General primer polymerase chain reaction in combination with sequence analysis for identification of potentially novel human papillomavirus genotypes in cervical lesions

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Abstract
We recently described the detection of potentially novel human papillomaviruses (HPV) genotypes (HPV types X [HPV X]) in cervical smears (A. J. C. van den Brule, C. J. L. M. Meijer, V. Bakels, P. Kenemans, and J. M. M. Walboomers, J. Clin. Microbiol. 28:2739-2743, 1990) by using the general primer-mediated polymerase chain reaction method (GP-PCR). In this study, the HPV specificities of GP-PCR products were determined by sequence analyses. M13 bacteriophage clones of PCR products derived from cloned unsequenced HPV genotypes 13, 32, 35, 43, 44, 45, 51, and 56 were subjected to dideoxy sequencing. Analyses of the putative amino acid sequences of these HPV types in addition to published HPV sequence data revealed stretches of highly conserved amino acid residues present in all HPV types, resulting in an HPV amino acid consensus sequence. Subsequently, HPV X-specific PCR products found in premalignant cervical lesions (n = 3), carcinomas in situ (n = 6), and invasive cancer (n = 6) were analyzed for their nucleotide sequences. Comparison of these sequences with published HPV nucleotide sequences and data obtained in this study revealed three HPV type 35, two HPV type 45, one HPV type 51, two HPV type 56, and six unique HPV X sequences, of which three types were present in four cases of carcinomas (in situ). The nucleotide sequences determined appeared to be unique after a data bank search. Furthermore, the sequences of all HPV X isolates matched the HPV amino acid consensus sequence, thus confirming HPV specificity. This study illustrates the power of GP-PCR in combination with sequence analysis to determine HPV specificity and genotyping of PCR products derived from sequenced as well as unsequenced HPVs, including novel, not yet identified HPV types.