Hpv detection and quantification by real-time multiplex amplification

Abstract

The present invention relates to amplification primers and detection probes, which are useful for the detection of human papillomaviruses (HPV), and more particularly of HPV, which can be oncogenic for the mucosal epithelia. The amplification and detection systems provided by the present invention are group-targeted systems, namely A5-, A6- A7-, and A9-targeted systems. The amplification and detection systems of the invention allow for an amplification of HPV in multiplex as well as for a real-time detection, whereby at least the thirteen HR HPV can be detected in a single-tube assay. The invention further allows for a reliable quantitation of HPV viral loads in real-time multiplex amplification.

We claim: 
     
         1 . A process for detecting in a sample at least one HPV, which can be oncogenic for the mucosal epithelia, which comprises contacting said sample or nucleic acid material thereof with HPV amplification primers to amplify at least one HPV nucleic acid from said sample or nucleic acid material by means of said HPV amplification primers and detecting the HPV amplicon(s) thereby produced, if any, wherein the production of at least one HPV amplicon indicates that at least one HPV, which can be oncogenic for the mucosal epithelia, is present in said sample, wherein said HPV amplification primers comprise
 i/ primers of 14-30 nucleotides, which are suitable for use as primer pair(s) in the amplification of at least one template polynucleotide, wherein one primer of a primer pair anneals to the 5′ terminal end of said at least one template polynucleotide and the other primer of the same primer pair anneals to the 5′ terminal end of the polynucleotide that is complementary to said at least one template polynucleotide, wherein said at least one template polynucleotide is selected from the group consisting of the HPV18 fragments, which comprise one of the sequences of SEQ ID NOs: 46-51 or the complementary sequence thereof or a fragment of said SEQ ID or complementary sequence, wherein said HPV18 fragments differ by at most 5 nucleotides in length from said SEQ ID or complementary sequence;   
       and
 ii/ primers of 14-30 nucleotides, which are suitable for use as primer pair(s) in the amplification of at least one template polynucleotide, wherein one primer of a primer pair anneals to the 5′ terminal end of said at least one template polynucleotide and the other primer of the same primer pair anneals to the 5′ terminal end of the sequence that is complementary to said at least one template polynucleotide, wherein said at least one template polynucleotide is selected from the group consisting of the HPV16 fragments, which comprise one of the sequences of SEQ ID NOs: 177-180, 189-192 and 195-198 or the complementary sequence thereof or a fragment of said SEQ ID sequence or complementary sequence, wherein said HPV16 fragments differ by at most 5 nucleotides in length from said SEQ ID or complementary sequence; 
 
       wherein said primers of i. are suitable for nucleic acid amplification from one or several HPV selected from the group consisting of HPV18, HPV45, HPV39, HPV59 and HPV68; and 
       wherein said primers of ii. are suitable for nucleic acid amplification from one or several HPV selected from the group consisting of HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58. 
     
     
         2 . The process of  claim 1 , wherein the primers of i. are suitable for nucleic acid amplification from at least HPV18 and HPV45. 
     
     
         3 . The process of  claim 1 , wherein the primers of i. are suitable for nucleic acid amplification from HPV18, HPV45, HPV39, HPV59 and HPV68. 
     
     
         4 . The process of  claim 1 , wherein the primers of ii. are suitable for nucleic acid amplification from at least HPV16, HPV31 and HPV33. 
     
     
         5 . The process of  claim 1 , wherein the primers of ii. are suitable for nucleic acid amplification from HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58. 
     
     
         6 . The process of  claim 1 , wherein the primers of i. are suitable for use as primer pairs in the amplification of several of said template polynucleotides. 
     
     
         7 . The process of  claim 1 , wherein the at least one template polynucleotide of the primers of ii. is selected from the group consisting of the polynucleotides of SEQ ID NOs: 169-210 and the complementary sequences thereof. 
     
     
         8 . The process of  claim 1 , wherein the primers of ii. are suitable for use as primer pairs in the amplification of several of said template polynucleotides. 
     
     
         9 . The process of  claim 1 , wherein a primer of the primer pair of i., or of at least one of the primer pairs of i., is at least 85% identical to the sequence of the same length that is the 5′ terminal end of said at least one template polynucleotide, and the other primer of the same primer pair is at least 85% identical to the sequence of the same length that is the 5′ terminal end of the polynucleotide that is complementary to said at least one template polynucleotide. 
     
     
         10 . The process of  claim 1 , wherein a primer of the primer pair of i., or of at least one of the primer pairs of i., is selected from the group consisting of the oligonucleotides of SEQ ID NOs: 68-70, and the other primer of the same primer pair is selected from the group consisting of the oligonucleotides of SEQ ID NOs: 79-81. 
     
     
         11 . The process of  claim 1 , wherein a primer of the primer pair of ii., or of at least one of the primer pairs of ii., is at least 85% identical to the sequence of the same length that is the 5′ terminal end of said at least one template polynucleotide, and the other primer of the same primer pair is at least 85% identical to the sequence of the same length that is the 5′ terminal end of the polynucleotide that is complementary to said at least one template polynucleotide. 
     
     
         12 . The process of  claim 1 , wherein a primer of the primer pair of ii., or of at least one of the primer pairs of ii., is selected from the group consisting of the oligonucleotides of SEQ ID NOs: 231-239, and the other primer of the same primer pair is selected from the group consisting of the oligonucleotides of SEQ ID NOs: 256-265. 
     
