Amplification with molecular beacon primers and reverse line blotting for the detection and typing of human...

A novel method for the detection and typing of human papillomavirus (HPV) was developed using molecular beacon primers. The method is based on the use of HPV-specific primers containing a hairpin loop structure in which fluorescent donor and quencher groups are held in close proximity such that fluorescence is quenched. Amplification of the target sequence results in the opening of the loop and the resulting fluorescence can be detected on a sequence detector system (SDS) 7700 (Applied Biosystems), as used for TaqMan assays. Fluorescent amplicons were identified on the SDS 7700 and then typed by a single hybridisation with specific probes immobilised in lines on a nylon membrane and detected on a fluorescent scanner. This novel beacon primer method compared well with conventional PCR for cervical scrape specimens. The combination of the beacon primer method and reverse line blotting should enable large-scale population studies of HPV infection.