MX2008001751A - In vitro diagnostic kit for identification of human papillomavirus in clinical samples - Google Patents

Abstract

A method and kit for detection and typing of HPV in a sample are described, as is a reaction vessel for use in the method. Universal HPV primers are used to amplify a sample by PCR;the amplified sample is then hybridised to an array of HPV type-specific probes to determine the HPV type.

Description

IN VITRO DIAGNOSTIC EQUIPMENT FOR IDENTIFICATION OF HUMAN PAPILLOMA VIRUSES IN CLINICAL SAMPLES Field of the Invention The present invention relates to an in vitro diagnostic equipment and a method for the identification of Human Papilloma Virus (HPV) in clinical samples. The present invention also relates to an apparatus for use in the equipment and method. More specifically, in preferred embodiments the present invention relates to an in vitro diagnostic equipment for the specific detection of human papilloma virus genotypes in chemical samples using probes for genotyping HPV, a platform in which acid formation is combined nucleic that includes the probes and a standard laboratory reaction bottle, an apparatus for automatic processing of the results and a method of diagnosis of infection, using the in vitro diagnostic equipment. BACKGROUND OF THE INVENTION To date, about 100 types of Human Papilloma Virus (HPV) have been described. An HPV type is considered a new type when at least 10% of the genetic sequences in the HPV regions E6, E7 and L1 differ from any previously known type. The subtypes, or variants, differ from the primary type in less than 2 to 5%. These viruses have tropism for human epithelium and have been linked to severe human diseases, especially carcinomas of the genital and oral mucosa. Approximately 50 HPV types have been isolated from the anogenital mucosa. They have been divided into low risk types (for example, HPV types 6, 11, 42, 43 and 44) and high risk types (for example types 16, 18, 31m 33 and 45) depending on their association with cervical cancer. The detection and identification of HPV types is very important since persistent infection with high-risk HPV types is the main etiologic factor for cervical cancer. The detection and identification of HPV genotypes is carried out through the elaboration of HPV DNA tests. These methods can be carried out by direct detection of HPV DNA or by detection of amplified HPV DNA. Among the HPV DNA direct detection methods is the Hybrid Capture (HC) method from Corp., Gaithersburg, Md., USA and in situ hybridization techniques. The HC is an FDA approved technique that is based on a method of signal amplification hybridization. The hybridization probes that are used are HPV specific RNA sequences. After incubation of these probes with denatured HPV DNA from the clinical sample, RNA / DNA hybrids are formed that can be detected using an antibody specific. The HC method allows the differentiation between HPV types of high and low risk, although they can not identify the HPV type. A further disadvantage of this test method is that the use of the probe cocktail often results in cross-reactions between HPV types of the two classes. Methods of identification of the HPV type by amplification of the viral genome can be carried out mainly by polymerase chain reaction (PCR). HPV genotyping can be carried out by type-specific PCR using primers that recognize only one specific type. An alternative method is the use of universal-primer PCR for the amplification of all types of HPV. Papilloma viruses are typed by subsequent analysis of the sequence of the amplified gene fragment. The analysis of this sequence can be carried out through different methods, such as DNA sequencing, restriction fragment length polymorphism (RFLP) or nucleic acid hybridization. Hybridization techniques such as reverse stain hybridization have been considered as the most suitable for diagnostic purposes (Kleter et al J Clin Microbiol 1999, 37: 2508-2517; Van den Brule and associates J Clin Microbiol. 40: 779-787). Recently, the technology of microformation (see for example U.S. Patent Number 5,445,934). The term microformation is meant to indicate the analysis of many small spots to facilitate large-scale nucleic acid analysis allowing the simultaneous analysis of hundreds of DNA sequences. As is known in the art, reverse staining is usually carried out on membranes, while microformation is normally carried out on a solid support and can also be carried out on smaller scales. Microform technology has been successfully applied to the field of HPV diagnostics (patent publications WO0168915 and No. CA2484681). However, there is still a drawback with the use of microformation technology, in that they require expensive equipment and laborious management. This inconvenience is addressed by the patent application No. US2005064469, where a training "tube" is provided). The term "forming tube" describes a reaction container having a typical shape and size of a laboratory reaction container (e.g., a 1.5 ml Eppendorf tube) with a microformation arranged at its base where it can be carried Microformation-based tests. Brief Description of the Invention In light of the foregoing, it is an object of the present invention to provide a reliable method for identification specific HPV types possibly present in a clinical sample. It is more particularly an object of the present invention to provide a method of specific identification of HPV types using the "tube of formation" platform. It is also an object of the present invention to provide probes for detection and / or specific identification of different types of HPV. It is still a further object of the present invention to provide an HPV type detection and / or identification equipment comprising HPV-specific reagents, protocols and zones fitted in a "formation tube" enabling reliable specific detection and / or identification. of HPV types possibly present in a clinical sample. OBJECTIVES OF THE INVENTION In accordance with a first aspect of the present invention, there is provided an assay for detecting and typing human papilloma virus (HPV) in a sample, wherein the assay comprises: carrying out a nucleic acid amplification reaction in a sample the amplification reaction being projected to amplify an HPV target sequence in a non-specific form of the type; obtain single-stranded oligonucleotides from any amplification products; allow the single-stranded oligonucleotides to hybridize when possible with the plurality of specific probes of the HPV type provided on a solid support, the support being located within a suitable reaction container to contain the sample; and detect flanked h i oligonucleotides. The aspects of the present invention also provide an assay for detecting and typing human papilloma virus (HPV) in a sample, wherein the assay comprises: carrying out a nucleic acid amplification reaction in a sample, the sample being in contact with a solid support having a plurality of specific probes of the HPV type immobilized therein, the amplification reaction being projected to amplify an HPV target sequence in a non-specific form of the type; obtain single-stranded oligonucleotides from any amplification products; allow single-stranded oligonucleotides to hybridize when possible with HPV-type specific probes; and detect hybridized oligonucleotides. The amplification reaction is preferably PCR. Single-stranded oligonucleotides can be obtained by denaturing any double-stranded oligonucleotides present, for example by heating. The single-stranded oligonucleotides are preferably allowed to hybridize under stringent conditions; said conditions will be understood by those skilled in the art, although preferably they include denaturing oligonucleotides incubated at a temperature of 55 ° C with the target, in a regulator comprising 1 x SSC. In preferred embodiments, the sample and the solid support are contained within a reaction container; for example, the one described in patent US2005064469. Preferably, specific probes are used for at least 5, 10, 15, 20, 25, 30, 35, 40 or 42 types of HPV, which are preferably selected from HPV types 6, 11, 16, 18, 26, 30 , 31, 32, 33, 34/64, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 62, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85 and 89. The probes are conveniently 20 to 40 nt in length, preferably 25 to 35 nt, more preferably 28 to 32 nt and most preferably about 30 nt. Not all probes need to have the same length. The probes are conveniently specific for the L1 region of HPV. Each probe specific for the type may differ from specific probes of another HPV type in at least 1, 2, 3 or preferably more than 3 nt. Preferred probes are selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 133; several of these probes detect the same HPV type as the one described below. Preferably, a plurality of probes are specific for the same HPV type, and more preferably at least two specific probes are used for each HPV type which will be detected. The mixtures of these probes can be immobilized in the same place in the solid support, or each probe specific for the type can be immobilized in a different location. Each specific probe of the same HPV type, preferably detects a different part of the target HPV sequence. The probes can be duplicated on the solid support, to provide multiple detection locations for redundancy. One or more control sequences can also be detected; for example, an immobilized probe for the solid support that does not hybridize to the target sequence from any HPV type. A probe can be a human genomic target sequence; the assay may subsequently comprise amplification of the human target sequence from the sample and detect whether amplification has occurred. An additional control can be introduced using non-specific tagged sequences immobilized for the solid support; Label detection can ensure that the label is working properly. Still further control can be provided, including a control amplification sequence that can be amplified through the same primers as the human target, but which will be detected by a different oligonucleotide on the solid support. This control ensures that the amplification is working correctly. The present invention also provides a reaction package that includes a solid support having a plurality of HPV type specific probes immobilized therein. HPV detection and typing equipment comprising said reaction package is also provided in combination with a nucleic acid amplification mixture. The mixture may comprise HPV consensus primers such as MY09 and MY11; and optionally HMB01; primers for amplifying a human target sequence; and an objective control amplification sequence that includes sequences corresponding to flanking portions of the human target sequence, so that the amplification of both target sequences occurs using the same primers. The team can also include instructions for its use. Brief Description of the Drawings Figure 1 shows an array of probes on the surface of a microform with locations 12 x 11 = 132. The numbers correspond to SEQ ID NO of the sequence listing. Simple probes were set in two different locations for detection of 21 different types of HPV, DNA sample quality control and amplification control. LR = location reference probes (SEQ ID NO 140 + SEQ ID NO 141).

