization platforms such as the exemplified hybriSpot 12 or 24 platforms, thermocyclers, thermal blocks or constant temperature baths / heaters, and cold plates.

[0101] Preferably, the means for identifying each specific region of the solid support to which a probe is attached and a sequence is hybridized comprises an image capture device, preferably included in a hybridization platform as described above, such as the exemplified hybriSpot 12 or 24 platform.

[0102] The multiplex real-time PCR instrument, hybridization platform, and image capture device may each include a computer program product.

[0103] A computer program product may include a computer-readable storage medium (or multiple storage media) having computer-readable program instructions thereon for causing a processor to perform aspects or steps of the present invention.

[0104] A computer-readable storage medium may be a tangible device capable of retaining and storing instructions by an instruction execution device. Computer-readable storage media may include, but are not limited to, electronic, magnetic, optical, electromagnetic, or semiconductor storage devices, or any suitable combination of the foregoing. A non-exclusive list of more specific examples of computer-readable storage media includes portable computer diskettes, hard disks, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), static random access memory (SRAM), portable compact disc read-only memory (CD-ROM), digital versatile disc (DVD), memory sticks, floppy disks, mechanically encoded devices such as punch cards or raised structures having grooves on which instructions are recorded, and any suitable combination of the foregoing.

[0105] The computer-readable program instructions described herein can be downloaded from a computer-readable storage medium to each computing / processing device or to an external computer or external storage device via a network, such as the Internet, a local area network, a wide area network, and / or a wireless network. The network may include copper transmission cables, optical fiber transmissions, wireless transmissions, routers, firewalls, switches, gateway computers, and / or edge servers. A network adapter card or network interface in each computing / processing device receives the computer-readable program instructions from the network and forwards the computer-readable program instructions for storage in a computer-readable storage medium within the respective computing / processing device.

[0106] The computer-readable program instructions for carrying out the operations of the present invention may be either assembler instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, context data, or source or object code written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Smalltalk, C++, and conventional procedural programming languages ​​such as the "C" programming language or similar programming languages. The computer-readable program instructions may execute 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 scenario, the remote computer may connect to an external computer (e.g., over the Internet using an Internet Service Provider), which may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN). In some embodiments, electronic circuitry including, for example, a programmable logic circuit, a field programmable gate array (FPGA), or a programmable logic array (PLA) may execute computer-readable program instructions by utilizing state information of the computer-readable program instructions to personalize the electronic circuitry to perform aspects or steps of the present invention.

[0107] Aspects or steps of the present 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 present invention (see, for example, Figures 1-1 and 1-2 and 4). It will be understood that each square or diamond shaped block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by biological techniques or computer readable program instructions, or a combination thereof.

[0108] These computer-readable program instructions may be provided to a processor of a general-purpose computer, special-purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, executed by the processor of the computer or programmable data processing apparatus, create means for performing the function / acts specified in the block or blocks of the flowcharts and / or block diagrams. These computer-readable program instructions may also be stored in a computer-readable storage medium that can direct a computer, programmable data processing apparatus, and / or other apparatus to function in a particular manner, such that the computer-readable storage medium having instructions stored therein comprises an article of manufacture containing instructions that perform an aspect of the function / acts specified in the block or blocks of the flowcharts and / or block diagrams.

[0109] The computer-readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device, causing a series of operational steps to be performed on the computer, other programmable apparatus, or other device to produce a computer-executed process, such that the instructions, which execute on the computer, other programmable apparatus, or other device, perform the function / acts specified in a block or blocks of the flowcharts and / or block diagrams.

[0110] The flowcharts and block diagrams in the figures illustrate possible implementation structures, functionality, and operation of systems, methods, and kits according to various embodiments of the present invention. In this regard, each block in a flowchart or block diagram may represent a module, segment, or portion of instructions, including one or more executable instructions for performing a specified logical function. In some alternative implementations, the functions noted in the blocks may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may possibly be executed in the reverse order, depending on the functionality involved. It should also be noted that each block of the block diagrams and / or flowchart illustrations, and combinations of blocks in the block diagrams and / or flowchart illustrations, may be executed by special-purpose hardware-based systems that perform the specified functions or acts or implement a combination of special-purpose hardware and computer instructions.

[0111] Corresponding structure, materials, acts, and equivalents of all means or step-plus-function elements in the following claims are intended to include any structure, material, or acts for performing the function as specifically claimed in combination with other claimed elements. The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or limited to the embodiments of the present invention in the form disclosed. Numerous modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the embodiments of the present invention. The embodiments have been chosen and described to best explain the principles and practical application of the embodiments of the present invention and to enable others skilled in the art to understand the embodiments of the present invention in various embodiments with various modifications suited to the particular use contemplated.

[0112] The present invention also relates to the following items:

[0113] Item 1. A method for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of human papillomavirus infecting the subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in the subject, comprising: (a) subjecting at least one sequence contained in DNA present in a biological sample obtained from said subject to multiplex real-time PCR, wherein said DNA is extracted from said biological sample before subjecting said biological sample to multiplex real-time PCR, and said multiplex real-time PCR comprises: (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker for a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; of, (iii) a set of probes, - a probe comprising a first fluorophore and a sequence, said sequence being complementary to and specific for a sequence of a marker of 16 genotypes of human papillomavirus; - a probe comprising a second fluorophore and a sequence that is complementary to and specific for a sequence of a marker of 18 genotypes of human papillomavirus; and - a pool of probes, each probe of the pool of probes comprising a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus having a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes. and a set of probes comprising: (iv) a probe comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; amplifying in a composition comprising Each probe that binds to a sequence of the marker that is complementary to and specific thereto is detected; - repeating step (a) for said subject if the fourth fluorophore of the probe that binds to said second sequence is not detected and the first or second or third fluorophore of the probe that binds to the first sequence is not detected; - if the fourth fluorophore of the probe that binds to the second sequence is detected and at least one of the first or second fluorophore of the probe that binds to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected; - detecting a fourth fluorophore of the probe that binds to the second sequence and a third fluorophore of the probe of the pool of probes that binds to the first sequence, whereby a human papillomavirus genotype associated with the development of cervical, oral, 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 each second sequence of the compositions selected in step (a) to hybridization on a solid support comprising a probe attached thereto, wherein each probe comprises a forward primer and a reverse primer that are complementary to and specific for a marker locus and a sequence that is complementary to and specific for the marker sequence provided in the multiplex real-time PCR of step (a); each probe containing 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 containing the same sequence; (c) identifying specific regions of each of said solid supports to which probes are attached and to which the first sequence or the second sequence is hybridized, (I) in step (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the first fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the occurrence of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 16; (II) in step (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the second fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 18; (III) if in step (a) a fourth fluorophore of the probe that binds to the second sequence is detected and a third fluorophore of the probe that binds to the first sequence is detected, and in step (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified, the genotype is (IIIa) identifying specific regions of each of said solid supports to which probes are attached and to which first sequences are hybridized; (IIIb) determining which genotype of human papillomavirus is identified by a marker containing a sequence that is complementary and specific to the sequence of the probe attached to each specific region identified in step (IIIa); The steps and A method comprising:

