Abstract

Mediator Probe PCR - Principle, Applications and Guidelines for Primer and Probe Design

Felix Von Stetten, Head, HSG IMIT

Mediator probe PCR (MP PCR) is a novel approach for universal sequence-dependent detection of real time PCR. Detection relies on label-free primary probes, referred to as mediator probes (MP), and secondary universal fluorogenic reporter (UR). In comparison to usage of target-specific fluorogenic probes, e.g. in hydrolysis probe (HP) PCR, MP PCR allows cost savings in oligonucleotide synthesis. The MP consists of a target-specific probe region, which anneals to the target, and a standardized sequence tag, the mediator, cleaved off by the nuclease activity of the polymerase during amplification. The mediator anneals to the fluorogenic UR, is extended and thus leads to dequenching of the UR. Guidelines for design of MP PCR and reverse transcription MP PCR are presented and detection of human papillomavirus 18 (HPV-18), influenza B virus, and human adenovirus B7 are reported. Limits of detection for amplification of HPV18 DNA with either MP or HP PCR were 78.3 and 85.1 copies per reaction, respectively. Co-amplification of different HPV18-DNA concentrations and an internal standard showed excellent linearity of back-calculated HPV18-DNA concentrations for MP PCR (R2 = 0.998) and HP PCR (R2 = 0.988). In conclusion, the MP PCR has equal performance to HP PCR and enables cost savings.