A real-time polymerase chain reaction assay detects Human Papillomavirus (HPV) enabling discrimination of all high-risk HPV types in a clinical high-throughput setting.

This real-time quantitative polymerase chain reaction assay (qPCR) can be routinely applied in a liquid-based cytology screening setting and can be used in many epidemiological and clinical studies.

Scientists at the University of Antwerp (Belgium) have described the laboratory workflow and the validation of a type-specific qPCR assay for high-throughput HPV detection, genotyping, and quantification. The optimization of each multiplex PCR was based on plasmids for each HPV type and female human DNA. Numerous trials were performed to evaluate the primers, probes, fluorochromes, cycling parameters and the effect of background DNA.

DNA was extracted from cervical cells that had been collected into the ethanol-based preservative Surepath TM (Tripath Imaging; Burlington, NC, USA) using the Cervex-Brush (Rovers Medical Devices B.V.; KV Oss, The Netherlands), and after manipulation, the DNA was amplified in the LightCycler 480 thermocycler (Roche Applied Science; Basel, Switzerland).

The TaqMan-based qPCR assay enables the detection of 17 HPV genotypes and beta-globin in seven multiplex reactions. These HPV types include all 12 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), three probably high-risk types (HPV53, 66 and 68), one low-risk type (HPV6) and one undetermined risk type (HPV67). The analytical sensitivity of less than 100 copies was obtained for all the HPV types. The analytical specificity of each primer pair was 100% and an intra- and inter-run variability of less than 6.4% was observed.

Worldwide, cervical cancer is the third most common female cancer. In Western Europe, it is only the 15th most common cause of cancer death in women as a result of cytology-based screening. However, the efficacy of cytological screening is hampered by the high inter-observer variability and high false positive and false-negative rates. The authors concluded that the type-specific real-time PCR approach enables detection of 17 HPV types, identification of the HPV type and determination of the viral load in a single sensitive assay suitable for high-throughput screening. The study was published online on April 5, 2012, in the journal Clinical Chemistry and Laboratory Medicine.

Related Links:
University of Antwerp
Rovers Medical Devices
Roche Applied Science