HPV DNA Detection and Typing in Cervical Scrapes

Abstract

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.

Presence of high‐risk human papillomavirus DNA in penile carcinoma predicts favorable outcome in survival

“…5 Subsequent genotyping for 14 high-risk HPV types (i.e., 16,18,31,33,35,39,45, 51, 52, 56, 58, 59, 66, 68) was performed by reverse line blot analysis, as previously described. 23,24 The HPV detection and typing data were in complete agreement with those of the frozen tissue of the 53 cases for which frozen material of adequate quality was tested before. 5 Follow-up and statistical methods…”

“…23 Various measures were taken during sample preparation to avoid contamination. Gloves were worn throughout the cutting and preparation procedures; new blades were used for cutting new specimens, and the microtome was cleaned each time in between different specimens.…”

“…23 Samples of 5 patients were negative in the b-globin PCR and excluded from further HPV DNA analysis. The latter included 2 of the 55 patients of whom frozen tissue was previously analyzed, which scored b-globin PCR negative as well.…”

Detection of human papillomavirus in women attending Pap cervical screening camp at a peripheral hospital of North-Eastern India

“…6 Extracted DNA was confirmed by performing PCR of housekeeping gene coding for human interleukin e 1b promoter. HPV PCR was performed by using HPV MY-PCR primers (MY09/MY11) and positive samples were further subjected to HPV type specific PCR for HPV 16 and HPV 18.…”

Detección y tipificación de virus papiloma humano en adenocarcinoma de cuello uterino mediante reverse line blot, Región de La Araucanía, Chile

“…La técnica Reverse Line Blot utilizada para la tipificación de los genotipos virales, presenta ventajas, entre las cuales se destaca que permite discriminar entre HPV de alto y bajo riesgo oncogénico, en forma específica, permitiendo llevar a cabo un estudio de buena calidad experimental 20,27 . En este estudio sólo se registraron HPV de alto riesgo oncogénico.…”