[0114] Item 2 The multiplex real-time PCR is (i) at least one first sequence, each of which is a sequence of a marker derived from a human papillomavirus genotype present in a conserved region of an HPV L1 or HPV L2 gene; (ii) the second sequence; and The method according to item 1, wherein the

[0115] Item 3 The multiplex real-time PCR (i) at least one first sequence, each first sequence being a sequence of a marker derived from a human papillomavirus genotype selected from 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 genotypes of human papillomavirus; (ii) the second sequence; and 3. The method of any one of items 1 and 2, wherein the gene encoding the target gene is amplified.

[0116] Item 4 The multiplex real-time PCR is (i) - a first sequence which is a sequence of markers derived from 16 genotypes of human papillomavirus; - a first sequence, which is a sequence of markers derived from 18 genotypes of human papillomavirus; and - at least one other first sequence, each of which is a sequence of a marker derived from a genotype of human papillomavirus selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus; and (ii) the second sequence; and The method according to item 3, wherein the

[0117] Item 5: The DNA is extracted from the biological sample by lysis before being subjected to multiplex real-time PCR; - a first control is also subjected to multiplex real-time PCR as defined in step (a), when at least one sequence comprised in DNA present in said biological sample is subjected thereto, said first control being a blank subjected to said extraction, and optionally - when at least one sequence comprised in DNA present in said biological sample is subjected thereto, a second control is also subjected to multiplex real-time PCR as defined in step (a), said second control comprising DNA comprising a first sequence which is a sequence of a marker derived from a genotype of human papillomavirus selected from genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus, and optionally - the first and second sequences amplified in step (a) and the solid support further comprise molecules attached thereto, the molecules attached to the solid support being attached to specific regions of the solid support that are different from other specific regions of the solid support to which probes are attached, and the first and second sequences amplified in step (a) and the molecules attached to the solid support have the same chemical structure.

[0118] Item 6. The method of any one of Items 1 to 5, wherein if a probe that binds to the second sequence is not detected and a probe that binds to the first sequence is not detected, step (a) is repeated for the subject using a new sample obtained from the subject.

[0119] Item 7. The method further comprises detecting the absence of a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in the subject; 7. The method of any one of items 1 to 6, wherein if the fourth fluorophore of the probe that binds to the second sequence is detected and the first, second, or third fluorophore of the probe that binds to the first sequence is not detected, then the human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is not detected.

[0120] Item 8: A specific region of the solid support that is different from other specific regions of the solid support to which probes are attached has a pool of probes attached thereto, each probe of the probe pool having a sequence that is complementary to and specific for a sequence of a conserved region of an HPV L1 or HPV L2 gene, and at least two probes in the probe pool have sequences that are different from each other; - identifying specific regions of the solid support to which the pool of probes is attached and to which sequences hybridize; - identifying specific regions of the solid support to which probes are attached and to which second sequences are hybridized; - the specific region of the solid support to which the probe is attached and to which the first sequence hybridizes is not identified; 8. The method according to any one of items 1 to 7, wherein the genotype of the human papillomavirus is determined as each genotype identifiable by the markers provided in the multiplex real-time PCR of step (a) by using forward and reverse primers complementary to and specific for the loci of the markers.

[0121] Item 9. The method of any one of Items 1 to 8, wherein the method is further for determining whether the subject is at risk for developing cervical or anogenital cancer, and the subject is determined to be at risk for developing cervical or anogenital cancer if any one of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 is determined.

[0122] Item 10. The method of any one of Items 1 to 8, wherein the method is further for determining whether the subject is at risk for developing a benign lesion, and the subject is determined to be at risk for developing a benign lesion if any one of human papillomavirus genotypes 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82, and 84 is determined.

[0123] Item 11. The method of any one of items 1 to 10, wherein the composition selected in step (a) is subjected to heating to 60°C for at least 10 minutes before being subjected to cooling and hybridization according to step (b).

[0124] Item 12. The method of any one of Items 1 to 11, wherein the biological sample obtained from the subject is obtained by the subject obtaining the sample himself / herself.

[0125] Item 13. A system for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of human papillomavirus infecting the subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in the subject, comprising: (a) a means for subjecting at least one sequence contained in DNA present in a biological sample obtained from said subject to multiplex real-time PCR, said multiplex real-time PCR comprising: (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker for a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; of, (iii) a set of probes, - a probe comprising a first fluorophore and a sequence, said sequence being complementary to and specific for a sequence of a marker of 16 genotypes of human papillomavirus; - a probe comprising a second fluorophore and a sequence that is complementary to and specific for a sequence of a marker of 18 genotypes of human papillomavirus; and - a pool of probes, each probe of the pool of probes comprising a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus having a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes. and a set of probes comprising: (iv) a probe comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; amplifying in a composition comprising Each probe that binds to a sequence of the marker that is complementary to and specific thereto is detected; - repeating (a) for said subject if the fourth fluorophore of the probe that binds to said second sequence is not detected and the first or second or third fluorophore of the probe that binds to the first sequence is not detected; - if the fourth fluorophore of the probe that binds to the second sequence is detected and at least one of the first or second fluorophore of the probe that binds to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected; - detecting a fourth fluorophore of the probe that binds to the second sequence and a third fluorophore of the probe of the pool that binds to the first sequence, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected, and selecting a composition comprising the amplified first sequence and the second sequence; (b) means for subjecting each amplified first sequence and each second sequence of the compositions selected in (a) to hybridization on a solid support comprising probes attached thereto, wherein each probe comprises a forward primer and a reverse primer complementary to and specific for a marker locus, the forward primer and the reverse primer comprising sequences complementary to and specific for the marker sequences provided in the multiplex real-time PCR of (a); each probe containing 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 containing the same sequence; (c) means for identifying the specific regions of each of said solid supports to which probes are attached and to which the first sequence or the second sequence is hybridized, said means comprising: (I) In (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the first fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the occurrence of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 16; (II) In (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the second fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 18; (III) if in (a) a fourth fluorophore of the probe that binds to the second sequence is detected and a third fluorophore of the probe that binds to the first sequence is detected, and in (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified, the genotype is (IIIa) identifying specific regions of each of said solid supports to which probes are attached and to which first sequences are hybridized; (IIIb) determining which genotype of human papillomavirus is identified by a marker containing a sequence that is complementary and specific to the sequence of the probe attached to each specific region identified in (IIIa); Determined by means and Including, the system.

