Comparison of variant-specific hybridization and single-strand conformational polymorphism methods for detection of mixed human papillomavirus type 16 variant infections

PCR-based variant-specific hybridization (VSH) and single-strand conformati onal polymorphism (SSCP) analyses were compared for their capacities to det ect mixed human papillomavirus type 16 (HPV-16) variant infections within c linical specimens. The SSCP assay used in this comparison targets a 682-bp fragment that spans nucleotides 7445 to 222 within the HPV-16 genome. This fragment includes portions of the HPV-16 long control region and the E6 ope n reading frame and identifies three categories of SSCP patterns: those ide ntical to the patterns of prototype HPV-16 (]P), those identical to the pat terns of Caski-derived HPV-16 (C), or those that are different from the P a nd C HPV-16 patterns and that are therefore classified as belonging to nove l (N) HPV-16 variants. VSH targets the entire HPV-16 E6-coding region (nucl eotides 56 to 640) and distinguishes previously described variant nucleotid es at positions 109, 131, 132, 143, 145, 178, 286, 289, 350, 403, and 532. Clinical samples used in VSH and SSCP analyses were subjected to multiple i ndependent amplification reactions. The resultant amplicons were cloned, an d 14 to 78 clones per clinical specimen were evaluated by VSH. VSH detected an HPV-16 variant that represented at least 20% of the amplified HPV-16 va riant population. In contrast, SSCP analysis detected HPV-16 variants that represented 36% of the amplified HPV-16 population. Comparison studies were conducted with mixed HPV-16 variant laboratory constructs. Again, VSH had a higher sensitivity than SSCP analysis in detecting mixed HPV-16 variant i nfections in these constructed amplicon targets. Accurate detection of HPV- 16 variants may enhance our understanding of the natural history of HPV-16 infections.