Detection and identification of human papillomavirus by PCR and type-specific reverse hybridization

Abstract

A method for detection and/or identification of HPV present in a biological sample comprising amplification of HPV polynucleic acids and of hybridization of said amplified polynucleic acids to a number of probes whereby a short fragment of the L 1 gene of HPV is amplified after which, the amplimers are contacted with probes that specifically hybridize to the said short fragment of the L 1 gene of at least one HPV type and a diagnostic kit to perform said method and primers and probes used in the said method.

1 . Method for detection and/or identification of HPV present in a biological sample, comprising the following steps: 
 (i) amplification of a polynucleic acid fragment of HPV by use of: 
 a 5′-primer specifically hybridizing to the A region or B region of the genome of at least one HPV type, said A region or B region being indicated in FIG. 1, and,  
 a 3′-primer specifically hybridizing to the C region of the genome of at least one HPV type, said C region being indicated in FIG. 1;  
   (ii) hybridizing the amplified fragments from step (i) with at least one probe capable of specific hybridization with the D region of at least one HPV type, said D region being indicated in FIG. 1.    
     
     
         2 . Method according to  claim 1 , characterized further in that: 
 the 3′-end of said 5′-primer specifically hybridizing to the A region of the genome of at least one HPV type, is situated at position 6572 of the genome of HPV 16, or at the corresponding position of any other HPV genome, as indicated in FIG. 1, and/or,    the 3′-end of said 5′-primer specifically hybridizing to the B region of the genome of at least one HPV type, is situated at position 6601 of the genome of HPV 16, or at the corresponding position of any other HPV genome, as indicated in FIG. 1, and/or,    the 3′-end of said 3′-primer specifically hybridizing to the C region of the genome of at least one HPV type, is situated at position 6624 of the genome of HPV 16, or at the corresponding position of any other HPV genome, as indicated in FIG. 1.    
     
     
         3 . A method according to  claim 2 , characterized further in that: 
 said 5′-primer specifically hybridizing to the A region is chosen from the following list: 
 SGP3 (SEQ ID NO 2), SGP3A (SEQ ID NO 3), SGP3B (SEQ ID NO 4), SGP3C (SEQ ID NO 10), SGP3D (SEQ ID NO 11), SGP3E (SEQ ID NO 12), SGP3F (SEQ ID NO 13), SGP3G (SEQ ID NO 14), and/or,  
   said 5-primer specifically hybridizing to the b region is chosen from the following list: 
 SGP1 (SEQ ID NO 6), SGP1A (SEQ ID NO 15), SGP1B (SEQ ID NO16), SGP1C (SEQ ID NO 17), SGP1D (SEQ ID NO 18), and/or,  
   said 3′-primer specifically hybridizing to the C region is chosen from the following list: 
 SGP2 (SEQ ID NO 9), SGP2A (SEQ ID NO 8), SGP2B (SEQ ID NO 19), SGP2C (SEQ ID NO 20), SGP2D (SEQ ID NO 21), SGP2E (SEQ ID NO 22), SGP2F (SEQ ID NO 23), SGP2H (SEQ ID NO 98), SGP2I (SEQ ID NO 154), SGP2J (SEQ ID NO 155), SGP2K (SEQ ID NO 156), SGP2L (SEQ ID NO 157), SGP2M (SEQ ID NO 158), SGP2N (SEQ ID NO 159), SGP2P (SEQ ID NO 160).  
   
     
     
         4 . Method according to any of  claims 1  to  3 , characterized further in that said probe mentioned in step (ii) is capable of specific hybridization with the D region of the genome of only one HPV type, and thus enables specific identification of this HPV type, when this type is present in a biological sample.  
     
     
         5 . Method according to any of  claims 1  to  3 , characterized further in that said probe mentioned in step (ii) is capable of specific hybridization with the D region of more than one HPV type, and thus enables detection of any of said more than one HPV type, when any of said types is present in a biological sample.  
     
     
         6 . Method according to  claim 4 , characterized further in that said probe capable of specific hybridization with the D region of the genome of only one HPV type, more particularly hybridizes to the E region, with said E region being a subregion of the D region, as indicated in FIG. 1.  
     
     
         7 . Method according to  claim 4 , characterized further in that said probe capable of specific hybridization with the D region of the genome of only one HPV type, more particularly specifically hybridizes to the 22 bp region situated between the B region and the C region, as indicated in FIG. 1.  
     
