Bead-Based Multiplex Genotyping of Human Papillomaviruses

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 genotyp...

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Bibliographic Details
Published in:Journal of Clinical Microbiology 2006-02, Vol.44 (2), p.504-512
Main Authors: Schmitt, Markus, Bravo, I. G, Snijders, Peter J. F, Gissmann, Lutz, Pawlita, Michael, Waterboer, Tim
Format: Article
Language:English
Subjects:
Biological and medical sciences
Cervix Uteri - virology
Female
Fundamental and applied biological sciences. Psychology
Genotype
Human papillomavirus
Humans
Infectious diseases
Medical sciences
Microbiology
Microspheres
Oligonucleotide Probes
Papillomaviridae - classification
Papillomaviridae - genetics
Papillomaviridae - isolation & purification
Papillomavirus Infections - virology
Polymerase Chain Reaction
Polystyrenes
Reproducibility of Results
Sensitivity and Specificity
Virology
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Summary: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.
ISSN:0095-1137
1098-660X
1098-5530
DOI:10.1128/jcm.44.2.504-512.2006