Genotyping of the 25 beta papillomavirus types.
Summary and explanation
Papillomaviruses (PV) constitute a group of double-stranded DNA viruses associated with benign and malignant lesions of cutaneous and mucosal epithelia. So far, 118 different PV genotypes have been identified of which approximately 48 HPV types are detected in human cutaneous lesions (de Villiers, Fauquet, et al. 2004). The HPV types infecting the cutaneous epithelium can roughly be subdivided in classic wart inducing types and the so-called epidermodysplasia verruciformis (EV) related HPV types. EV is a rare syndrome characterized by numerous EV-HPV-induced warts that display a highrisk of progression to squamous cell carcinoma (SCC) at sun-exposed skin. The EV-HPV types belong to the beta papillomavirus genus, formerly known as the B1 group, comprising papillomavirus types 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92*, 93 and 96*. (*=candidates). In contrast to the presence of multiple HPV types in the benign lesions, mostly HPV type 5 and 8 have been detected in the SCCs of EV-patients and may be regarded as high-risk types.
Although ultraviolet radiation is the most important causative factor of cutaneous SCC in the general population, recent (sero-) epidemiological and molecular-biological evidence suggest that specific high-risk EV-HPV types are also involved. The role that these viruses play in the etiology of cutaneous SCC seems to be different from the role that genital HPV types play in cervical cancer. Cutaneous SCC is probably caused by an interaction between the EV-HPV types and ultraviolet (UV) radiation. The E6 protein of some EV-HPV types has been shown to impair the process of DNA-repair and to prevent apoptosis of cells exposed to UV radiation. This may lead to survival of UV damaged cells. The resulting genetic instability of these cells in combination with HPV-induced extended cell proliferation due to the action of the E7 viral protein may lead to actinic keratoses, which are considered as premalignant lesions, and finally to SCC. At the later stages of carcinogenesis EV-HPV does not seem to play an important role. There is a strong need to perform large epidemiological studies to differentiate the role of individual HPV types in this early stage of cutaneous SCC. In the last two decades, several PCR-primer sets have been used detect EV-HPV types in skin biopsies, plucked hairs and skin swabs. EV-HPV genotyping is generally performed by broad spectrum PCR methods combined with cloning and sequencing and/or type-specific PCRs. However, when the detection and typing of all the 25 known beta papillomaviruses in a large epidemiological study is required, these methods are too laborious. Recently, a novel general primer set, designated PM has been developed. It targets the relatively conserved E1 open reading frame that is essential for DNA replication. The resulting PCR product is 117 base pairs long and suitable for the genotyping of the 25 known beta papillomavirus types.
Principles of the procedure
The RHA skin (beta) HPV genotyping test is based on the reverse hybridization principle. Part of the E1 region of the beta papillomavirus genome is amplified by PCR, and denatured biotinylated amplicons are hybridized with specific oligonucleotide probes, which are immobilized as parallel lines on membrane strips. After hybridization and stringent washing, streptavidin-conjugated alkaline phosphatase is added and bound to any biotinylated hybrid previously formed. Incubation with BCIP/NBT chromogen yields in a purple precipitate and the results can be visually interpreted.
Samples which are suitable for testing are: DNA purified from eyebrow hairs, (skin) biopsies, formalin-fixed paraffin embedded (skin) biopsies and skin swabs in saline solution.
REF: S-1014 RHA kit Skin (beta) HPV 20 tests
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