[0126] Item 14 The multiplex real-time PCR is (i) at least one first sequence, each of which is a sequence of a marker derived from a human papillomavirus genotype present in a conserved region of an HPV L1 or HPV L2 gene; (ii) the second sequence; and Item 14. The system according to item 13, wherein the signal is amplified.

[0127] Item 15 The multiplex real-time PCR is (i) at least one first sequence, each first sequence being a sequence of a marker derived from a human papillomavirus genotype selected from 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 genotypes of human papillomavirus; (ii) the second sequence; and 15. The system of any one of items 13 and 14, wherein the system amplifies

[0128] Item 16 The multiplex real-time PCR is (i) - a first sequence which is a sequence of markers derived from 16 genotypes of human papillomavirus; - a first sequence, which is a sequence of markers derived from 18 genotypes of human papillomavirus; and - at least one other first sequence, each of which is a sequence of a marker derived from a genotype of human papillomavirus selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus; and (ii) the second sequence; and Item 16. The system according to item 15, wherein the system amplifies

[0129] Item 17. The method of claim 17, wherein the DNA is extracted from the biological sample by lysis before subjecting it to multiplex real-time PCR. - a first control is also subjected to a multiplex real-time PCR as defined in (a) when at least one sequence contained in the DNA present in said biological sample is subjected thereto, said first control being a blank subjected to said extraction, and optionally - when at least one sequence comprised in DNA present in said biological sample is subjected thereto, a second control is also subjected to multiplex real-time PCR as defined in (a), said second control comprising DNA comprising a first sequence which is a sequence of a marker derived from a genotype of human papillomavirus selected from genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus, and optionally 17. The system of any one of items 13 to 16, wherein the first and second sequences amplified in (a) and the solid support further comprise molecules attached thereto, and 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, and the first and second sequences amplified in (a) and the molecules attached to the solid support have the same chemical structure.

[0130] Item 18. The system of any one of items 13 to 17, wherein if a probe that binds to the second sequence is not detected and a probe that binds to the first sequence is not detected, step (a) is repeated for the subject using a new sample obtained therefrom.

[0131] Item 19. The system further detects the absence of a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in the subject; 19. The system of any one of items 13 to 18, wherein if the fourth fluorophore of the probe that binds to the second sequence is detected and the first, second, or third fluorophore of the probe that binds to the first sequence is not detected, then the human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is not detected.

[0132] Item 20: A specific region of the solid support that is different from other specific regions of the solid support to which probes are attached has a pool of probes attached thereto, each probe of the probe pool having a sequence that is complementary to and specific for a sequence of a conserved region of an HPV L1 or HPV L2 gene, and at least two probes in the probe pool have sequences that are different from each other; - identifying specific regions of the solid support to which the pool of probes is attached and to which sequences hybridize; - identifying specific regions of the solid support to which probes are attached and to which second sequences are hybridized; - the specific region of the solid support to which the probe is attached and to which the first sequence hybridizes is not identified; 20. The system of any one of items 13 to 19, wherein the genotype of the human papillomavirus is determined as each genotype identifiable by the markers provided in the multiplex real-time PCR of (a), wherein forward primers and reverse primers complementary to and specific for the marker loci are used.

[0133] Item 21. The system of any one of Items 13 to 20, wherein the system is further configured to determine whether the subject is at risk for developing cervical or anogenital cancer, and the subject is determined to be at risk for developing cervical or anogenital cancer if any one of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 is determined.

[0134] Item 22 The system of any one of Items 13 to 20, wherein the system is further configured to determine whether the subject is at risk for developing a benign lesion, and the subject is determined to be at risk for developing a benign lesion if any one of human papillomavirus genotypes 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82, and 84 is determined.

[0135] Item 23. The system of any one of items 13 to 22, wherein the composition selected in (a) is subjected to heating to 60°C for at least 10 minutes before being subjected to cooling and hybridization according to (b).

[0136] Item 24. The system of any one of Items 13 to 23, wherein the biological sample obtained from the subject is obtained by the subject obtaining the sample themselves.

[0137] Item 25 - the means for subjecting DNA present in said biological sample to multiplex real-time PCR comprises a multiplex real-time PCR instrument and multiplex real-time PCR reagents; - the means for subjecting each of the first and second sequences amplified in the composition selected in (a) to hybridization on a solid support comprises a solid support having probes attached thereto, each probe having a forward primer and a reverse primer complementary to and specific for a marker locus, the forward primer and the reverse primer comprising sequences complementary to and specific for the marker sequences provided in the multiplex real-time PCR of (a), and a hybridization reagent; 25. The system according to any one of items 13 to 24, wherein the means for identifying each specific area of ​​the solid support to which the probe is attached and the sequence is hybridized comprises an image capture device.

