A novel strategy for human papillomavirus detection and genotyping with SybrGreen and molecular beacon polymerase chain reaction.
Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV types found in (pre)malignant lesions, a robust methodology is needed that combines general HPV detection with HPV genotyping. We have developed for formaldehyde-fixed samples a strategy that, in a homogeneous, real-time fluorescence polymerase chain reaction (PCR)-based assay, accomplishes general HPV detection by SybrGreen reporting of HPV-DNA amplicons, and genotyping of seven prevalent HPV types (HPV-6, -11, -16, -18, -31, -33, -45) by real-time molecular beacon PCR. The false-positive rate of the HPV SybrGreen-PCR was 4%, making it well suited as a prescreening, general HPV detection technology. The type specificity of the seven selected HPV molecular beacons was 100% and double infections were readily identified. The multiplexing capacity of the HPV molecular beacon PCR was analyzed and up to three differently labeled molecular beacons could be used in one PCR reaction without observing cross talk. The inherent quantitation capacities of real-time fluorescence PCR allowed the determination of average HPV copy number per cell. We conclude that the HPV SybrGreen-PCR in combination with the HPV molecular beacon PCR provides a robust, sensitive, and quantitative general HPV detection and genotyping methodology.
Szuhai
K
K
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
Sandhaus
E
E
Kolkman-Uljee
S M
SM
Lemaître
M
M
Truffert
J C
JC
Dirks
R W
RW
Tanke
H J
HJ
Fleuren
G J
GJ
Schuuring
E
E
Raap
A K
AK
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D005456:Fluorescent Dyes; D018628:Gene Dosage; D016678:Genome; D005838:Genotype; D006367:HeLa Cells; D006801:Humans; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D005456:Fluorescent Dyes