Cost-effective real-time analysis by mediator probe (RT-)PCR

Abstract
Probe-based real-time PCR is used in molecular diagnostics due to its superior specificity and clinical sensitivity. High synthesis costs for sequence-specific dual-labelled detection probes are still one reason why researchers are reluctant when larger numbers of probes need to be ordered. In order to reduce costs we suggested a novel real time PCR method, the mediator probe PCR [1, 2]. It replaces fluorescently labeled hydrolysis probes by sequence-specific label free mediator probes (MP). Cleavage of the MP during amplification results in release of a mediator which is detected by a universal fluorogenic reporter (UR) oligonucleotide. The key to cost savings is that the same UR can be used for all assays and therefore can be ordered in large scale. This way oligonucleotide synthesis costs can be reduced to less than 40 % compared to the synthesis costs in hydrolysis probe based assays. In this work, performance characteristics of mediator probe PCR (MP PCR) were compared to hydrolysis probe PCR (HP PCR). Method: Nucleic acids were extracted from two viral DNA targets (human papilloma virus-18, human adenovirus B7) and one viral RNA target (influenza virus B). MPs and the UR were designed according to design rules provided in [1]. Probe sequences of the MPs were based on the corresponding HP sequences and comprise the following differences: (1) HP: fluorescence quencher at the 3’-terminus; MP: phosphate group at the 3′ terminus to avoid extension by polymerase activity; (2) HP: fluorophore at the 5’-terminus; MP: mediator sequence at the 5′ terminus. As the mediator and the corresponding mediator hybridization sites at the UR can be standardized the same fluorogenic UR could be used for all assays. Results: Analysis of serial dilutions of the DNA- and RNA targets revealed good agreement between MP and HP PCR: HPV18 (MP PCR r2 = 0.999/HP PCR r2 = 0.975), HAdV B7 (0.992/0.983), FluB (0.992/0.991). Amplification of 102 to106 copies HPV 18 DNA per reaction gave CVs of 55.1% to 9.9% / 38.3% to 10.7% and accuracies in the range of +21.6% to -8.1% / +19.4% to -9.8%. Amplification of 102 to105 copies HAdV B7 DNA per reaction gave CVs of 29.4% to 3.4% / 51.3% to 5.4% and accuracies ranging from +30.6% to -18.3% / +49.3% to -18.1%. Amplification of 104 to 107 copies FluB RNA per reaction gave CVs of 46.9% to 4.0% / 42.4% (4 out of 5 detectable) to 4.2% and accuracies ranging from +5% to -5% / +11% to -5%. Using the novel MP (RT-)PCR detection of 3 different targets with one universal detection oligonucleotide, the UR, showed same performance characteristics as HP (RT-)PCRs. MP PCR can reduce oligonucleotide synthesis costs and is recommended where a multitude of probes with low batch size is required. This can be in research laboratories, during assay development or in low resource settings. Our current research aims at (1) extending the design rules and (2) increasing the degree of multiplexing.