[0138] Item 26. A kit for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of human papillomavirus infecting the subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in the subject, comprising: (a) a means for subjecting at least one sequence contained in DNA present in a biological sample obtained from said subject to multiplex real-time PCR, said multiplex real-time PCR comprising: (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker for a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; of, (iii) a set of probes, - a probe comprising a first fluorophore and a sequence, said sequence being complementary to and specific for a sequence of a marker of 16 genotypes of human papillomavirus; - a probe comprising a second fluorophore and a sequence that is complementary to and specific for a sequence of a marker of 18 genotypes of human papillomavirus; and - a pool of probes, each probe of the pool of probes comprising a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus having a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes. and a set of probes comprising: (iv) a probe comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; amplifying in a composition comprising Each probe that binds to a sequence of the marker that is complementary to and specific thereto is detected; - repeating (a) for said subject if the fourth fluorophore of the probe that binds to said second sequence is not detected and the first or second or third fluorophore of the probe that binds to the first sequence is not detected; - if the fourth fluorophore of the probe that binds to the second sequence is detected and at least one of the first or second fluorophore of the probe that binds to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected; - detecting a fourth fluorophore of the probe that binds to the second sequence and a third fluorophore of the probe of the pool that binds to the first sequence, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected, and selecting a composition comprising the amplified first sequence and the second sequence; (b) means for subjecting each amplified first sequence and each second sequence of the compositions selected in (a) to hybridization on a solid support comprising probes attached thereto, wherein each probe comprises a forward primer and a reverse primer complementary to and specific for a marker locus, the forward primer and the reverse primer comprising sequences complementary to and specific for the marker sequences provided in the multiplex real-time PCR of (a); each probe containing 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 containing the same sequence; (c) means for identifying the specific regions of each of said solid supports to which probes are attached and to which the first sequence or the second sequence is hybridized, said means comprising: (I) In (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the first fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the occurrence of cervical or anogenital cancer is determined to be human papillomavirus genotype 16; (II) In (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the second fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 18; (III) if in (a) a fourth fluorophore of the probe that binds to the second sequence is detected and a third fluorophore of the probe that binds to the first sequence is detected, and in (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified, the genotype is (IIIa) identifying specific regions of each of said solid supports to which probes are attached and to which first sequences are hybridized; (IIIb) determining which genotype of human papillomavirus is identified by a marker containing a sequence that is complementary and specific to the sequence of the probe attached to each specific region identified in (IIIa); Determined by means and Includes a kit.

[0139] Item 27 The multiplex real-time PCR is (i) at least one first sequence, each of which is a sequence of a marker derived from a human papillomavirus genotype present in a conserved region of an HPV L1 or HPV L2 gene; (ii) the second sequence; and 27. The kit according to Item 26, wherein the kit amplifies

[0140] Item 28 The multiplex real-time PCR is (i) at least one first sequence, each first sequence being a sequence of a marker derived from a human papillomavirus genotype selected from 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 genotypes of human papillomavirus; (ii) the second sequence; and 28. The kit of any one of items 26 and 27, wherein the kit amplifies

[0141] Item 29 The multiplex real-time PCR is (i) - a first sequence which is a sequence of markers derived from 16 genotypes of human papillomavirus; - a first sequence, which is a sequence of markers derived from 18 genotypes of human papillomavirus; and - at least one other first sequence, each of which is a sequence of a marker derived from a genotype of human papillomavirus selected from genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 of human papillomavirus; and (ii) the second sequence; and Item 29. The kit according to Item 28, wherein the

[0142] Item 30: The DNA is extracted from the biological sample by lysis before subjecting it to multiplex real-time PCR; - a first control is also subjected to a multiplex real-time PCR as defined in (a) when at least one sequence contained in the DNA present in said biological sample is subjected thereto, said first control being a blank subjected to said extraction, and optionally - when at least one sequence comprised in DNA present in said biological sample is subjected thereto, a second control is also subjected to multiplex real-time PCR as defined in (a), said second control comprising DNA comprising a first sequence which is a sequence of a marker derived from a genotype of human papillomavirus selected from genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 of human papillomavirus, and optionally 30. The kit of any one of items 26 to 29, wherein the first and second sequences amplified in (a) and the solid support further comprise molecules attached thereto, the molecules attached to the solid support being attached to specific regions of the solid support that are different from other specific regions of the solid support to which probes are attached, and the first and second sequences amplified in (a) and the molecules attached to the solid support have the same chemical structure.

[0143] Item 31. The kit of any one of Items 26 to 30, wherein if a probe that binds to the second sequence is not detected and a probe that binds to the first sequence is not detected, (a) is repeated for the subject using a new sample obtained therefrom.

[0144] Item 32. The kit further comprises detecting the absence of a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in the subject; 32. The kit of any one of items 26 to 31, wherein a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is not detected if the fourth fluorophore of the probe that binds to the second sequence is detected and the first, second, or third fluorophore of the probe that binds to the first sequence is not detected.

[0145] Item 33: A specific region of the solid support that is different from other specific regions of the solid support to which probes are attached has a pool of probes attached thereto, each probe of the probe pool having a sequence that is complementary to and specific for a sequence of a conserved region of an HPV L1 or HPV L2 gene, and at least two probes in the probe pool have sequences that are different from each other; - identifying specific regions of the solid support to which the pool of probes is attached and to which sequences hybridize; - identifying specific regions of the solid support to which probes are attached and to which second sequences are hybridized; - the specific region of the solid support to which the probe is attached and to which the first sequence hybridizes is not identified; 33. The kit of any one of items 26 to 32, wherein the genotype of the human papillomavirus is determined for each genotype identifiable by the markers provided in the multiplex real-time PCR of (a), wherein forward and reverse primers complementary to and specific for the marker loci are provided in (a).

[0146] Item 34. The kit of any one of Items 26 to 33, wherein the kit is further for determining whether the subject is at risk for developing cervical or anogenital cancer, and the subject is determined to be at risk for developing cervical or anogenital cancer if any one of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 is determined.

[0147] Item 35: The kit of any one of Items 26 to 33, wherein the kit is further for determining whether the subject is at risk for developing a benign lesion, and the subject is determined to be at risk for developing a benign lesion if any one of human papillomavirus genotypes 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82, and 84 is determined.

[0148] Item 36. The kit of any one of Items 26 to 35, wherein the composition selected in (a) is subjected to heating to 60° C. for at least 10 minutes before being subjected to cooling and hybridization according to (b).

[0149] Item 37. The kit of any one of Items 26 to 36, wherein the biological sample obtained from the subject is obtained by the subject obtaining the sample themselves.

[0150] Item 38 - the means for subjecting DNA present in the biological sample to multiplex real-time PCR comprises a multiplex PCR instrument and multiplex PCR reagents; - the means for subjecting each of the first and second sequences amplified in the composition selected in (a) to hybridization on a solid support comprises a solid support having probes attached thereto, each probe having a forward primer and a reverse primer complementary to and specific for a marker locus, the forward primer and the reverse primer comprising sequences complementary to and specific for the marker sequence provided in the multiplex PCR in (a), and a hybridization reagent; 38. The kit according to any one of items 26 to 37, wherein the means for identifying each specific area of ​​the solid support to which a probe is attached and a sequence is hybridized comprises an image capture device.

[0151] Item 39. The method of any one of items 1 to 18, the system of any one of items 13 to 25, or the kit of any one of items 26 to 38, wherein the forward and / or reverse primer comprises a molecular tag.

