Bead-based multiplex genotyping of human papillomaviruses

Abstract

Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.

FIG. 1.

Schematic overview of HPV genotyping of GP5+/6+-PCR products by bead-based multiplex HPV genotyping. ORF, open reading frame. (Picture of the Luminex analyzer reproduced from a tutorial on the Luminex Corp. website with permission.)

FIG. 2.

Detection limit analysis of the HPV-16 probe on twofold dilutions of HPV-16 GP5+/6+-PCR products. The graph displays the fit of the experimental values to a hyperbola. The inset shows the linear fitting (R² = 0.991) of the five smallest amounts of PCR products in double-logarithmic scale. The detection limit was determined using the cutoff of 10 MFI.