     
         13 . The process of  claim 1 , wherein a primer of the primer pair of ii., or of at least one of the primer pairs of ii., is selected from the group consisting of the oligonucleotides of SEQ ID NOs: 232, 234 and 235, and the other primer of the same primer pair is selected from the group consisting of the oligonucleotides of SEQ ID NOs: 258, 261, 264 and 265. 
     
     
         14 . The process of  claim 1 , wherein the primers of i. and the primers of ii. are in the same amplification container. 
     
     
         15 . The process of  claim 1 , wherein said detecting comprises contacting said amplicon or at least one of said amplicon(s) with at least one HPV-specific probe in real-time amplification. 
     
     
         16 . The process of  claim 1 , wherein said detecting comprises contacting said amplicon or at least one of said amplicon(s) with at least one HPV-specific probe, and wherein the oligonucleotide sequence of said at least one HPV-specific probe is at least one of SEQ ID NOs: 88-92 and 277-282, or one of the complementary sequences thereof, or a sequence, which differs by at most 5 nucleotides in length from said SEQ ID or complementary sequence and which is at least 90% identical to said SEQ ID or complementary sequence, said at least one HPV-specific probe being optionally linked to at least one detection label and/or to two complementary nucleotide sequences of 3 to 10 nucleotides, one of said two complementary nucleotide sequences of 3 to 10 nucleotides being linked at the 5′ end of said probe, the other of said two complementary nucleotide sequences of 3 to 10 nucleotides being linked at the 3′ end of said probe. 
     
     
         17 . The process of  claim 1 , wherein said detecting comprises contacting said amplicon or at least one of said amplicon(s) with at least one HPV-specific probe, and wherein said at least one HPV-specific probe consists of at least one of SEQ ID NOs: 102-106 and 304-319, or of one of the complementary sequences thereof, said at least one HPV-specific probe being optionally linked to at least one detection label. 
     
     
         18 . The process of  claim 1 , wherein said HPV amplification primers further comprise primers of 14-30 nucleotides, which are suitable for use as primer pair(s) in the amplification of at least one HPV selected from the group consisting of the oncogenic HPV belonging to HPV phylogenic groups A5 and A6. 
     
     
         19 . The process of  claim 1 , wherein said HPV amplification primers further comprise primers of 14-30 nucleotides, which are suitable for use as primer pair(s) in the amplification of at least one template polynucleotide, wherein one primer of a primer pair anneals to the 5′ terminal end of said at least one template polynucleotide and the other primer of the same primer pair anneals to the 5′ terminal end of the polynucleotide that is complementary to said at least one template polynucleotide, wherein said at least one template polynucleotide is selected from the group consisting of the HPV51 fragments, which comprise the sequence of SEQ ID NO: 5 or the complementary sequence thereof or a fragment of said SEQ ID or complementary sequence, wherein said HPV51 fragments differ by at most 5 nucleotides in length from said SEQ ID or complementary sequence. 
     
     
         20 . The process of  claim 1 , wherein said HPV amplification primers further comprise primers of 14-30 nucleotides, which are suitable for use as primer pair(s) in the amplification of at least one template polynucleotide, wherein one primer of a primer pair anneals to the 5′ terminal end of said at least one template polynucleotide and the other primer of the same primer pair anneals to the 5′ terminal end of the polynucleotide that is complementary to said at least one template polynucleotide, wherein said at least one template polynucleotide is selected from the group consisting of the HPV56 fragments, which comprise the sequence of SEQ ID NO: 25 or the complementary sequence thereof or a fragment of said SEQ ID or complementary sequence, wherein said HPV56 fragments differ by at most 5 nucleotides in length from said SEQ ID or complementary sequence. 
     
     
         21 . A process of production of HPV primers, which comprises producing at least two oligonucleotides, the respective sequences of which consist of 14-30 nucleotides each and are suitable for use as primer pair(s) in the amplification of at least one template polynucleotide, wherein said at least one template polynucleotide is at least one of the HPV18 fragments, which comprise one of the sequences of SEQ ID NOs: 46-51 or the complementary sequence thereof or a fragment of said SEQ ID or complementary sequence, wherein said HPV18 fragments differ by at most 5 nucleotides in length from said SEQ ID or complementary sequence, wherein one primer of a primer pair anneal to the 5′ terminal end of said at least one template polynucleotide and the other primer of the same primer pair anneal to the 5′ terminal end of the polynucleotide that is complementary to said at least one template polynucleotide, and wherein said primer pair(s) anneals(anneal) to at least two HPV selected from the group consisting of HPV16, HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67. 
     
     
         22 . A process of production of HPV primers, which comprises producing at least two oligonucleotides, the respective sequences of which consist of 14-30 nucleotides each and are suitable for use as primer pair(s) in the amplification of at least one template polynucleotide, wherein said at least one template polynucleotide is at least one the HPV16 fragments, which comprise one of the sequences of SEQ ID NOs: 177-180, 189-192 and 195-198 or the complementary sequence thereof or a fragment of said SEQ ID sequence or complementary sequence, wherein said HPV16 fragments differ by at most 5 nucleotides in length from said SEQ ID or complementary sequence, wherein one primer of a primer pair anneal to the 5′ terminal end of said at least one template polynucleotide and the other primer of the same primer pair anneal to the 5′ terminal end of the polynucleotide that is complementary to said at least one template polynucleotide, and wherein said primer pair(s) anneals(anneal) to at least two HPV selected from the group consisting of HPV18, HPV45, HPV39, HPV59 and HPV68.