Figure 2 shows an adjustment of probes on the surface of a microformation with locations 12 x 11 = 132. The numbers correspond to SEQ ID NO from the sequence listing. The simple probes or probe mixes were fixed in two different locations for detection of 23 different types of HPV, DNA sample quality control and amplification control. LR = location reference probes (SEQ ID NO 140 + SEQ ID NO 141). Figure 3 shows an adjustment of probes on the surface of a microformation with locations 12 x 11 = 132. The numbers correspond to SEQ ID NO of the sequence listing. The mixture of the probes was fixed in two different locations for the detection of 42 different types of HPV and quality control of the DNA sample. LR = location reference probe (SEQ ID NO 140 + SEQ ID NO 141); M1 = SEQ ID NO 76 + SEQ ID NO 77 + SEQ ID NO 78; M2 = SEQ ID NO 122 + SEQ ID NO 123 + SEQ ID NO 124; M3 = SEQ ID NO 116 + SEQ ID NO 117 + SEQ ID NO 118 + SEQ ID NO 119. Figure 4 shows an adjustment of probes on the surface of a microform with locations 12 x 10 = 120. The numbers correspond to the SEQ ID NO from the sequence listing. The single probe or probe mixes were set at three different locations for the detection of 35 different types of HPV, DNA sample quality control and amplification control. LR = probes for reference of location (SEQ ID NO 140 + SEQ ID NO 141); M1 = SEQ ID NO 76 + SEQ ID NO 77 + SEQ ID NO 78; M2 = SEQ ID NO 122 + SEQ ID NO 123 + SEQ ID NO 124. Figure 5 shows an array of probes on the surface of a microform with the locations 12 x 10 = 120. The numbers correspond to SEQ ID NO of the sequence listing. Single probes or probe mixes were set at two different locations for the detection of 14 different types of HPV, DNA sample quality control and amplification control. LR = location reference probes (SEQ ID NO 140 + SEQ ID NO 141); M4 = SEQ ID NO 100 + SEQ ID NO 101 + SEQ ID NO 102. Figure 6 shows a schematic representation of the recombinant plasmid pPG44 used in the PCR reaction as a positive amplification control. Figure 7 shows a photograph of a "forming tube" used in the present invention. Detailed Description of the Invention The method for specific detection and / or identification of types of HPV, comprises the following steps: (i) Amplification of sample DNA: the DNA obtained from the clinical samples is amplified, preferably by PCR, using universal primers for all known types of HPV flanking a genome region variable enough to allow genotyping additional. Although PCR is the preferred amplification method, the amplification of target sequences in a sample can be achieved by any other method known in the art (ligase chain reaction, transcription based amplification system, strand displacement amplification). , etc.). In one embodiment of the present invention, the primers MY11 and MY09 (Hands and Associates, Molecular Diagnostics of Human Cancer, Furth M, Greaves MF, eds, Cold Spring Harbor Press, 1989, vol.7: 209-214) have been used. ), which amplify the variable region L1. A tag is introduced into the amplified DNA during its amplification to allow additional detection. Preferably, a label providing a signal can be detected by colorimetric methods. In a preferred embodiment, at least one of the primers used labels the 5 'end with biotin. However, any other type of label known in the art can be used (for example digoxigenin). In addition, labeling of amplified DNA can alternatively be achieved by adding modified nucleotides containing a tag (eg biotinylated dUTP or dioxigenin derivatives) in the PCR mixture. Radioactive labels, or fluorophores can be used in certain modalities. (ii) Hybridization: it is denatured (for example by heat) the DNA of step (i) and is applied to a "training tube" with one or more probes of those shown in Table 1 (SEQ ID NO: 1-133). Other forms can also be used to prepare single-stranded DNA after amplification. Each probe shown in Table 1 (SEQ ID NO: 1-133) has the ability of specific hybridization with the amplified L1 region of step (i) of only one type of HPV, and therefore specific identification of this type is allowed. type of HPV, when this type is present in a biological sample. The different types of HPV in a sample can be identified by hybridization of amplified DNA from the HPV types of at least one, preferably more than one probe. (iii) Detection: DNA hybrids can be detected by recognition of the tag by specific binding to a ligand or by immunodetection. In the preferred embodiment, the biotin label is detected by specific binding to streptavidin conjugated to horseradish peroxidase (HRP) and the subsequent conversion of tetramethylbenzidine (TMB) to a blue pigment that precipitates at the particular location where the probe was bound corresponding specific Another type of conjugates well known in the art may also be suitable for purposes of the present invention (for example streptavidin-Au conjugate). Detection systems can be used labeled either directly or indirectly. Alternatively, other enzyme-based systems can be used. (iv) Analysis and processing of the results: you can read "already processed training tubes using simple optical devices, such as an optical microscope or readers ATR01 and ATS manufactured by chip technologies CLONDIAG GmbH (Jena, Germany). In an alternative embodiment, amplification and hybridization steps can be carried out in the same forming tube; that is, a sample is added to the forming tube, where the sample is amplified and hybridized to probes within the tube. In Patent Application Number US2005064469, a process for preparing the "forming tube" is described. In a preferred embodiment of the present invention, oligonucleotide probes linked with 5 'amine are linked to the surface of a solid support at various known locations. The probes can be immobilized individually or as mixtures for locations delineated on the solid support. In a preferred embodiment, two type-specific probes are used for each HPV type, which provides additional assurance that all HPV will be correctly typed including variants where nucleotide changes have occurred in the region of a specific probe of the type . Two are preferably used type-specific probes that have the ability to hybridize in separate regions of the amplified product. The probes or probe mixes can be immobilized at a single location of the solid support, preferably at two different locations of the solid support and more preferably at three different locations of the solid support. Figures 1 through 5 exemplify schematic representations of different probe settings on the microformation surface. The "forming tube" used in the present invention may comprise one or more HPV probes selected from nucleotide sequences from the sequence listing (SEQ ID NO: 1-133). In addition, it may comprise one or more probes for the specific detection of controls such as PCR reaction control and DNA adaptation of the control sample. In addition, it may also comprise one or more labeled oligonucleotides (e.g., biotin-modified oligonucleotides) for the positive control of the detection reaction and the positioning reference, so that all remaining probes can be localized. The specific probes of the HPV type identification were designed as indicated below. Sequences of all reference HPVs deposited in GenBank, including known variants of the amplified L1 region, were aligned using an alignment programional nucleic acid, such as BioEdit (4.8.6, version, Nucí Acids Symp Ser. 1999, 41: 95-98) and most of the regions of variable sequences within different types of HPV were localized. Potential oligonucleotide sequences that will be used as specific probes were selected from these variable sequence regions, which preferably have the following characteristics: length from 20 to 40 bases, preferably an approximate length of 30 bases; preferably without secondary structures or chains of the same consecutive nucleotides greater than 4; preferably with a G + C ratio of 50% and a Tm as similar as possible among all the selected probes; and preferably with the nucleotides without correspondence between the different HPV types sequences, as much as possible in the center of the oligonucleotide sequence. Each potential probe sequence selected as indicated above, was compared against all known HPV sequences in the amplified L1 region using the BLAST program of the NCBI Web page (Altschul et al. Nucleic Acid Res. 1997, 25: 3389-3402). Finally, probes that have at least three nucleotide mismatches when compared to all known HPV types (except when compared to the HPV time for which the oligonucleotide probe is specific) were chosen, with preference of the probes with greater than three faults of correspondence. The present invention provides probes for the specific detection of the 42 clinically most important HPV types: 6, 11, 16, 18, 26, 30, 31, 32, 33, 34/64, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85 and 89 (table 1; SEQ ID NO 1-133). Probe sequences are represented as single-stranded DNA oligonucleotides at the 5 'and 3P end. In a preferred embodiment of the present invention, the probe sequences correspond to the antisense strand, although it is obvious to anyone skilled in the art that these probes they can be used as they are, or in their complementary form or in their RNA form (where T is replaced by U). The probes of the present invention can also be prepared by adding or changing one or more nucleotides of their sequence without dramatically affecting their functionality.

