A Novel and Rapid PCR-based Method for Genotyping Human Papillomaviruses in Clinical Samples

Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovativetechnique for genotyping of HPV in cervical tissue samples. Thismethod provides an accurate means of identification of the specificHPV genotypes present in clinical specimens. By using the MY09-MY11and the GP5+-GP6+ consensus primer pairs, HPV sequences were amplified by nestedPCR from DNA isolated from cervical smear samples. This led tothe production of an approximately 140-bp PCR product from theL1 (major capsid) gene of any of the HPVs present in the sample.PCR was performed with a deoxynucleoside triphosphate mixturewhich resulted in the incorporation of deoxyuridine into the amplifiedDNA product at positions where deoxythymidine would normally beincorporated at a frequency of about once or twice per strand.Following the PCR, the product was treated with an enzyme mixthat contains uracil N-glycosylase (UNG) and endonuclease IV.UNG removes the uracil base from the nucleotide, and endonucleaseIV cleaves the phosphodiester bond at this newly formed abasicsite, producing fragments of various sizes. By having end labeledone of the amplification primers, a DNA ladder which is analogousto a T-sequencing ladder was produced upon electrophoresis ofthe products. By comparing this T-sequencing ladder to the knownsequences of HPVs, the genotypes of unknown HPV isolates in sampleswere assigned. Data showing the utility of this technique forthe rapid analysis of clinical samples arepresented