[0152] Item 40: Use of forward and / or reverse primers to obtain amplified sequences containing molecular tags by multiplex real-time PCR, The multiplex real-time PCR amplifies sequences contained in DNA present in a biological sample obtained from a subject, and the DNA is extracted from the biological sample before being subjected to the multiplex real-time PCR; The forward and / or reverse primers are used to identify the specific region on the solid support to which the probe is attached. - a sequence complementary to and specific for one or more loci of markers of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68; and - Molecular tags and wherein the probe comprises a sequence that is complementary to and specific for the sequence of a marker to which the forward and / or reverse primer is complementary to and specific and to which the amplified sequence comprising a molecular tag is hybridized.

[0153] Item 41. Use of forward and / or reverse primers, Use of the method according to any one of items 1 to 18 or 39, the system according to any one of items 13 to 25 or 39, or the kit according to any one of items 26 to 39 for obtaining amplified sequences comprising molecular tags by multiplex real-time PCR, as defined in step (a); and 40. Use 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, for identifying a specific region on a solid support to which a probe is attached, as defined in step (b) and identified as defined in step (c), wherein the probe comprises a sequence that is complementary to and specific for the sequence of a marker to which the forward and / or reverse primer is complementary to and specific and to which the amplified sequence comprising a molecular tag is hybridized.

[0154] Item 42. Use of an amplified sequence comprising a molecular tag, wherein the amplified sequence is obtained by 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, the DNA is extracted from the biological sample before being subjected to multiplex real-time PCR, and amplification is performed using forward and / or reverse primers, wherein the forward and / or reverse primers are used to identify specific regions on a solid support to which probes are attached. - a sequence complementary to and specific for one or more loci of markers of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68; and - Molecular tags and wherein the probe comprises a sequence that is complementary to and specific for the sequence of a marker to which the forward and / or reverse primer is complementary to and specific and to which the amplified sequence comprising a molecular tag is hybridized.

[0155] Item 43. Use of an amplified first and / or second sequence, wherein the amplified first and / or second sequence is obtained by 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; 39。 Use 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, wherein the amplified first and / or second sequences comprise molecular tags and are hybridized to a solid support as defined in step (b) 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, for identifying a specific region on the solid support to which a probe is attached, as defined in step (c) 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.

[0156] Item 44. 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, or the use of any one of items 40 to 43, wherein the molecular tag is biotin.

[0157] Item 45. The method of any one of items 1 to 18, 39, or 44, the system of any one of items 13 to 25, 39, or 44, the kit of any one of items 26 to 39, or 44, or the use of any one of items 40 to 44, wherein the molecular tag biotin is identified by streptavidin containing a reporter.

[0158] Item 46. The method of any one of items 1 to 18, 39, 44, or 45, the system of any one of items 13 to 25, 39, 44, or 45, the kit of any one of items 26 to 39, 44, or 45, or the use of any one of items 40 to 45, wherein the reporter is horseradish peroxidase or alkaline phosphatase.

[0159] Item 47. The method of any one of items 1 to 18, 39, 44, 45, 46, the system of any one of items 13 to 25, 39, 44, 45, 46, the kit of any one of items 26 to 39, 44, 45, 46, or the use of any one of items 40 to 46, wherein the sample is obtained by a sample collection device.

[0160] Item 48. The method of any one of items 1 to 18, 39, 44, 45, 46, 47, the system of any one of items 13 to 25, 39, 44, 45, 46, 47, the kit of any one of items 26 to 39, 44, 45, 46, 47, or the use of any one of items 40 to 47, wherein the sample collection device is a swab, brush, or bale device.

[0161] Item 49. The system of any one of items 13 to 25, 39, 44, 45, 46, 47, or 48, or the kit of any one of items 26 to 39, 44, 45, 46, 47, or 48, wherein the system and / or kit comprises a means for extracting human papillomavirus DNA present in a biological sample obtained from the subject in step (aa).

[0162] Item 50. The system of any one of items 13 to 25, 39, 44, 45, 46, 47, 48, or 49, or the kit of any one of items 26 to 39, 44, 45, 46, 47, 48, or 49, wherein step (aa) is before step (a). [Example]

[0163] The following examples are illustrative of the present invention and should not be considered limiting, but rather exemplary of the present invention.

[0164] The following example is based on the amplification of a fragment in the viral region L1 of papillomavirus by PCR, followed by hybridization onto a membrane with a DNA-specific probe, using techniques suitable for analysis on both automated and manual hybriSpot platforms. [Example]

[0165] Multiplex real-time PCR of samples obtained from subjects infected with human papillomavirus. Example 1 is a specific embodiment of (a) the methods, uses, systems, and kits of the present invention.

[0166] reagent: Samples were obtained from 24 subjects infected with human papillomavirus (HPV) by use of swabs of the anogenital area (self-swabs).

[0167] DNase / RNase-free double-distilled water was used for handling clinical samples.

[0168] A lyophilized multiplex real-time PCR mix was prepared for analysis of 24 clinical samples on the hybriSpot 24 platform. The lyophilized multiplex real-time PCR mix contained PCR buffer, dNTPs (U / T), DNase / RNase-free water, biotin-labeled primers, DNA polymerase and uracil-DNA glycosylase (Cod-UNG), and a fluorescent probe. The biotin-labeled primers included a pair of primers specific for amplifying a fragment of the HPV L1 region, which detects at least 35 HPV genotypes. Additionally, the remaining biotin-labeled primers included a pair of primers for amplifying a human housekeeping gene (beta-globin gene) to serve as an internal control for the multiplex real-time PCR. The fluorescent probes used were ROX (for detection of HPV16), Cy5 (for detection of HPV18), FAM (for detection of HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 = HPV HR genotypes), and JOE (for use as an internal control).

[0169] Sequences of markers derived from human papillomavirus genotypes selected from 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus were used as positive controls (PCs), and DNase / RNase-free DEPC-treated water was used as a negative control (NTC).

[0170] The reagents were transported and stored at 2-8°C in the dark in an area that was not contaminated with DNA or PCR products.

[0171] method: Purified genetic material from various types of biological samples, such as liquid-based cytology and vaginal and rectal swabs, is used. DNA / RNA purification kits and extraction equipment *DNA was extracted using the Opentrons, starting with 200 μl of biological sample and eluting in 100 μl of elution buffer (for purification using the Opentrons, it was started with 92 μl of clinical specimen and eluted in 60 μl of elution solution).

