A novel and rapid PCR-based method for genotyping human papillomaviruses in clinical samples

Many human papillomavirus (HPV) genotypes are associated with cervical carc inoma, We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specime ns. By using the MY09-MY11 and the GP5(+)-GP6(+) consensus primer pairs, HP V sequences were amplified by nested PCR from DNA isolated from cervical sm ear samples. This led to the production of an approximately 140-bp PCR prod uct from the L1 (major capsid) gene of any of the HPVs present in the sampl e, PCR was performed with a deoxynucleoside triphosphate mixture which resu lted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequen cy of about once or twice per strand, Following the PCR, the product was tr eated with an enzyme mb that contains uracil N-glycosylase (UNG) and endonu clease IV. UNG removes the uracil base from the nucleotide, and endonucleas e IV cleaves the phosphodiester bond at this newly formed abasic site, prod ucing fragments of various sizes. BF having end labeled one of the amplific ation primers, a DNA ladder which is analogous to a "T-sequencing ladder" w as produced upon electrophoresis of the products. By comparing this T-seque ncing ladder to the known sequences of HPVs the genotypes of unknown IBV is olates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.