Multiplexed PCR genotyping of HPVs from plantaris verrucae

Abstract

Background Plantaris verrucae are a common diagnosis in childhood, consume a significant amount of health-care resources, have many painful treatment options and many recurrences. Objectives The objective of this study was to design and test a single site-anchored, multiplexed and expandable PCR assay for common types of cutaneous HPVs. Study design Common forward and unique reverse primers were selected from the E2 open reading frames of five cutaneous HPV genotypes. These were analyzed for sensitivity and selectivity using pHPV plasmids and several control DNAs in an optimized multiplexed assay. This standardized assay was used to analyze human verruca plantaris tissue for genome type and to evaluate the effect of a commonly used treatment protocol. Results A sensitive, multiplexed PCR assay for human cutaneous HPV genotypes 1a, 2a and 4 was developed. Specific-unique primers and a consensus anchor primer were selected within the HPV E2 region to produce amplicons varying by greater than 100 bp. In analytical sensitivity studies, fewer than 100 genome copies of HPV1a and 2a were detected, and fewer than 1000 copies of HPV4 were detected. The multiplexed assay did not amplify regions of human placenta, calf thymus, CaSki or SiHa DNA and E. coli, pBR322 or non-HPV virus DNAs. In combination with a forensic DNA extraction procedure, the multiplexed HPV assay detected and identified HPV types in 23 of 51 (45%) deep plantaris verrucae. Two patients were found with two different genotypes in single deep plantaris verruca. Detection of the HPV genome was followed as a function of tissue ablation and Mediplast treatment in one patient. In healing tissue, the genome content was reduced but had not totally disappeared. Conclusions The multiplexed HPV assay can be used to determine genotype prevalence that may correlate with treatment efficacy.