Quantification of beta-human papillomavirus DNA by real-time PCR.
Quantitative PCR with hybridization probes allows the reliable quantification of viral DNA sequences in clinical samples with a dynamic range and sensitivity that cannot be achieved with other methods. The technical background for the establishment of protocols is described and established protocols are presented to estimate the viral load per cell of frequently occurring betapapillomaviruses (HPV5, -8, -15, -20, -23, -24, -36 and -38) in skin tumors, healthy skin and hair bulbs. This approach accurately adjusts dilution series of reference DNA of different viral types relative to pUC18, which is crucial for comparative analyses and for interlaboratory standardization. The type-specific determination of beta-HPV DNA loads is an important research tool toward discrimination between low-level persistence and activated possibly pathologically relevant infections. The analysis of 24 samples, starting with DNA extraction and followed by HPV typing and quantification of-on average-three of the described HPV types takes about 2 d.
Weissenborn
Sönke J
SJ
Institute of Virology, German National Reference Centre for Papilloma and Polyomaviruses, University of Cologne, Koeln, Germany.
Wieland
Ulrike
U
Junk
Monika
M
Pfister
Herbert
H
D001483:Base Sequence; D052686:Betapapillomavirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics*; D018859:Hair Follicle / Q000821:virology; D006801:Humans; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012867:Skin / Q000821:virology; D012878:Skin Neoplasms / Q000821:virology; D019562:Viral Load / Q000235:genetics
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral