A sensitive, type-specific, fluorogenic probe assay for detection of human papillomavirus DNA

Abstract

A simple method for the detection of a number of human papillomavirus (HPV) genotypes associated with cervical cancer has been developed. The assay exploits the 5' → 3' exonucleolytic activity of Taq DNA polymerase to increase the signal from fluorescent dyes by releasing them from genotype- specific probes during PCR. The probes are oligonucleotides with a 5' reporter dye (6-carboxyfluorescein), a quencher dye (6-carboxytetramethyl- rhodamine), and a phosphate-blocked 3' end. In the intact 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 downstream of either primer during PCR, the 5' → 3' exonucleolytic activity of Taq polymerase degrades it, allowing the reporter to diffuse away from the quencher, which results in an increase in reporter fluorescence. The increased fluorescence is directly related to the amount of target 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 sensitive assay can detect as few as two copies of HPV DNA in human cervical specimens.