A multiplex real-time PCR-platform integrated into automated extraction method for the rapid detection and...

The persistent infection with most frequent high-risk (HR)-HPV types (HPV-16, -18, -31, -33, -45, -52, and -58) is considered to be the true precursor of neoplastic progression. HR-HPV detection and genotyping is the most effective and accurate approach in screening of the early cervical lesions and cervical cancer, although also the HR-HPV DNA load is considered an ancillary marker for persistent HPV infection. Here, it is described an in-house multiplex quantitative real-time PCR (qPCR)-based typing system for the rapid detection and quantitation of the most common HR-HPV genotypes from cervical cytology screening tests. First, a separate qPCR assay to quantify a single-copy gene is recommended prior to screening (prescreening assay) to verify the adequate cellularity of the sample and the quality of DNA extracted and to normalize the HPV copy number per genomic DNA equivalent in the sample. Subsequently, to minimize the number of reactions, two multiplex qPCR assays (first line screening) are performed to detect and quantify HPV-16, -18, -31, -33, -45, -52, and -58 (HPV-18 and -45 are measured together by single-fluorophore). In addition, a multiplex qPCR assay specific for HPV-18 and HPV-45 is also available to type precisely the samples found to be positive for one of the two strains. Finally, two nucleic acid extraction methods are proposed by using a 96-well plate format: one manual method (supported by centrifuge or by vacuum) and one automated method integrated into a robotic liquid handler workstation to minimize material and hands-on time. In conclusion, this system provides a reliable high-throughput method for the rapid detection and quantitation of HR-HPV DNA load in cervical samples.