     
         8 . Method according to  claim 7 , characterized further in that said probe specifically hybridizing to said 22 bp region of only one HPV type is chosen from the following list: 
 HPV6 Pr1, HPV6 Pr2, HPV6 Pr3, HPV6 Pr4, HPV6 Pr5, HPV11 Pr1, HPV11 Pr2, HPV11 Pr3, HPV11 Pr4, HPV11 Pr5, HPV16 Pr1, HPV16 Pr2, HPV16 Pr3, HPV16 Pr4, HPV16 Pr5, HPV18 Pr1, HPV18 Pr2, HPV18 Pr3, HPV18 Pr4, HPV18 Pr5, HPV31 Pr1, HPV31 Pr2, HPV31 Pr3, HPV31 Pr4, HPV31 Pr5, HPV31 Pr21, HPV31 Pr22, HPV31 Pr23, HPV31 Pr24, HPV31 Pr25, HPV31 Pr26, HPV31 Pr31, HPV31 Pr32, HPV33 Pr1, HPV33 Pr2, HPV33 Pr3, HPV33 Pr4, HPV33 Pr5, HPV33 Pr21, HPV33 Pr22, HPV33 Pr23, HPV33 Pr24, HPV33 Pr25, HPV33 Pr26, HPV40 Pr1, HPV45 Pr1 (=SGPP68), HPV45 Pr2, HPV45 Pr3, HPV45 Pr4, HPV45 Pr5, HPV45 Pr11, HPV45 Pr12, HPV45 Pr13, HPV52 Pr1, HPV52 Pr2, HPV52 Pr3, HPV52 Pr4, HPV52 Pr5, HPV52 Pr6, HPV56 Pr1, HPV56 Pr2, HPV56 Pr3, HPV56 Pr11, HPV56 Pr12, HPV58 Pr1, HPV58 Pr2, HPV58 Pr3, HPV58 Pr4 (SEQ ID NOs 24 to 91), and, SGPP35, SGPP39, SGPP51 (=HPV51 Pr1), SGPP54, SGPP59, SGPP66, SGPP70 (=HPV70 Pr1), SGPP13, SGPP34, SGPP42, SGPP43, SGPP44, SGPP53, SGPP55, SGPP69, SGPP61, SGPP62, SGPP64, SGPP67, SGPP74 (=HPV74 Pr13), MM4 (=HPVM4 Pr11), MM7, MM8 (SEQ ID NOs 92 to 115), and,    HPV18b Pr1, HPV18b Pr2, HPV31 Vs40-1, HPV31 Vs40-2, HPV31 Vs40-3, HPV34 Pr1, HPV35 Pr1, HPV35 Pr2, HPV35 Pr3, HPV39 Pr1, HPV42 Pr1, HPV42 Pr2, HPV43 Pr1, HPV43 Pr2, HPV43 Pr3, HPV44 Pr1, HPV44 Pr2, HPV44 Pr3, HPV44 Pr4, HPV51 Pr2, HPV53 Pr1, HPV54 Pr1, HPV54 Pr11, HPV54 Pr11as, HPV54 Pr12, HPV55 Pr1, HPV55 Pr11, HPV55 Pr12, HPV55 Pr13, HPV56 Vs74-1, HPV59 Pr1, HPV59 Pr11, HPV59 Pr12, HPV59 Pr13, HPV66 Pr1, HPV67 Pr1, HPV67 Pr11, HPV67 Pr12, HPV67 Pr13, HPV67 Pr21, HPV67 Pr22, HPV67 Pr23, HPV68 Pr1, HPV68 Pr2, HPV68 Pr3, HPV68 Vs45-1, HPV68 Vs45-2, HPV70 Pr1, HPV70 Pr12, HPV70 Pr13, HPV74 Pr1, HPV74 Pr11, HPV74 Pr12, HPV74 Pr2, HPV74 Pr3, HPVM4 Pr1, HPVM4 Pr12, HPVM4 Pr21, HPVM4 Pr22 (SEQ ID NOs 161 to 219).    
     
     
         9 . Method according to  claim 5 , characterized further in that said probe capable of specific hybridization with the D region of the genome of more than one HPV type, more particularly hybridizes to the E region, with said E region being a subregion of the D region, as indicated in FIG. 1.  
     
     
         10 . Method according to  claim 9 , characterized further in that said probe specifically hybridizing to said E region of more than one HPV type, is chosen from the following list: 
 HPVuni1, HPVuni2, HPVuni3, HPVuni4, HPVuni5, HPVuni6, HPVuni7, HPVuni2L2, HPVuni2L3, HPVuni2L4, HPVuni2L5, HPVuni2L6, HPVuni2L7, HPVuni4L1, HPVuni4L2, HPVuni4L3, HPVuni4L4, HPVuni4L5, HPVuni4L6 (SEQ ID NOs 116 to 134), and,    HPVuni1A, HPVuni1B, HPVuni1C, HPVuni2A, HPVuni3A (SEQ ID NOs 220 to 224), and,    HPV G1, HPV G1A1, HPV G1A2, HPV G1A3, HPV G1A4, HPV G2, HPV G3, HPV G4, HPV G5, HPV G6, HPV R1, HPV R10, HPV R11, HPV R2, HPV R3, HPV R4, HPV R5, HPV R6, HPV R7, HPV R8, HPV R9 (SEQ ID NOs 225 to 245).    
     
     
         11 . A primer as defined in any of  claims 1  to  3 , for use in the detection and/or identification of HPV present in a biological sample.  
     
     
         12 . A primer combination consisting of a 5′-primer as defined in any of  claims 1  to  3  and of a 3′-primer as defined in any of  claims 1  to  3 , for use in the detection and/or identification of HPV present in a biological sample.  
     
     
         13 . A probe as defined in any of claims  1  and  4  to  10 , for use in the detection and/or identification of HPV present in a biological sample.  
     
     
         14 . A diagnostic kit for detection and/or identification of HPV, possibly present in a biological sample, comprising the following components: 
 (i) at least one suitable primer, with said primers being defined in any of  claims 1  to  3 ;    (ii) at least one suitable probe, with said probes being defined in any of claims  1  and  4  to  10 .    
     
     
         15 . An isolated HPV polynucleic acid, defined by SEQ ID NO 135 to 153, or any fragment thereof, that can be used as a primer or as a probe in a method for detection and/or identification of HPV present in a sample.