Quantitative multiplex PCR assay for the detection of the seven clinically most relevant high-risk HPV types

BackgroundHigh-risk HPV DNA detection has become a valuable tool for the triage of borderline, questionable and abnormal cytologic findings in cervical carcinoma screening programs. This knowledge is largely based on studies which could only discriminate between low-risk (LR-) and high-risk (HR-) HPV groups. However, it is becoming increasingly clear that HPV genotyping may allow further risk stratification and may offer different treatment options in the future.ObjectivesTo establish a fast and cost-effective system not only for genotyping but also for quantification of viral DNA.Study designDevelopment and validation of a 5′ exonuclease fluorescent probe multiplex real-time PCR assay (TaqMan format) for the detection and quantification of the 7 most frequent HR-HPV types (16, 18, 31, 33, 45, 52 and 58) which account for over 87% of cervical carcinomas world-wide. Two PCR reactions are required to detect the designated HPV types.ResultsExperiments with plasmid constructs of all 18 HR-HPV DNA showed that the multiplex real-time PCR assay was highly sensitive and specific. Evaluation of DNA extracted from archived cell pellets of cervical scrapes by the multiplex assay and the GP5+/6+-EIA showed identical genotyping for 234 of 261 (89.6%) samples and an almost perfect agreement when considering all typing results (kappa 0.901). Viral load did not correlate with disease progression within the CIN spectrum but significant differences were evident when comparing all CIN with the group lacking CIN (p = 0.0028) or with the cancer group (p = 0.0001).ConclusionOur multiplex assay will be useful to address questions related to viral persistence at the genotype level, the kinetics of viral load and disease recurrence.