Table 1 Name of SEQ ID NO Type HPV Sequence (5 * -> 3 ') 1 6Al-AS 6 TGTATGTGGAAGATGTAGTTACGGATGCAC 2 6B4 6 CATGACGCATGTACTCTTTATAATCAGAATT 3 11Al-AS 11 11 TGTATGTAGCAGATTTAGACACAGATGCAC i UBI CATGGCGCATGTATTCCTTA7AATCTGAAT 5 16A 16 GTAGATATGGCAGCACATAATGACATATTT 5 16E-AS 16 TTCTGAAGTAGATATGGCAGCACATAATGA 7 16C3 16 16C4 16 CGTCTGCAGTTAAGGTTATTTTGCACAGTT 8 CAAAATAGTGGAATTCATAGAATGTATGTAG_9_16C5 16 CTATAAGTATCTTCTAGTGTGCCTCCTGGG 10 1BA1-AS 16 ATCATATTGCCCAGGTACAGGAGACTGTGT 11 18B3 18 CTTATTTTCAGCCGGTGCAGCATCCTTTT 12 18C2 18 AAGTTCCAATCCTCTAAAATACTGCTATTC 13 18C3 18 TCCTTTAAATCCACATTCCAAAACTTTAACT 14 26A1-AS 26 TGGTTTAAATGGAGTGGATGCAGATGCTGC 15 26B2 26 ATCTTCCTTTGGCACAGGAGGGGCGTTACG 16 26C1 26 CATATTCTTCGCCATGTCTTATAAATTGTT 17 26C3 26 TCCTCCAATA7G6AGGCATTCATTAAATGT 18 26C4 26 GATCTTCCTTTGGCACAGGAGGGGCGTTAC 19 30A1 30 CT? GTGGCAGCTGGGGGTGACAATCCAATA 20 30b1 30 GATAACGTTTGTGTGGTTGCAGATATAGTC 21 30C1 30 TTCCAGCCCTCAAGTAAAGTGGAGTTCATA 22 31A-AS 31 TGTAGTATCACTGTTTGCAA? TGCAGCACA 23 31B5 31 AGAACCTGAGGGAGGTGTGGTCAATCCAAA 24 31C2 31 TCAAWTCCtCACCATGTCTTAAATACTCTTTA 31C5 31 AAAATAGCAGGATTCATACTGTGAATATATG 26 32A1 32 AGTTAGTAGACTTGTATGTGTCTTCAGTTGTT 32b1 TTCCCAAAATGAATAGTCAGAAAAAGGAIC 27 28 33 2 32 33 29 33B1 TACTGTCACTAGTTACTTGTGTGCATAAAG-AS 33 30 33C1 33 GTATATTTACCTAAGGGGTCTTCCTTTTCC AATGTATATTTACCTAAGGGGTCTTCCTTT 31 33C3 33 TTCTGCAGTTAAGGTAACTTTGCA? AGTIG 32 34 / 64A1 ATATGGTGGAGTTGTACTTGTGGATTGTGT 34/64 33 34/34/64 TCCTTAGGAGGTTGCGGACGCTGACATGTA 64B1 35A1-AS 35 34 35 35b1 GTCACTAGAAGACACAGCAGAACACACAGA 35 AATGGATCATCTTTAGGTTTTGGTGCACTG 36 35C4 35 TTACATAGCGATATGTGTCCTCTAAGGTAC 37 35C7 35 TTTGACAAGTTACAGCCTGTGATGTTACAT 38 39A1-AS 39 GGTATGGAAGACTCTATAGAGGTAGATAATGGTATCTGTAAGTGTCTACCAAACTGGCAGA 40 39C2 39 TAGAGGTAGATAATGTAAAGTTGGTACTAC 41 39C3d 39 GTAAATCATACTCCTCCACGTGCCTGRTAT 42 39C4 39 CATCAGTTGTTAATGTGACAGTACACAGTT 43 39C4d 39 CATCAGTTGTTAATGTKACAGTACACAGTT 44 0A1-AS 40 CTTGAAATTACTGTTATTATATGGGGTTGG 45 40B1 40 CCTCCAACAACGTAGGATCCATTGCATGAA 46 2A1 42 GTATATGTATCACCAGATGTTGCAGTGGCA 47 42B1-AS 42 TAGCCTGACAGCGAATAGCTTCTGATTGTA 48 42C1 42 TGACAGCGAATAGCTTCTGATTGTACATAC 49 42C5 42 TCCTCTAATATGTTAGGATTCATATTGTGTA 50 42C6 42 TTCTAAAGTTCCTGAAGGTGGTGGTGCAAC 51 43A1 43 ATATGTACTGGGCACAGTAGGGTCAGTAGA 52 43B1-AS 43 AAGCAGAGGCAGGTGGGGACACACCAAAAT 53 44A1 44 TATATGTAGACGGAGGGGACTGTGTAGTGG 54 4 B1-AS 44 CGCATGTATTGCTTATATTGTTCACTAGTAT 55 45B1 45 CTGCTTTTCTGGAGGTGTAGTATCCTTTT 56 45B4-AS 45 GGCACAGGATTTTGTGTAGAGGCACATAAT 57 45Cld 45 TACTATASTGCTTAAACTTAGTAGGRTCAT 58 45C3d 45 CCACYAAACTTGTAGTAGGTGGTGGAGGKA 59 45C4 45 CAGGTAACAGCAACTGATTGCACAAAACGA 60 51A1-AS 51 TAAATGTTGGGGAAACCGCAGCAGTGGCAG 61 51B1 51 TGGAGGGGTGTCCTTTTGACAGCTAGTAGC 62 51C3 51 ATGGTAGGATCCATTGTGTGTAAATAAGCC 63 51C4 51 CCACTGTTCAAGAATGGTAGGATCCATTGT 64 51C5 51 CCAAACTAGCAGACGGAGGTAATGTTAATC 65 52A1-AS 52 TTATATGTGCTTTCCTTTTTAACCTCAGCA 66 52B2 52 GTGTCCTCCAAAGATGCAGACGGTGGTGG 67 53A1 53 AACCTCAGCAGACAGGGATATTTTACATAG_68_53B1 53 AAGCTAGTGGCAACAGGAGGCGACAAACCT 69 54A1 54 GCTATCCTGCGTGGATGCTGTAGCACACAA 70 54B1 54 AACTACTTGTAGCTGGGGGGGTTATACCAA 71 54C1 54 ATCTGCTGTAAGGGTTATGGTACATAACTG 72 56A1 56 TGTCTAAGGTACTGATTAATTTTTCGTGCA 73 56B1 56 TTTATCTTCTAGGCTGGTGGCCACTGGCGG 74 57Ald 57 TATAATTAGTTTCTGTGKTTACAGTGGCAC 75 57B1 57 AGTCCTCTAGCAACCGCGCATCCATGTTAT 76 58A1 58 ATATTCTTCAACATGACGTACATATTCCTT 77 58Bla 58 TCTTCTTTAGTTACTTCAGTGCATAATGTC 78 58Blb 58 CCTTCGTTAGTTACTTCAGTGCATAATGTC 79 59A2 59 CTGGCATATTCTTTAAAACTGGTAGGTGTG 80 59B1-AS 59 TCCTGTTTAACTGGCGGTGCGGTGTCCTTT 81 59C3_3d 59 GAAGVAGTAGTAGAAGCACACACAGAAAGA 82 59C4 59 TTCCTCCACATGTCTGGCATATTCTTTAAA 83 59C6 59 GTGGTATTCATATTATGAATGTATGACATT 84 61Al 61 TATATTCAGATACAGGGGGGGATGTAGCAG 85 61B1 61 ATAACTTGGCATAGCGATCCTCCTTGGGCG 86 61B2 61 ATATTCCCTAAAGCTTGTGGCTTTATATTC 87 62A1 62 GTGGAAGGGGGAGGTAAAACCCCAAAGTTC 88 62B1 62 ATACGGGTCCACCTTGGGACGGGTAGGCAG 89 62B2 62 AAATGTCATTTGCGCATACGGGTCCACCTT 90 66A1 66 GTTAATGTGCTTTTAGCTGCATTAATAGTC 91 66B1 66 TGGCGAAGGTATTGATTGATTTCACGKGCA 92 66B2 66 CACATGGCGAAGGTATTGATTGATTTCACG 93 66C3d 66 CCAATRTTCCAATCGTCTAATAAAGTATTA 94 66C4 66 CTGTGCTTTTAATATACCTATATTTATCCT 95 67A1 67 TCTTCCTTTGCTGTTGGAGGGGATGTTTTT 96 67B1 67 TGGTGTGTATGTATTGCATAACATTTGCAG 97 67B2 67 GTTTTCATTTTTGTATGTAGCCTCTGATTT 98 68A1-AS 68 AGGTGCAGGGGCGTCTTTTTGACATGTAAT 99 68B1 68 AGCGGTATGTATCTACAAGACTAGCAGATG 100 68C4b 68 TACATCAGTTGACAATGTTATAGTACACAAC 101 68C4c 68 TACATCAGTGGATAATGTTATAGTACACAAC 102 68C7 68 CAAGACTAGCAGATGGTGGAGGGGCAACAC 103 69A1 69 ATGGTTTAAAAGTGGCAGATGCAGATTGTG 104 69B1 69 TGTGCAGGGGCATCGCGTTGACATGTAGTA 105 70A1-AS 70 AAACTTTGTAGGGCTATATACAGCAGGTAT 106 70B1 70 TGGTGGAGGGGTAACTCCTATATTCCAATT 107 71A1 71 AATATTCCATGAAACTAGAGGCTTTATATG 108 71B1 71 TTTTTCTGCAGGAGGAGGACTGTTTTTCTG 109 72A1 72 TCTGATACAGAGGACGCTGTGGCAGTACAA 110 72B1 72 GTGGCGAAGATACTCACGAAAATTAGAAGC 111 73A1 73 TAGAGTTGGCATACGTTGTAGTAGAGCTAC 112 73A2 73 GAGTTGGCATACGTTGTAGTAGAGCTACTA 113 73B1 73 AGGAGGTTGAGGACGTTGGCAACTAATAGC 114 73C3 73 TCCACTCTTCCAATATAGTAGAATTCATAG_115_73C4 73 TTCCTCTAAAGTACCTGACGGTGGTGGGGT 116 74Ala 74 TTAAATTTGCATAGGGATTGGGCTTTGCTT 117 74Alb 74 TTAAATTGGCATAGGGATTAGGCTTTGCTT 118 74Bla 74 AGCAGAAGGCGATTGTGAGGTAGGAGCACA 119 74Blb 74 AGCAGGAGGGGATTGTGTAGTAGGCGCACA 120 81A1 81 TTCTGCAGCAGCAGATGTAGCTGTGCAAAT 121 81B1 81 CTGTCCAAAATGACATGTCGGCATAAGGGT 122 82A2a-AS 82 TGCAACAGATGGAGTAACAGCAGTGCTAAT 123 82A2b-AS B2 TGCAACTGATGGAGTAGCAGCAGTGCTAAT 124 82B1 82 TGTAGAATCCATGGTGTGCAGGTAAGCCAT 125 83A1 83 TTCATTAGCCTGTGTAGCAGCAGCTGAAAT 126 83Bld 83 CACTCATCYAATAAATGTTCATTCATACTAT 127 84A1 84 ATATTCTGATTCGGTGTTGGTAGCAGCACT 84B1 128 129 84 AAATAGGACATGACCTCTGGAGTCAGACGG 85A1 85B1 85 85 130 ATATAGATGGAACTGGATTAGTAGTTGCAG CCTTTTTTTGTGGAACAACCACATCCTTCT 131 8 A1 132 89 TCCTTAAAGCGTGTAGAACTGTATTCTGTG 89B1 89C1 89 133 B9 ATCTCAGGCGTTAGGTGTATCTTACATAGT AATGGCCCGAGAGGTAAGAAAGCGATAGGT The nucleotides of the sequences are designated as follows: G for Guanine, A for Adenine, T for Thymine, C for Cytosine, R for G or A, Y for T or C, M for A or C, K for G or T, S for G or C, W for A or T, H for A or C or T, B for G or T or C, V for G or C or A, D for G or A or T and finally, N for G or A or T or C. The nucleotides as used in the present invention can be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups that do not essentially alter their hybridization characteristics. The probes of the present invention can be obtained through different methods, such as chemical synthesis (for example through the conventional phosphotriester method) or genetic engineering techniques, for example by molecular cloning of recombinant plasmids wherein the nucleotide sequences corresponding ones have been inserted and can be obtained later by