Detection and quantitation of human papillomavirus by using the fluorescent 5' exonuclease assay.
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 10(1) to 10(6) 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 beta-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.
Josefsson
A
A
Department of Genetics and Pathology, Unit of Medical Genetics, University of Uppsala, S-751 23 Uppsala, Sweden.
Livak
K
K
Gyllensten
U
U
D002584:Cervix Uteri / Q000821:virology*; D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood / Q000302:isolation & purification*; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis*; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D019914:Taq Polymerase; D014412:Tumor Virus Infections / Q000175:diagnosis*; D014626:Vaginal Smears