Detection and typing of human papillomavirus by single hybridization.
A rapid and non-radioactive molecular hybridization test was developed which simultaneously detects and types different human papillomaviruses (HPV) DNA in fresh and paraffin-embedded clinical specimens. The method includes reverse blot hybridization between different recombinant HPV plasmids immobilized on nylon membrane and probe of cellular DNA amplified and biotin- or digoxigenin-labeled by the polymerase chain reaction (PCR). PCR protocol using consensus primers includes the mixing of Taq polymerase at high temperature (Hot-Start) and the addition of the hapten-conjugated nucleotide after the first ten cycles of amplification. The sensitivity level of this method resulted in detecting about 50 copies of HPV 16 for sample, independently of hapten used. The specificity of the typing method was also validated by more laborious and conventional analyses such as Southern-blot or PCR followed by several hybridizations with specific probes. Using this test HPV-11, -16, -18, -31 and -35 were typed in a number of samples from patients attending hospital. The method appears suitable for the handling of clinical samples in a selected population screening for type specific infections by HPV.
Venuti
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A
Regina Elena Institute for Cancer Research, Laboratory of Virology, Rome, Italy.
Badaracco
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G
Marcante
M L
ML
D002584:Cervix Uteri / Q000821:virology; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000175:diagnosis; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014412:Tumor Virus Infections / Q000175:diagnosis; D002583:Uterine Cervical Neoplasms / Q000821:virology; D014773:Virology / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data
D004279:DNA, Viral