digestion with nucleases. For some types of HPV, the probes were designed from a sequence region containing different nucleotides in a specific position of different variants of the aforementioned HPV type. In these cases, degenerate probes were used, that is, a mixture of oligonucleotides each containing alternative nucleotides in the aforementioned position. This is the case of the probes 39C3d [SEQ ID NO 41], 39C4d [SEQ ID NO 43], 45C1d [SEQ ID NO 57], 45C3d [SEQ ID NO 58], 57A1d [SEQ ID NO 74], 59C3_3d [SEQ ID NO 81], 66B1 [SEQ ID NO 91], 66C3d [SEQ ID NO 93] and 83B1d [SEQ ID NO 126]. As an alternative, equimolar mixtures were used of two oligonucleotides comprising exactly the same sequence region but different in composition for certain positions, in the form of a simple probe (mixture of nucleotides 58B1a [SEQ ID NO 77] and 58B1b [SEQ ID NO 78]; 68C4b [SEQ ID NO 100] and 68C4c [SEQ ID NO 101]; 74Ala [SEQ ID NO 116] and 74A1b [SEQ ID NO 117]; 74B1a [SEQ ID NO 118] and 74B1b [SEQ ID NO 119]; and mixture of oligonucleotides 82A2a -AS [SEQ ID NO 122] and 82A2b-AS [SEQ ID NO 123] All the probes described in the present invention have been approved to specifically hybridize to their target sequences under the same conditions of hybridization in the platform of " training tube. "This fact makes possible the use of these probes for the simultaneous identification of 42 different types of HPV using this microformation platform.The high number of HPV types identified through the use of the" tube of formation "developed in the present invention, makes this methodology also be considered as a direct detection method, since the remaining types of HPV are clinically irrelevant. One of the weak points of the diagnostic methods is the appearance of false negatives. In the case of the method of the present invention, false negatives can be caused by DNA samples of poor quality or by the presence of DNA polymerase inhibitors in the samples that will be analyzed. The present invention illustrates the manner of eliminating These false negatives through the use of two types of controls. A control consists in the amplification of the patient's own DNA which is preferably used to ensure a good quality of the DNA sample. Any fragment of human DNA sequence can be used as an objective for this purpose. A fragment of a single copy gene, such as the CFTR gene, was considered an especially suitable target for the positive control of DNA quality in the present invention. The primers CFTR-F4 (SEQ ID NO 134) and CFTR-R5 (SEQ ID NO 135) were designed for the amplification of a fragment of 892 base pairs of the CFTR gene. The use of a single copy versus multiple copy target and the larger size of the amplified product with DNA quality control was compared with the amplified HPV fragment, this is 892 base pairs versus approximately 450 base pairs, respectively, allowing the inclusion of primers for CFTR amplification in the same reaction mixture that was used for the amplification of the L1 region of the HPV genome with minimal competition effects. Therefore, DNA quality control can be run simultaneously in the same reaction tube where samples were analyzed without affecting the sensitivity of HPV detection. A second control can be used as a positive PCR amplification control that detects PCR reaction failures, due, for example, to the presence of DNA polymerase inhibitors. In a preferred embodiment, the positive amplification control consists of a recombinant plasmid that can be amplified using the same primers and the same PCR conditions as those used for amplification of the CFTR gene fragment. Both the size and the internal sequence of the primers are different between the PCR products resulting from the amplification of the CFTR gene and the amplification of the recombinant plasmid. In this way, both types of amplification products can be easily distinguished through gel electrophoresis or through hybridization, with specific probes. Figure 6 shows a schematic representation of recombinant plasmid pPG44 having these characteristics. Plasmid pPG44 was constructed by molecular cloning techniques. In short, a DNA insert consisting of a fragment of 1162 base pairs from position 124 to position 1285 of the pBiuescript® II SK + vector (Stratagene, La Jolla, CA, USA) flanked by the CFTR, CFTR-F4 primers and CFTR-R5, was cloned into the pGEM®-T Easy vector using commercially available equipment from Promega Corporation, Madison, Wl, USA. A purified preparation of the recombinant plasmid obtained pPG44 was further characterized through the use of restriction enzymes and by sequence analysis. Plasmid pPG44 was used as a positive control of the amplification process in a linearized form. The presence of a positive control in the form of the aforementioned recombinant plasmid in the same PCR amplification mixture in which the sample was analyzed, avoids the occurrence of false negative results, that is, prevents a negative result from being provided even in the presence of the genome. HPV target in the sample, because when the amplification products are not generated, it must be assumed that the PCR amplification was not operated properly and a conclusion can not be obtained regarding the presence or absence of the HPV genome in the sample. Probes for specific detection of two types of positive controls described, namely, DNA quality control and amplification reaction control, are provided in Table 2 (SEQ ID NO 136-139 and SEQ ID NO 145-147). Oligonucleotide sequences without significant homology to any of the amplified products of the present invention are also provided in this table 2 (SEQ ID NO 140-141). When immobilized on the surface of the microformation, the biotinylated oligonucleotides SEQ ID NO 140 and SEQ ID NO 141 serve as a positive control of the PCR product detection reaction and as a positioning reference, so that all of them can be located the remaining probes.