[0172] [Table 6]

[0173] Alternatively, DNA was extracted using the RNA / DNA Pathogen Extraction Kit (for Robot Nextractor® NX-48S), reference number MAD-003955M-EX.

[0174] 8 μL of eluted DNA was added to 12 μL of PCR master mix. Thus, PCR reactions were carried out in each tube containing multiplex real-time PCR reaction mix in a final volume of 20 μL to obtain template DNA for the PCR reaction. A negative control was included by adding 8 μL of water to 12 μL of PCR master mix. Additionally, a positive control was included by adding 8 μL of positive DNA control HPV screening to 12 μL of PCR master mix.

[0175] Each tube was placed in a thermocycler and the following steps of PCR amplification shown in Table 6 were used.

[0176] [Table 7]

[0177] Table 7 below was used to interpret the fluorescence data obtained for the positive and negative controls (PC and NTC, respectively), and detection of a signal (amplified signal) was determined by a rapid and steady increase in fluorescence data values, rather than by a peak phenomenon or a gradual increase in background signal (irregular background or increased background noise) (Figures 2A and 2B).

[0178] [Table 8]

[0179] PCR runs were considered valid if the number of detections (Ct) values ​​obtained for all reaction controls according to the aforementioned criteria and for positive controls for the various targets were within the expected range of 20 ± 2 for HPV16 (ROX), 20 ± 2 for HPV18 (Cy5), 20 ± 2 for HPV HR (FAM), and 20 ± 2 for beta-globin (JOE).

[0180] The results obtained for each of the clinical samples were interpreted according to Table 8 below.

[0181] [Table 9]

[0182] A default threshold line was established automatically by the VitroCycler (Vitro SA) instrument or, if necessary, adjusted manually until it was within the exponential phase of the fluorescence curve and above any background signal.

[0183] Samples were considered positive if the resulting Ct value was ≤40, even if the internal control showed no amplification (in some cases, the internal control was not amplified correctly due to the presence of high initial copy numbers of the target bacterial nucleic acid, which may cause preferential amplification of the latter).

[0184] A sample was considered negative if no amplification above the threshold was detected as indicated by the internal control. Inhibition of the PCR reaction can be excluded by amplification of the internal control.

[0185] The method of the present invention has been established to have a detection limit of 10 copies / reaction for HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68 genotypes. [Example]

[0186] Hybridization of amplicons from Example 1. Example 2 is a specific embodiment (c) of the methods, uses, systems and kits of the present invention.

[0187] reagent: Biotin-labeled amplicons generated during PCR from Example 1 were used for hybridization.

[0188] The following reagents were prepared for the analysis of 24 clinical samples obtained from human papillomavirus (HPV)-infected subjects on the hybriSpot 24 platform: 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). Reagent A was preheated to 41°C in a thermostatic bath or heater immediately before use, while the remaining reagents B through F were used at room temperature (20–25°C).

[0189] The solid supports used were hybriSpot chips (membranes) containing probes attached to their specific regions. Each chip contained four specific region subsets, where: - each specific region of a subset of said specific regions comprises a probe, each probe having a sequence selected from SEQ ID NO: 20 to SEQ ID NO: 54, which is complementary to and specific for a sequence of a marker of human papillomavirus, and each probe having the same sequence is attached to a specific region of said solid support; - each specific region of one subset of said specific regions comprises a probe having the sequence of SEQ ID NO: 55, which is complementary to and specific for the sequence of a marker of the human HBB gene (endogenous amplification control, C), and each probe comprising the same sequence is attached to a specific region of said solid support; - Biotin protein serves as a hybridization control, - each specific region of one subset of said specific regions comprised a degenerate universal probe having a sequence selected from SEQ ID NO: 56;

[0190] The reagents were transported and stored at 2-8°C in an area that was not contaminated with DNA or PCR products.

[0191] method: The biotin-labeled amplicons generated during the PCR of Example 1 were hybridized onto a membrane containing an array of specific probes for each HPV genotype associated with the development of cervical, oral, and / or anogenital cancer, as well as amplification and hybridization control probes. The hybridization process was performed semi-automatically on a hybriSpot (HS12) platform, following instructions provided by the platform's wizard. Specifically, reagents were added manually, and incubation time, temperature, and reagent discharge were controlled by firmware. However, hybridization was also fully automated in parallel on a hybriSpot HS12a or HS24 platform, achieving the same results (not shown here).

[0192] This technique allowed for much faster binding of PCR products to their specific probes in the three-dimensional porous environment of the membrane compared to conventional surface hybridization. After binding between a specific amplicon and its corresponding probe occurred, the signal was visualized by an immunoenzymatic colorimetric reaction using streptavidin-phosphatase and a chromogen (NBT-BCIP), which produced an insoluble precipitate in the membrane at the specific region where hybridization occurred.

[0193] Sample management, image capture, and analysis and reporting of results were automated by hybriSoft™ software.

[0194] Before starting the hybridization process, the following steps were performed: (i) denaturing the biotinylated amplicons generated during the PCR of Example 1 by heating to 95°C for 10 minutes in a thermocycler and quickly cooling in ice for at least 2 minutes; (ii) preheating Reagent A to 41°C; (iii) placing each HPV chip (one for each valid clinical sample that tested positive for HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68 in Example 1) into the indicated position on the platform (HS12).

[0195] Notably, the PCR products added to the array contained remnants of Taqman probes from the real-time PCR that had not been consumed during amplification, resulting in two complementary probes in the hybridization device, one in the liquid phase and the other in the solid phase, which could compete to hybridize with the amplicon of interest. To prevent this from happening and to ensure that the generated amplicons fully hybridize with the solid-phase probes, i.e., those in the array, we performed a first hybridization step using temperature switching. The temperature was initially set to 60°C. This separated the remnants of the Taqman probes from the amplicons in the liquid phase, preventing their hybridization and improving the sensitivity of the technique. This occurs because the temperature is higher than the melting temperatures of all probes, both arrayed and real-time. If this initial temperature increase did not occur, some of the Taqman probe fragments could hybridize with the amplicon of interest and prevent it from binding to the chip probe.

[0196] After all samples were added to the array, they were incubated at 60° C. for 1 minute, and then the temperature was reduced to 41° C. Specifically, the samples were incubated at 41° C. for an additional 6 minutes.

[0197] By setting the temperature at 41° C., the PCR products (amplicons, amplified DNA) bind to the array probes according to the melting temperatures of these probes.

