Type-specific sensitivity versus detection breadth in HPV diagnostics: improved analytical detection of HPV16/18 by nested PCR

Accurate detection of high-risk human papillomavirus (HPV) is central to cervical cancer screening. Current diagnostic strategies increasingly prioritize broad-spectrum assays to maximize genotype coverage. However, theoretical genotype breadth does not necessarily translate into effective sample-level detection when clinically dominant targets are present at low copy number. Here, we evaluated this analytical trade-off using conventional PCR as a controlled experimental framework. Type-specific nested PCR assays targeting HPV16 and HPV18 were developed and applied to 41 cervical samples. Nested PCR showed markedly higher relative analytical sensitivity than single-round amplification and produced a greater proportion of specific and interpretable detections than the broad-spectrum consensus systems tested here (MY09/11 and GP5/6). HPV16 and/or HPV18 DNA was detected by nested PCR in 22/41 samples, whereas MY09/11 and GP5/6 detected 11/41 and 2/41 samples, respectively, under the tested conditions. These findings indicate that broad genotype coverage and effective detection of positive samples are distinct analytical properties. In settings where prevalent HPV targets occur at low abundance, a highly sensitive genotype-focused assay may reveal more target-positive samples than a broader but less sensitive consensus approach. The results are not intended to replace clinically validated broad-spectrum screening assays, but they support explicit preservation of per-target analytical sensitivity in reflex testing and future multi-target HPV assay design.