Table 2: I KNOW THAT ID NO Probe Name Control Type Sequence (5 '- 3") 136 CFTR- A 1 -AS DNA Quality Sample T CTCCACCCACTACGCACCCCCGCCAGCA 137 CFT -E ^ DNA Quality Sample GGGCTCAAGCTCCTAATGCCAAAGACCTACT? CTCTG 145 CFTR- Bl -AS Sample of DNA Quality CAB.GCTCCTAATSCCAAAGACC.'ACTACTC l-Jf. CGTP-82 DNA Quality Sample GGGCTCAAGC7CR: TAATGCCAAAGACCTACTACT <J 138 CIA1-AS PCR Reaction C? CATTAGGCACCCC? GCCTTTAC? CTTT? T 139 CI A2 - AS Reaction PCR TCACTCATTAGGCACCCG /? GGCTTTACA TTTATI '? 147 CTA3-AS PCR reaction GAGT5AGCR3ATACCCCRC3CCGCAGCCGAACGAC 140 Marker-1 Detection and location GCAGTATAAGAT? ATTGATGCCGGAAC 141 Marker-2 Detection and location 3TCAA_ACCrGGC-¡¡TATAGrtS "? TTTACC The present invention also relates to an in vitro diagnostic equipment for the specific detection of HPV types in clinical samples. Preferably, the aforementioned equipment can include any or all of the following components: amplification mixture, including amplification buffer, dNTPS, primers and control plasmid; wash regulator; detection reagent; training tube including a solid support that includes specific probes of the HPV type; reagents to obtain and prepare a sample. The particular components will depend on the exact conditions under which the equipment is intended to be used, although those skilled in the art have the ability to determine the appropriate equipment components and regulation compositions. Examples The examples below are only illustrate the present invention, and do not limit in any way the scope of the appended claims. Example 1: Preparation of "forming tubes" The "forming tubes" of the present invention were manufactured at CLONDIAG chip Technologies GmbH (Jena, Germany) as indicated below. A standard Eppendorf reaction test tube made of polypropylene and having a nominal reception volume of 1.5 ml was modified by recasting, so that an open recess for the microformation support with an edge of the tube was modeled in the tube. adhesive. The microformations that will be inserted into these tubes were produced using a MicroGrid II Arrayer (BioRobotics, Cambridge, Great Britain). Probes consisting of 5-end amine-modified oligonucleotides having a sequence from the sequence list were deposited at defined sites on a slice-epoxied glass surface (slice size: 75 mm x 25 mm) and immobilized covalently. A simple microformation included 12 x 10 = 120, or 12 x 11 = 132 specific locations in which the oligonucleotides can be deposited. These locations have a separation of 0.2 mm, so that the DNA library included in each microformation covered an area of 2.4 mm x 2.4 mm and in total, more than 100 identical DNA libraries can be produced in This form by slice. Depending on the type of experiment, either a simple probe or a mixture of them can be deposited in each of these locations. Normally, simple probes were deposited at each location when specificity and sensitivity experiments were carried out for the selection of probes. Once the probes have been validated, mixtures of the probes with the ability to hybridize in separate regions of the amplified product of a specific HPV type can be deposited at the same location when identification of HPV genotype assays was carried out. Figures 1 to 5 show different alignments of probes within the microformations used for the present invention. Two or three replicates of each probe or mixture of probes were included in each microformation. In addition to the specific probes for genotyping HPV and for the detection of amplification control and adequacy of DNA control, the microformations included reference markers in various locations consisting of oligonucleotides modified with biotin 5 (marker-1 [SEQ ID NO. 140] and marker-2 [SEQ ID NO 141]) without significant homology to any of the amplified sequences of the present invention. These reference markers served both to verify the adequate performance of the detection reaction and for the optical orientation of the image by the reader, so that all remaining probes can be located and analyzed in terms of data. All the oligonucleotides were deposited in the slice of a 1x QMT Spotting Solution I (Quantifoil Micro Tools GmbH, Jena, Germany). The total concentration of oligonucleotides in each staining solution ranged from 2.5 μM for reference markers to 20 μM for specific probes. Subsequently, the oligonucleotides were covalently bound to the epoxide groups on the glass surface, baking at a temperature of 60 ° C for 30 minutes followed by a multistep washing process. The dried slices were cut into glass pieces of 3.15 mm x 3.15 mm which, strictly speaking, are what are called microformations. In the final step of manufacturing "forming tubes", these microformations were subsequently inserted into modified Eppendorf tubes mentioned above and stuck to the edge of the adhesive. Figure 7 shows a photograph of a "formation tube" produced as specified in this example. Example 2: preparation of DNA samples 2.1. DNA standards The HPV DNAs used to evaluate the specificity and sensitivity of type-specific probes were either recombinant plasmids containing the L1 region amplified (types of HPV 6, 11, 13, 16, 18, 26, 31, 33, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 61, 62, 66 , 68, 70, 71, 72, 73, 81, 82, 83, 84, 85 and 89) or DNAs extracted from clinical samples, wherein the amplified L1 region was additionally characterized by DNA sequencing. The recombinant plasmids were constructed by molecular cloning techniques. Briefly, the amplified L1 region of each HPV type was cloned into a pGEM®-T Easy Vector using commercially available equipment from Promega Corporation, Madison, Wl, E.U.A. Subsequently, a purified preparation obtained from each recombinant plasmid was further characterized by sequence analysis. 1 to 10 pg of plasmid DNA was used in the evaluation of specificity experiments. The DNA of the K562 cell line (catalog number DD2011, Promega Corporation, Madison, Wl, E.U.A.) served to evaluate the specificity and sensitivity of the specific CFTR probes. 2.2. Clinical samples For the purpose of deteg HPV, first of all it is necessary to separate the DNA from the rest of the biological material. The preparation of DNA procedures varies according to the source of the sample. Specific examples are provided for the preparation of DNA from samples from a variety of sources: A. Cottons: the samples were taken with a cotton wool. cotton, dry and clean. The cells of the clinical cottons were recovered through the addition of 1.5 ml of saline solution directly to the container containing the sample and by vigorous vortexing. The sample material was transferred to a 1.5 ml Eppendorf tube and pelleted by centrifugation. The supernatant was discarded and the precipitated cells were suspended in 100 μl of lysis buffer containing 10 mM Tris-HCl (pH 9.0 at a temperature of 25 ° C), 50 mM KCl, 0.15 mM MgCl 2, 0.1% Triton® X- 100, 0.5% Tween 20 and 0.25 mg / ml Proteinase K. This mixture was incubated at a temperature of 56 ° C for approximately 2 hours and proteinase K was deaated with heat incubating the mixture at a temperature of 100 ° C for 10 minutes. . Detritus was pelleted by centrifugation and the supernatant was transferred to a clean, sterile tube. Subsequently, an