[0198] After this incubation at 41°C for 6 minutes, the hybridization protocol continues as described below.

[0199] The manual hybridization protocol involves the following steps: (a) setting the temperature of the instrument to 60°C, adding 300 μL of Reagent A (hybridization solution) preheated to 41°C to each chip, and incubating until the temperature reaches 60°C; (b) removing Reagent A (hybridization solution) by activating a vacuum pump after the temperature reaches 60°C; (c) mixing 20 μL of each PCR sample (previously denatured and stored on ice) with 270 μL of Reagent A (hybridization solution at 41° C.) and dispensing the mixture onto the corresponding HVP chip; (d) incubating at 60°C for 1 minute; (e) setting the temperature of the instrument to 41°C and incubating for 6 minutes after reaching that temperature; (f) running 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); (g) washing three times with 300 μL of Reagent A (hybridization solution at 41° C.); (h) setting the temperature to 29°C; (i) adding 300 μL of Reagent B (Blocking Solution) and incubating for 5 minutes; (j) activating a pump to remove Reagent B; (k) adding 300 μL of Reagent C (streptavidin-alkaline phosphatase) to each chip after the temperature reaches 29° C.; (l) incubating for 5 minutes at 29°C; (m) activating a pump to remove the reagent; (n) setting the temperature to 36°C; (o) washing the membrane four times with 300 μL of Reagent D (Wash Buffer I); (p) after the temperature reaches 36°C, adding 300 μL of Reagent E to each chip and incubating for 10 minutes at 36°C; (q) activating the pump to remove reagent E containing the substrate and chromogen to perform the enzymatic colorimetric reaction; (r) washing the membrane twice with 300 μL of Reagent F (Wash Buffer II); (s) activating the pump to remove reagent F (a buffer solution with a very low salt concentration) and stop the enzymatic colorimetric reaction; (t) performing image capture, analysis, and reporting of results according to the instructions in the HS12 user manual; [Example]

[0200] Determining the 56 genotype of the human papillomavirus infecting the subject. Example 3 is a specific embodiment of (c) of the methods, uses, systems, and kits of the present invention. This was performed on one of 24 samples obtained from subjects that tested positive for HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68 after being subjected to multiplex real-time PCR according to Example 1, as per the third column of Table 8. Subsequent hybridization on a solid support according to Example 2 resulted in the visualization of hybridization as occurring in the specific regions indicated by: (i) specific regions 1A, 1B, 2I, 5E, and 8A in FIG. 3B, which correspond to the specific regions to which hybridization control probes (B) are attached according to the schematic diagram in FIG. 3C (specific regions are identified by numbers and alphabetical letters, respectively, counting from specific region 1A in the upper left corner of the solid support); (ii) specific regions 1C and 5F in Figure 3B, which correspond to the specific regions to which endogenous amplification control probes (C) were attached according to the schematic diagram in Figure 3C; and (iii) Specific regions 1D and 5G in Figure 3B, which correspond to the specific regions to which the HPV degenerate universal probe (U) was attached according to the schematic diagram in Figure 3C. , thereby indicating that hybridization was effective. Furthermore, hybridization was visualized as occurring in specific regions indicated by: (iv) Specific regions 2H and 7C in Figure 3B , corresponding to the specific regions to which the HPV56 genotype probe was attached and the degenerate HPV universal probe (U) was attached according to the schematic diagram in Figure 3C .

[0201] Thus, the subject from whom the sample was obtained was determined to be infected with an HPV56 genotype that is associated with the development of cervical, oral, or anogenital cancer in the subject. [Example]

[0202] Comparison of various hybridization conditions. The same group of samples (liquid cytology samples with previous positive results for HR HPV genotypes by real-time PCR) was assayed using a variety of assay conditions, and the results are shown in Figures 5-1 and 5-2.

[0203] Twelve clinical samples were used that had previously had positive results for HPV by hybridization-free real-time PCR screening. The results of the previous hybridization-free real-time PCR are shown in Figures 5-1 and 5-2.

[0204] Extraction of genetic material from the samples was performed using an RNA / DNA Pathogen Extraction Assay (VITRO SA, Granada, Spain), resulting in a total sample volume of 60 μl. Three hybridization conditions were tested using these samples.

[0205] 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 required for multiplex real-time PCR using the Vitro HPV Screening Assay. The real-time PCR products were denatured by heating at 95°C for 10 minutes and hybridized onto a membrane with HPV-specific probes at 41°C using DNAFlow technology on the hybriSpot platform.

[0206] Hybridization condition 2: Amplification of the sample by end-point PCR without a fluorescent probe, followed by hybridization of the PCR product at a temperature of 41°C. This is considered the gold standard method. 30 μl of each sample was subjected to PCR amplification using a mix that did not contain any fluorescent probe (lyophilized PCR mix). Under these conditions, there should be no interference with the probe on the solid surface. The real-time PCR product was denatured by heating at 95°C for 10 minutes and hybridized to a membrane with an HPV-specific probe at a temperature of 41°C using DNAFlow technology on the hybriSpot platform.

[0207] Hybridization condition 3: Real-time PCR according to the method of the present invention, followed by hybridization of the PCR products at a temperature of 60°C, followed by cooling to 41°C. 8 μl of each sample was mixed with 12 μl of the master mix required for multiplex real-time PCR using the Vitro HPV Screening Assay. The real-time PCR products 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, using a temperature gradient starting at 60°C and ending at 41°C. The products were kept at 60°C for 1 minute, after which the temperature began to gradually decrease to 41°C over 4 minutes, and finally, the products were kept at 41°C for 6 minutes.

[0208] For all conditions, the entire hybridization process was performed automatically, with sample management, image capture, and analysis and reporting of results performed through hybriSoft™ software. The same reagent lot was used for all conditions.

[0209] When real-time PCR is followed by hybridization of the PCR products at a temperature of 41°C (not according to the present invention), some genotypes are not identified (see hybridization condition 1 in Figures 5-1 and 5-2): genotype 18 is not identified in sample M1, genotype 16 is not identified in sample M2, genotypes 18, 53, and 67 are not identified in sample M4, genotypes 16 and 58 are not identified in sample M5, genotype 16 is not identified in sample M6, genotype 16 is not identified in sample M7, genotype 16 is not identified in sample M8, genotype 18 is not identified in sample M9, and genotype 18 is not identified in sample M11. According to the method of the present invention, all these genotypes are correctly identified when real-time PCR is followed by hybridization of the PCR products at a temperature of 60°C and cooling to 41°C (see hybridization condition 3 in Figures 5-1 and 5-2), and in the gold standard method (see hybridization condition 2 in Figures 5-1 and 5-2).

