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 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.
Schmitt
Markus
M
Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. markus.schmitt@dkfz.de
Bravo
I G
IG
Snijders
Peter J F
PJ
Gissmann
Lutz
L
Pawlita
Michael
M
Waterboer
Tim
T
D002584:Cervix Uteri / Q000821:virology; D005260:Female; D005838:Genotype; D006801:Humans; D008863:Microspheres; D015345:Oligonucleotide Probes; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000821:virology*; D016133:Polymerase Chain Reaction; D011137:Polystyrenes; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015345:Oligonucleotide Probes; D011137:Polystyrenes