A SENSITIVE, TYPE-SPECIFIC, FLUOROGENIC PROBE ASSAY FOR DETECTION OF HUMAN PAPILLOMAVIRUS DNA

A simple method for the detection of a number of human papillomavirus (HPV) genotypes associated with cervical cancer has been developed, Th e assay exploits the 5'-->3' exonucleolytic activity of Taq DNA polyme rase to increase the signal from fluorescent dyes by releasing them fr om genotype-specific probes during PCR. The probes are oligonucleotide s with a 5' reporter dye (6-carboxyfluorescein), a quencher dye (6-car boxy-tetramethyl-rhodamine), and a phosphate-blocked 3' end, In the in tact probe, the proximity of the reporter and the quencher results in suppression of reporter fluorescence by Forster-type energy transfer ( V. T. Forster. Ann, Phys. 2:55-75, 1948). If the probe is bound downst ream of either primer during PCR, the 5'-->3' exonucleolytic activity of Taq polymerase degrades it, allowing the reporter to diffuse away f rom the quencher, which results in an increase in reporter fluorescenc e, The increased fluorescence is directly related to the amount of tar get DNA and can be detected with an automated fluorometer. Probes for the L1 region of the cervical-cancer-associated HPV types 16, 18, 31, 33, and 35 were synthesized and the assays were optimized, The most se nsitive assay can detect as few as two copies of HPV DNA in human cerv ical specimens.