aliquot of 5 μl was used in the PCR rean. B. Cell suspensions: this type of sample refers to the one used in cervicovaginal fluid-based cytology tests. Cervical specimens were taken with a brush or spatula and resuspended in PreservCyt solution (Cytyc Corp., Marlborough, MA, E.U.A.). A 1 ml aliquot was centrifuged and the pellet resuspended in 1 ml of saline. After a new centrifugation step, the pellet was resuspended in 100 μl of lysis buffer as used with the samples of swabs in paragraph A, and the protocol continued in the same way as in this sen. C. Formalin-fixed and paraffin-embedded biopsies: several sens of 5 μm wide tissue were used in this method, usually 2 to 5 sens, depending on the surface area of the biopsy. The sens were placed in a sterile 1.5 ml tube and 100 μl of lysis buffer was added as was used with the swab samples in paragraph A. The protocol continued in the same way as in that sen, except that the incubation with proteinase K was carried out for 3 hours. As an alternative, commercial equipment (NucleoSpin® Tissue equipment catalog number 635966 from BD Biosciences Clontech, Palo Alto, CA, USA) was used, designed for the isolation of DNA from samples from a variety of sources to process swabs, cell suspensions and samples of biopsies fixed in formalin and embedded in paraffin. In this case, the beginning of the DNA isolation protocol was as specified in sens A, B and C. Instead of 100 μl of the lysis buffer, 180 μl of T1 regulator was added to the sample. The protocol was continued following the manufacturer's specifications for the isolation of genomic DNA from cells and tissues. Whatever was the type of clinical sample or method of DNA preparation, negative controls were run on parallel with each batch of samples. These negative controls, which were composed of 1 ml of saline solution, were processed in the same manner as in section A. Example 3: PCR amplification PCR amplification was carried out using the consensus primers MY11 and MY09 (Manos y asociados, Molecular Diagnostics of Human Cancer, Furth M, Greaves MF, eds, Cold Spring Harbor Press, 1989, vol 7: 209-214). A third primer, HMB01, was also included in the PCR reaction, which was often used in combination with MY09 and MY11 to amplify HPV type 51 which was not efficiently amplified with MY09 and MY11 alone (Hildesheim et al. 3 Infecí Dis. 1994, 169; 235-240). In synthesis, PCR amplification was carried out in a final volume reaction of 50 μl containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1 mM MgCl 2, 0.3 μM of each primer MY09 and MY11 (SEQ ID NO 142 and 143), 0.03 μM of primer HMB01 (SEQ ID NO 144), of each 200 μM, 4 units of AmpMTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) and 5 μl of each HPV DNA standard of the example 2.1 or clinical sample DNA of example 2.2. To test the amplification capacity of the DNA sample, 0.08 μM of each primer CFTR-F4 and CFTR-R5 (SEQ ID NO 134 and 135) were also added to the reaction mixture. In addition, to review the amplification process and eliminate the false negative results due to the 20 fg reaction failure of the internal control, pPG44 was included in the same reaction tube in which the samples were analyzed. All direct primers used in the PCR reaction (MY11 [Seq ID NO 143] and CFTR-F4 [Seq ID NO 134]) were modified with biotin at the 5-terminus, so that any amplified DNA could be detected subsequently. Negative controls containing 5 μl of blank samples from example 2.2 or 5 μl of deionized water were processed in parallel with the DNA of the samples. The use of these types of negative controls serves to check that contamination does not occur at any point in the handling of the sample or in the preparation of the PCR reaction and all positive results represent the actual presence of DNA in the sample. PCR reactions were run in a Mastercycler thermocycler (Eppendorf, Hamburg, Germany) programmed with the client cycling profile: an initial denaturation cycle at a temperature of 95 ° C for 9 minutes, 45 cycles of 30 seconds at a temperature of 94 ° C, 60 seconds at a temperature of 55 ° C and 90 seconds at a temperature of 72 ° C, and a final extension cycle at a temperature of 72 ° C for 8 minutes. After an amplification, 5 μl of each reaction was used for subsequent detection with specific probes.

Example 4: Simultaneous identification of HPV genotypes using "forming tubes" Previously "forming tubes" were pre-washed just before being used through the addition of 300 μl 0.5X PBS-Tween 20 regulator to each tube and reversed several times . All the liquid inside each tube was removed using a Pasteur pipette connected to a vacuum system. The amplification reactions of Example 3 were denatured by heating them at a temperature of 95 ° C for 10 minutes, and immediately afterwards, cooling them for 5 minutes on ice. Five microliters of denatured amplification reaction were applied to the "forming tube" prepared in Example 1 together with 100 μl of hybridization solution (250 mM sodium phosphate buffer, pH 7.2, 1 × SSC, 0.2% Triton® X- 100; 1 mM EDTA, pH 8.0). The hybridization reaction was carried out in a Thermomixer Comparat (Eppendorf, Hamburg, Germany), incubating the "forming tubes" at a temperature of 55 ° C for one hour with shaking at 550 rpm. After the incubation period, the hybridization reaction was eliminated using a pipette from Pasteur connected with a vacuum system and a wash step was carried out with 300 μl of 0.5X PBS-Tween 20 regulator. The hybridized DNA was detected by incubation in 100 μl of a 0.075 μg / ml Poly-HRP Streptavidin solution (Pierce Biotechnology Inc., Rockparad, IL, E.U.A.) to a temperature of 30 ° C for 15 minutes with stirring at 550 rpm. Subsequently, all the liquid from the "forming tube" was rapidly removed and two washing steps were carried out as mentioned above. The color development reaction was carried out on 100 μl of True Blue ™ substrate Peroxidase Substrate (KPL, Gaithersburg, MD, USA), which consists of a regulated solution containing 3,3 ', 5,5'-tetramethylbenzidine (TMB) and H2O2, by incubation at a temperature of 25 ° C for 10 minutes. The color precipitates produced in this way cause changes in the optical transmission at specific microformation locations that can be read using an ATR01 reader or an ATS reader manufactured by CLONDIAG chip technologies GmbH (Jena, Germany). Optionally, the ATS reader can have a specific software installed for the automatic processing of the result of the analysis of the sample obtained with the "tube of formation" developed in the present invention.

