HPV DNA detection and typing in cervical scrapes.
Polymerase chain reaction (PCR)-based assays that use consensus primers to detect DNA of a broad spectrum of human papillomavirus (HPV) types in a single assay belong to the most frequently used methods to detect HPV in clinical specimens. Here, we describe in detail one of these assays, the so-called GP5+/6+ PCR method, which can be used to detect and type HPV DNA in crude extracts of cervical scrapes and biopsy specimens. Following PCR with GP5+ and GP6+ primers, the latter of which is biotinylated at its 5' end, the presence of DNA of any of the high-risk genotypes can easily be determined by an enzyme immunoassay (EIA). In this assay, PCR products are captured in streptavidin-coated wells of a microtiter plate, denatured by alkaline treatment, and hybridized to cocktails of digoxigenin-labeled oligonucleotides specific for high-risk or low-risk HPV types. The resulting hybrids can then be detected by alkaline phosphatase conjugated anti-digoxigenin polyclonal antibodies and substrate followed by optical density reading. Subsequently, EIA-positive PCR products can be typed by a reverse line blot genotyping procedure that, using a miniblotter device, enables typing of up to 39 samples with specific oligonucleotide probes for 37 different HPV (sub)types in a single assay.
Snijders
Peter J F
PJ
Section Molecular Pathology, Department of Pathology, VU University Medical Center, The Netherlands.
van den Brule
Adriaan J C
AJ
Jacobs
Marcel V
MV
Pol
René P
RP
Meijer
Chris J L M
CJ
D001483:Base Sequence; D002584:Cervix Uteri / Q000821:virology*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005260:Female; D005914:Globins / Q000235:genetics; D006801:Humans; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral; D005914:Globins