DNA extraction: An understudied and important aspect of HPV genotyping using PCR-based methods

Testing for the group of ∼15 carcinogenic human papillomavirus (HPV) genotypes is an important adjunct to cytology. Because carcinogenic strengths of HPV types differ greatly, assays that permit identification of individual HPV genotypes are being introduced. Most HPV genotyping systems proposed for clinical use are PCR-based, depending heavily for validity on careful attention to numerous details. One understudied detail is the effect of different sample preparation methods including DNA extraction. This study examines the influence of DNA extraction on performance of a new PCR-based genotyping kit, the Roche LINEAR ARRAY® HPV assay. When volume of sample extracted, DNA extraction methods and/or amount of DNA tested were varied, the HPV type results were reproducible for strong viral bands but not weak ones. Moreover, although the experiments were not comprehensive, they showed that the manufacturer-approved DNA extraction method might not be the best method for use in this assay. Because different “front end” protocols introduce variability into genotyping results, the authors urge laboratories not to vary methods for this assay without due consideration. The results suggest that companies carefully optimize DNA extraction methods prior to commercial introduction of their PCR-based genotyping assays destined for widespread clinical use.