Claims (87)

1. CLAIMS 1. An assay for the detection and typing of human papilloma virus (HPV) in a sample, wherein the assay comprises: carrying out a nucleic acid amplification reaction in a sample, the amplification reaction being projected to amplify an objective sequence of HPV in a non-specific form of the type; obtain single-stranded oligonucleotides from any amplification products; allowing the single-stranded oligonucleotides to hybridize where possible with a plurality of HPV-type specific probes provided on a solid support, the support being located within a reaction container adapted to contain the sample; and detect hybridized oligonucleotides.
2. 2. The assay as described in claim 1, characterized in that the HPV-type specific probes comprise DNA.
3. The assay as described in claims 1 or 2, characterized in that the nucleic acid amplification step is carried out in the sample inside the reaction vessel in contact with the specific probes of the HPV type in the solid support .
4. 4. The test as described in claims 1 or 2, characterized in that the step of Nucleic acid amplification is carried out in the sample before the introduction of the amplified sample into the reaction container to contact the HPV type specific probes in the solid support.
5. The assay as described in any of the preceding claims, characterized in that the probes are selected to specifically bind to the HPV target sequence under the same hybridization conditions for all samples.
6. 6. The assay as described in any of the preceding claims, characterized in that specific probes are used for at least 20 types of HPV.
7. The assay as described in any of the preceding claims, characterized in that specific probes are used for at least 20 of the HPV types 6, 11, 16, 18, 26, 30, 31, 32, 33, 34 / 64, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85 and 89.
8. The test as described in any of the preceding claims, characterized in that the probes are 20 to 40 nt in length.
9. 9. The assay as described in any of the preceding claims, characterized in that the probes have from 25 to 35 nt.
10. 10. The test as described in any of the previous claims, characterized in that the probes have from 28 to 32 nt.
11. 11. The assay as described in any of the preceding claims, characterized in that the probes are about 30 nt.
12. 12. The assay as described in any of the preceding claims, characterized in that the probes are specific to the HPV L1 region.
13. The assay as described in any of the preceding claims, characterized in that the probe differs from the probes specific for another type of HPV by at least 2 nt.
14. The assay as described in any of the preceding claims, characterized in that each probe differs from the probes specific for another type of HPV by at least 3 nt.
15. The assay as described in any of the preceding claims, characterized in that one or more of the probes are selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 133.
16. 16. The assay as described in any of the preceding claims, characterized in that all the probes are selected from the group comprising SEQ ID NO 1 to SEQ ID NO 133.
17. 17. The assay as described in any of the preceding claims, characterized in that a plurality of probes are selected from one or more of the following groups of SEQ IDs: 1 or 2; 3 or 4; 5 to 9; 10 to 13; 14 to 18; 19, 20, or 21; 22 to 25; 26 or 27; 28 to 31; 32 or 33; 34 to 37; 38 to 43; 44 or 45; 46 to 50; 51 or 52; 53 or 54; 55 to 59; 60 to 64; 65 or 66; 67 or 68; 69, 70 or 71; 72 or 73; 74 or 75; 76, 77, or 78; 79 to 83; 84, 85, or 86; 87, 88, or 89; 90 to 94; 95, 96 or 97; 98 to 102; 103 or 104; 105 or 106; 107 or 108; 109 or 110; 111 to 115; 116 to 119; 120 or 121; 122, 123, or 124; 125 or 126; 127 or 128; 129 or 130; 131, 132 or 133.
18. 18. The test as described in claim 17, characterized in that the probe is selected from each of said groups.
19. 19. The assay as described in claim 17, characterized in that the probe is selected from the groups, and at least one probe is selected from each of the groups.
20. 20. The assay as described in claim 17, characterized in that two or more probes are selected from each of said groups.
21. The assay as described in any of the preceding claims, characterized in that the probes are selected from the following SEQ IDs: 2, 4, 7, 8, 9, 12, 13, 16, 17, 18, 19, 20 , 21, 24, 25, 26, 27, 30, 31, 32, 33, 36, 37, 40, 41, 42, 43, 45, 48, 49, 50, 51, 52, 53, 54, 57, 58 , 59 61, 62, 63, 64, 66, 67, 68, 70, 71, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 87, 88, 89, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 112, 114, 115, 116, 117, 118, 119, 120, 121, 124, 126, 128, 129, 130, 131, 132, 133.
22. 22. The assay as described in any of the preceding claims, characterized in that a plurality of probes are specific to the same type of HPV.
23. 23. The assay as described in any of the preceding claims, characterized in that a plurality of probes are specific to each type of HPV that will be detected.
24. 24. The assay as described in any of claims 22 or 23, characterized in that each plurality of probes is immobilized in the same region of the solid support.
25. 25. The assay as described in any of claims 22 to 23, characterized in that each plurality of probes is immobilized in a region other than the solid support.
26. 26. The assay as described in any of claims 23 to 25, characterized in that each specific probe of the same HPV type detects a different part of the HPV target sequence.
27. 27. The test as described in any of the the preceding claims, characterized in that at least one probe is in the solid support in at least two different locations.
28. 28. The assay as described in any of the preceding claims, characterized in that all the probes are in the solid support in at least two different locations.
29. 29. The assay as described in any of the preceding claims, characterized in that it further comprises detecting one or more control sequences.
30. 30. The assay as described in claim 29, characterized in that the control sequence comprises a probe immobilized on the solid support, which does not hybridize to the target sequence of any type of HPV.
31. 31. The test as described in the claim 29, characterized in that the control sequence comprises a human genomic target sequence.
32. 32. The assay as described in claim 31, characterized in that the human target sequence comprises at least a part of the CFTR gene.
33. 33. The assay as described in any of the preceding claims, characterized in that it further comprises amplifying a known control sequence, and detecting the product of the amplification.
34. 34. The test as described in any of the the preceding claims, characterized in that it comprises combining an amplification reaction mixture with the sample to carry out the amplification reaction.
35. 35. The assay as described in any of the preceding claims, characterized in that the amplification reaction is PCR.
36. 36. The assay as described in any of the preceding claims, characterized in that single-stranded oligonucleotides are obtained by denaturing any double-stranded oligonucleotides present.
37. 37. The assay as described in claim 29, characterized in that the denaturation step is carried out in a sample that is contained within the reaction vessel.
38. 38. The assay as described in any of the preceding claims, characterized in that the single-stranded oligonucleotides are allowed to hybridize under stringent conditions.
39. 39. An assay for detecting and typing human papilloma virus (HPV) in a sample, wherein the assay comprises: carrying out a nucleic acid amplification reaction in a sample in a reaction package comprising a solid support having a plurality of specific probes of the HPV type immobilized therein, the amplification reaction being projected to amplify an HPV target sequence in a non-specific form of the type; obtain single-stranded oligonucleotides from any amplification products; allow single-stranded oligonucleotides to hybridize when possible with HPV-type specific probes; and detect hybridized oligonucleotides; wherein the amplification reaction takes place in the sample in contact with the solid support.
40. 40. A reaction vessel for carrying out an assay for detecting and typing HPV in a sample, wherein the package comprises a solid support having a plurality of HPV-type specific probes immobilized therein, and which is adapted for contain a sample in contact with the solid support.
41. 41. The package as described in claim 40, characterized in that the package is suitable for carrying out a nucleic acid amplification reaction in a sample in contact with the solid support.
42. 42. The package as described in the claim 40 or 41, characterized in that the probes are selected to bind specifically to HPV target sequences under the same hybridization conditions for all probes.
