Comparison of variant-specific hybridization and single-strand conformational polymorphism methods for detection of...

PCR-based variant-specific hybridization (VSH) and single-strand conformational polymorphism (SSCP) analyses were compared for their capacities to detect mixed human papillomavirus type 16 (HPV-16) variant infections within clinical 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 open reading frame and identifies three categories of SSCP patterns: those identical to the patterns of prototype HPV-16 (P), those identical to the patterns of Caski-derived HPV-16 (C), or those that are different from the P and C HPV-16 patterns and that are therefore classified as belonging to novel (N) HPV-16 variants. VSH targets the entire HPV-16 E6-coding region (nucleotides 56 to 640) and distinguishes previously described variant nucleotides 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 independent amplification reactions. The resultant amplicons were cloned, and 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 variant 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 infections in these constructed amplicon targets. Accurate detection of HPV-16 variants may enhance our understanding of the natural history of HPV-16 infections.