[0210] Because the PCR products added to the array contained probes remaining from the real-time PCR that were not consumed during amplification, two complementary probes were present in the hybridization device, one in the liquid phase and the other in the solid phase, and both may compete to hybridize with the target amplicon. This is prevented by the hybridization of the PCR products at a temperature of 60°C, which promotes the generated amplicon to preferentially hybridize to the solid-phase probe, i.e., the one in the array, and not to the probe remaining from the PCR in the liquid phase, improving the sensitivity and specificity of the method. This occurs because the temperature is higher than the melting temperature of all probes, both in the array and in the real-time PCR. If the heating temperature is lower than 60°C, some of the probes remaining from the PCR may hybridize to the target amplicon and prevent it from binding to the chip probe.

[0211] This example demonstrates that the method of the present invention correctly detects and identifies genotypes present in a sample when real-time PCR is followed by hybridization of the PCR products at a temperature of 60° C., followed by cooling to 41° C. When real-time PCR is followed by hybridization of the PCR products at a temperature of 41° C., some genotypes are not identified.

Claims

1. 1. A method for detecting a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in a subject, and for determining one or more genotypes of human papillomavirus infecting said subject when a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected in said subject, comprising: (a) subjecting at least one sequence contained in DNA present in a biological sample obtained from the subject to multiplex real-time PCR, wherein the DNA is extracted from the biological sample before being subjected to multiplex real-time PCR, and the multiplex real-time PCR comprises: (i) at least one first sequence contained in the DNA, each first sequence being a sequence of a marker of a human papillomavirus genotype, and each sequence being amplified using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; (ii) a second sequence contained in the DNA, the second sequence being a sequence of a marker for a human housekeeping gene, and the amplification of the second sequence is carried out using a forward primer and a reverse primer that are complementary to and specific for the locus of the marker, respectively; of, (iii) a set of probes, a probe comprising a first fluorophore and a sequence, said sequence being complementary to and specific for the sequence of a marker of 16 genotypes of human papillomavirus, - a probe comprising a second fluorophore and a sequence that is complementary to and specific for the sequence of a marker of 18 genotypes of human papillomavirus, and a pool of probes, each probe of which comprises a third fluorophore and a sequence that is complementary to and specific for a sequence of a marker of a genotype selected from the 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 genotypes of human papillomavirus, each of which has a marker comprising a sequence that is complementary to and specific for the sequence of at least one of the probes of the pool of probes; and a set of probes comprising: (iv) a probe comprising a fourth fluorophore and a sequence that is complementary to and specific for a sequence of a marker of the human housekeeping gene; amplifying in a composition comprising Each probe that binds to a sequence of the marker that is complementary to and specific thereto is detected; - repeating step (a) for said subject if the fourth fluorophore of the probe that binds to said second sequence is not detected and the first or second or third fluorophore of the probe that binds to the first sequence is not detected; - if the fourth fluorophore of the probe that binds to the second sequence is detected and at least one of the first or second fluorophore of the probe that binds to the first sequence is detected, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected; - detecting a fourth fluorophore of the probe that binds to the second sequence and a third fluorophore of the probe of the pool of probes that binds to the first sequence, a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is detected, and selecting a composition comprising the amplified first sequence and the second sequence; (b) subjecting each amplified first sequence and each second sequence of the compositions selected in step (a) to hybridization on a solid support comprising probes attached thereto, wherein during hybridization, the compositions selected in step (a) are subjected to heating to at least 60°C followed by cooling, and each probe comprises a sequence complementary to and specific for the marker locus, and a forward primer and a reverse primer complementary to and specific for the marker locus provided in the multiplex real-time PCR of step (a); each probe containing 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 containing the same sequence; (c) identifying specific regions of each of said solid supports to which probes are attached and to which the first sequence or the second sequence is hybridized, (I) in step (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the first fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the occurrence of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 16; (II) in step (a), if the fourth fluorophore of the probe that binds to the second sequence is detected and the second fluorophore of the probe that binds to the first sequence is detected, the detected human papillomavirus genotype associated with the occurrence of cervical, oral, or anogenital cancer is determined to be human papillomavirus genotype 18; (III) if in step (a) the fourth fluorophore of the probe that binds to the second sequence is detected and the third fluorophore of the probe that binds to the first sequence is detected, and in step (c) at least one specific region of the solid support to which a probe is attached and to which the first sequence is hybridized is identified, the genotype is (IIIa) identifying specific regions of each of said solid supports to which probes are attached and to which first sequences are hybridized; (IIIb) determining which genotype of human papillomavirus is identified by a marker comprising a sequence complementary and specific to the sequence of the probe attached to each specific region identified in step (IIIa); The steps and A method comprising:

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

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

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

5. The multiplex real-time PCR (i) at least one first sequence, each first sequence being a sequence of a marker derived from a human papillomavirus genotype selected from the group consisting of 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 genotypes of human papillomavirus; (ii) the second sequence; and The method of any one of claims 1 and 4, the system of any one of claims 2 and 4, or the kit of claim 3 or 4, which amplifies

6. the method is further for detecting the absence of said human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer in said subject; 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 a human papillomavirus genotype associated with the development of cervical, oral, or anogenital cancer is not detected if the fourth fluorophore of the probe that binds to the second sequence is detected and the first, second, or third fluorophore of the probe that binds to the first sequence is not detected.

7. 10. 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 further for determining whether the subject is at risk of developing cervical, oral, or anogenital cancer, and wherein the subject is determined to be at risk of developing cervical, oral, or anogenital cancer if any one of human papillomavirus genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 is determined.

8. The method, system, or kit of any one of claims 1 or 4 to 7, the system of claims 2 or 4 to 7, or the kit of any one of claims 3 to 7, further for determining 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 if any of human papillomavirus genotypes 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 61, 62, 67, 69, 70, 71, 72, 73, 81, 82, and 84 is determined.

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 at 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 at 60 to 65°C.

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

12. 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. 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 at least 10 minutes.

14. 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 comprises cooling to 41°C.

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

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

17. 17. The method of any one of claims 1, 4 to 16, the system of any one of claims 2, 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. 20. The method, system, or kit of claim 17, wherein the sample collection device is a swab, brush, or bail device.

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

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

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