Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters

Abstract

BACKGROUND The majority of established techniques for monitoring real-time PCR amplification involve individual target-specific fluorogenic probes. For analysis of numerous different targets the synthesis of these probes contributes to the overall cost during assay development. Sequence-dependent universal detection techniques overcome this drawback but are prone to detection of unspecific amplification products. We developed the mediator probe PCR as a solution to these problems. METHODS A set of label-free sequence-specific primary probes (mediator probes), each comprising a target-specific region and a standardized mediator tag, is cleaved upon annealing to its target sequence by the polymerases' 5′ nuclease activity. Release of a mediator triggers signal generation by cleavage of a complementary fluorogenic reporter probe. RESULTS Real-time PCR amplification of human papillomavirus 18 (HPV18), Staphylococcus aureus, Escherichia coli, and Homo sapiens DNA dilution series showed exceptional linearity when detected either by novel mediator probes (r2 = 0.991–0.999) or state-of-the-art hydrolysis probes (TaqMan probes) (r2 = 0.975–0.993). For amplification of HPV18 DNA the limits of detection were 78.3 and 85.1 copies per 10-μL reaction when analyzed with the mediator probe and hydrolysis probe, respectively. Duplex amplification of HPV18 target DNA and internal standard had no effects on back calculation of target copy numbers when quantified with either the mediator probe PCR (r2 = 0.998) or the hydrolysis probe PCR (r2 = 0.988). CONCLUSIONS The mediator probe PCR has equal performance to hydrolysis probe PCR and has reduced costs because of the use of universal fluorogenic reporters.