Low-stringency single specific primer PCR, DNA sequencing and single-strand conformation polymorphism of PCR products for identification of genetic variants of human papillomavirus type 16

Abstract

Two fragments from within the long control region of the genome of human papillomavirus type 16 (HPV16) were amplified using the polymerase chain reaction (PCR). Putative genetic variation among the parent viruses was assessed by complete sequence analysis and single-strand conformation polymorphism (SSCP) analysis of one of the fragments and by application of the recently described low-stringency single specific primer (LSSP) PCR to both PCR products. The study comprised 34 HPV16 positive samples, derived from seventeen different individuals. It is demonstrated that under experimentally standardised conditions the LSSP PCR-, sequencing- and SSCP-data display differing degrees of resolution. Based on combined LSSP PCR analyses, 33 out of 34 samples can be discriminated, whereas SSCP and direct sequencing identify 4 and 8 types, respectively. Although the variability observed among LSSP PCR patterns may be the consequence of small quantities of mutated viral amplimers, no concordant grouping of strains, identical by sequencing and SSCP analysis, can be established by either of the four theoretically possible LSSP PCR assays. Results are discussed in the context of experimental variability of the procedures or genetic heterogeneity of HPV16 pools derived from cervical swabs.