Development of a Sensitive and Specific Multiplex PCR Method Combined with DNA Microarray Primer Extension To Detect Betapapillomavirus Types

Abstract

Emerging lines of evidence indicate that the cutaneous human papillomavirus (HPV) types that belong to the genusThe family of the epitheliotropic human papillomaviruses (HPVs) comprises approximately 100 different types that have been subgrouped into different genera according to their genomic DNA sequence (9). In addition, the HPVs can be subdivided into mucosal and cutaneous HPVs based on their tissue tropism. Members of the genus Alphapapillomavirus, referred to as mucosal high-risk HPV types, have been clearly linked to cervical cancer (23). Emerging lines of evidence indicate that another group of HPVs that belong to the genus Betapapillomavirus (beta HPV) may also be involved in human carcinogenesis, i.e., nonmelanoma skin cancer (NMSC) (reviewed in reference 16). They were first isolated in skin cancerprone patients suffering from a rare autosomal recessive genetic disorder called epidermodysplasia verruciformis, but it is now clear that they are commonly found in the skin of healthy individuals (16). Twenty-five beta HPV types have been identified and sequenced so far, namely, HPV type 5 (HPV5), 8, 9, 12, 14, 15, 17, 19 to 25, 36 to 38, 47, 49, 75, 76, 80, 92, 93, and 96. However, with the development of more sensitive HPV detection methods, it is now evident that many additional beta HPV types exist.Several methods have been developed for the detection of cutaneous HPV types. Shamanin et al. (19,20) described the degenerate primer pair, HPV2/B5, to detect HPVs from several genera with the exception of the phylogenetic clade comprising HPV4, HPV48, HPV50, HPV60, and HPV65, for which the primer pair F14/B15 was used (1). A nested PCRbased protocol implies the use of several primer sets and detects beta and non-beta cutaneous HPV types (5). Forslund et al. have designed a single pair of degenerate PCR primers in the two relatively conserved regions of the L1 open reading frame that can detect a large spectrum of cutaneous HPV types (10). The limitation of the methods described above is that the identification of the HPV types is acquired by DNA sequencing of the PCR amplimers directly or after cloning. This procedure is laborious and, in the case of multiple infections, may not allow the identification of all HPV types present in the skin specimen. Two new methods have recently been described that are still based on the use of consensus/degenerate primers, but the HPV typing is achieved by reverse line blotting (7) or a reverse hybridization assay (RHA) (8).All these methods have been used successfully to detect cutaneous HPV DNA in skin specimens from immunocompetent and immunosuppressed individuals (reviewed in references 6 and 16). However, there is no consensus on the prevalence and spectrum of HPV types in skin tumors across studies. The difference in the prevalence rate and diversity of HPV types detected in skin lesions in various studies may

Analysis of the presence of cutaneous and mucosal papillomavirus types in ductal lavage fluid, milk and colostrum to evaluate its role in breast carcinogenesis

“…Multiplex PCR and HPV typing Detection of HPV DNA was performed using two recentlydeveloped highly sensitive and specific assays [29,30] [29], which resulted to be less sensitive than the GP5+/6+ reverse line blot assay in detecting HPV16. We have modified the PCR forward primer for HPV16 E7 (5 0 -TTATGAGCAATTAAATGACAGCTCAG-3 0 ).…”

“…Two different detection assays for 25 cutaneous beta and 19 mucosal high-risk alpha HPV types were used [29,30].…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…4). This distinguishes the approach from standard allele specific PCR where the discrimination nucleotide is usually placed at the 3' end of the allele specific primer [42,43]. Pierik and co-workers [30] demonstrated the approach described by Kim et al [16] for real-time quantitative solid-phase hybridization performed along with PCR amplification.…”

Prevalência e diversidade de beta-papilomavírus em amostras anogenitais e orais de homens do estudo HIM

“…O desenvolvimento de 100 conjuntos de esferas com diferentes espectros de absorbância permite a utilização simultânea de 100 sondas tipo específicas diferentes numa única reação (Figura 5). Além disso, esta tecnologia é útil em estudos de grande escala uma vez que é possível analisar 96 produtos de PCR em cada reação simultaneamente, além da semi-quantificação da carga de DNA viral inferida indiretamente pela fluorescência medida (Gheit et al, 2007) (Figura 6).…”

“…Também tem sido utilizada eficazmente para detectar e genotipar β-HPV (Gheit et al, 2007). Estudos têm mostrado que a metodologia Luminex é mais sensível para a detecção de HPV em amostras clínicas em comparação com as metodologias baseadas em hibridação reversa , sendo assim adequada para aplicações diagnósticas e epidemiológicas.…”