43. 43. The package as described in any of claims 40 to 42, characterized in that the probes are selected to specifically bind target HPV sequences in a sample comprising a reaction mixture suitable for carrying out a nucleic acid amplification reaction.
44. 44. The package as described in any of claims 40 to 44, characterized in that the specific probes of the HPV type comprise DNA.
45. 45. The package as described in any of claims 40 to 44, characterized in that it comprises specific probes for at least 20 types of HPV.
46. 46. The package as described in any of claims 40 to 44, characterized in that it comprises specific probes for at least 20 of HPV types 6, 11, 16, 18, 26, 30, 31, 32, 33, 34 / 64, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72 , 73, 74, 81, 82, 83, 84, 85 and 89.
47. 47. The package as described in any of claims 40 to 46, characterized in that the probes are 20 to 40 nt in length.
48. 48. The package as described in any of claims 40 to 47, characterized in that the probes have from 25 to 35 nt.
49. 49. The package as described in any of claims 40 to 48, characterized in that the probes have from 28 to 32.
50. 50. The package as described in any of claims 40 to 49, characterized in that the probes are about 30 nt.
51. 51. The package as described in any of claims 40 to 50, characterized in that the probes are specific for the L1 region of HPV.
52. 52. The package as described in any of claims 40 to 51, characterized in that each probe of a specific HPV type differs from the probes specific for another type of HPV by at least 2 nt.
53. 53. The package as described in any of claims 40 to 52, characterized in that each probe of a specific HPV type differs from the probes specific for another type of HPV by at least 3 nt.
54. 54. The package as described in any of claims 40 to 53, characterized in that one or more of the probes are selected from the group comprising SEQ ID NO 1 to SEQ ID NO 133.
55. 55. The package as described in any of claims 40 to 54, characterized in that all the probes are selected from the group comprising SEQ ID NO 1 to SEQ ID NO 133.
56. 56. The package as described in any of claims 40 to 55, characterized in that HE selects a plurality of probes from one or more of the following groups of SEQ IDs: 1 or 2; 3 or 4; 5 to 9; 10 to 13; 14 to 18; 19, 20, or 21; 22 to 25; 26 or 27; 28 to 31; 32 or 33; 34 to 37; 38 to 43; 44 or 45; 46 to 50; 51 or 52; 53 or 54; 55 to 59; 60 to 64; 65 or 66; 67 or 68; 69, 70 or 71; 72 or 73; 74 or 75; 76, 77, or 78; 79 to 83; 84, 85, or 86; 87, 88, or 89; 90 to 94; 95, 96 or 97; 98 to 102; 103 or 104; 105 or 106; 107 or 108; 109 or 110; 111 to 115; 116 to 119; 120 or 121; 122, 123, or 124; 125 or 126; 127 or 128; 129 or 130; 131, 132 or 133.
57. 57. The package as described in the claim 56, characterized in that a probe is selected from each of said groups.
58. 58. The package as described in claim 56, characterized in that each probe is selected from said groups, and at least one probe from each of said groups is selected.
59. 59. The package as described in claim 56, characterized in that two or more probes are selected from each of said groups.
60. 60. The package as described in any of claims 40 to 59, characterized in that the probes are selected from the following SEQ IDs: 2, 4, 7, 8, 9, 12, 13, 16, 17, 18, 19 , 20, 21, 24, 25, 26, 27, 30, 31, 32, 33, 36, 37, 40, 41, 42, 43, 45, 48, 49, 50, 51, 52, 53, 54, 57 , 58, 59, 61, 62, 63, 64, 66, 67, 68, 7O7 71, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 87, 88, 89, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 112, 114, 115, 116, 117, 118, 119, 120, 121, 124, 126, 128, 129, 130, 131, 132, 133.
61. 61. The package as described in any of claims 40 to 60, characterized because a plurality of probes are specific for the same type of HPV.
62. 62. The package as described in any of claims 40 to 61, characterized in that a plurality of probes are specific for each type of HPV that will be detected.
63. 63. The package as described in any of claims 61 or 62, characterized in that the plurality of probes are immobilized in the same region of the solid support.
64. 64. The package as described in any of claims 61 or 62, characterized in that each plurality of probes is immobilized in a region other than the solid support.
65. 65. The package as described in any of claims 62 to 64, characterized in that each specific probe of the same HPV type detects a different part of the HPV target sequence.
66. 66. The package as described in any of claims 40 to 65, characterized in that it is at least one kind of probe in the solid support in at least two different locations.
67. 67. The package as described in any of claims 40 to 66, characterized in that all the species of probes are in the solid support in at least two different locations.
68. 68. The package as described in any of claims 40 to 67, characterized in that it also comprises one or more control sequences in the solid support.
69. 69. The package as described in the claim 68, characterized in that the control sequence comprises a probe immobilized on the solid support, which does not hybridize to the target sequence of any type of HPV.
70. 70. The package as described in claim 68, characterized in that the control sequence comprises a human genomic target sequence.
71. 71. The package as described in claim 70, characterized in that the human target sequence comprises at least a part of the CFTR gene.
72. 72. Equipment for the detection and typing of HPV, characterized in that it comprises the reaction vessel of any of claims 40 to 71, in combination with one or more of the following: i) reagents for extraction and / or purification of DNA; ii) a mixture of nucleic acid amplification; iii) reagents for use in visualizing the hybridization of nucleic acids in the probes of the reaction container.
73. 73. The equipment as described in claim 72, characterized in that the amplification mixture is provided in a reaction vessel separated from the reaction vessel comprising the solid support with specific probes of the HPV type.
74. 74. The equipment as described in claim 72, characterized in that the amplification mixture is provided in the reaction container comprising the solid support with the specific probes of the HPV type.
75. 75. The equipment as described in any of claims 72 to 74, characterized in that the amplification mixture comprises labeled dNTPs.
76. 76. The kit as described in any of claims 72 to 75, characterized in that the amplification mixture comprises HPV consensus primers which hybridize to parts of the HPV target sequence.
77. 77. The equipment as described in the claim 76, characterized in that the HPV consensus primers comprise MY09 and MY11; and optionally HMB01.
78. 78. The equipment as described in any of claims 72 to 77, characterized in that the amplification mixture comprises primers to amplify a human objective sequence.
79. 79. The equipment as described in claim 78, characterized in that the human target sequence is one of a length different from that of the HPV target sequence.
80. 80. The equipment as described in any of claims 78 or 79, characterized in that the human target sequence is at least a part of the CFTR gene.
81. 81. The kit as described in claim 80, characterized in that the primers comprise at least one of CFTR-F4 (SEQ ID NO 134) and CFTR-R5 (SEQ ID NO 135).
82. 82. The equipment as described in any of claims 76 to 81, characterized in that the primers are labeled primers.
83. 83. The equipment as described in any of claims 72 to 82, characterized in that it comprises an objective sequence of control amplification.
84. 84. The equipment as described in claim 83, characterized in that the target sequence of the control amplification includes sequences that correspond to flanking portions of the human target sequence, so that the amplification of both target sequences will occur using the same primers .
85. 85. A probe for detecting and typing HPV, the probe of SEQ ID NO 1 to 133. being selected.
86. 86. The probe as described in the claim 85, selected from the following SEQ IDs: 2, 4, 7, 8, 9, 12, 13, 16, 17, 18, 19, 20, 21, 24, 25, 26, 27, 30, 31, 32, 33 , 36, 37, 40, 41, 42, 43, 45, 48, 49, 50, 51, 52, 53, 54, 57, 58, 59, 61, 62, 63, 64, 66, 67, 68, 70 , 71, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 87, 88, 89, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103 , 104, 105, 106, 107, 108, 109, 110, 112, 114, 115, 116, 117, 118, 119, 120, 121, 124, 126, 128, 129, 130, 131, 132, 133.
87. 87 A primer for use in CFTR amplification, wherein the primer is selected from CFTR-F4 (SEQ ID NO 134) and CFTR-R5 (SEQ ID NO 135).