Detection and Quantitation of Human Papillomavirus by Using the Fluorescent 5′ Exonuclease Assay
AUTOR(ES)
Josefsson, Agnetha
FONTE
American Society for Microbiology
RESUMO
A method for the detection and quantitation of oncogenic human papillomavirus (HPV) was developed by using the fluorescent 5′ exonuclease assay. The method is based on the amplification of a 180-bp fragment from the 3′ part of the E1 open reading frame in a single PCR with type-specific probes for HPV types 16, 18, 31, 33, and 35. The probes can be used separately or in combinations of up to three probes per assay. Quantitation over a range of 101 to 106 initial HPV copies was possible by using real-time detection of the accumulation of fluorescence with cycle number. Reconstitution experiments, performed to mimic mixed infections, showed that individual HPV types can be detected down to a ratio of about 1% in a mixture. The performance of the assay depends on DNA quality, the presence of PCR inhibitors, and the number of different probes used simultaneously. This homogeneous assay provides a fast and sensitive way of screening for oncogenic HPV types in biopsy specimens as well as cervical smear samples. The closed-tube nature of the assay and the inclusion of uracil N′-glycosylase reduces cross contamination of PCR products to a minimum. A similar assay for β-actin was used in parallel for quantitation of genomic DNA. After normalizing the samples for genomic DNA content, the mean number of HPV copies per cell could be calculated.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=84442Documentos Relacionados
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