Multiplex Mediator Probe Real-Time PCR: Optimisation and Guideline Development through Systematic Characterisation of Label Free Mediator Probes and Fluorogenic Universal Reporters

Nadine Borst
Michael Lehnert1,2, Elena Kipf1, Franziska Schlenker1, Roland Zengerle1,2, Nadine Borst1,2, Felix von Stetten1,2
1) Hahn-Schickard, Freiburg, Germany;
2) Laboratory for MEMS Applications, Department of Microsystems Engineering – IMTEK, University of Freiburg, Germany;

Mediator Probe PCR is a powerful and robust real-time PCR technology for multiplex DNA detection and quantification. It uses label free mediator probes, for molecular detection of nucleic acids during DNA amplification, in combination with fluorogenic universal reporters for signal generation. During PCR, target sequence specific mediator probes are cleaved by the polymerase and a generic sequence, the mediator, is set free. In the second step the mediator binds to the universal reporter, where it is extended by the polymerase. This generates a strong fluorescence signal increase. Due to the separation of DNA detection and signal generation many advantages arise. Mediator probes are not limited in their design by properties of the target sequence and a standard set of highly optimised fluorogenic universal reporters can be used for multiplex Mediator Probe PCR, right from the start.1 In the last years Mediator Probe PCR evolved from an innovative new method to an optimised and robust multiplexing technology. This was achieved by systematic characterisation of its molecular processes, which again was advantaged by the separation of DNA detection and signal generation. A design of experiments (DoE) approach was used for the optimisation of Mediator Probes, focusing on their binding strengths.2 In parallel, a set of universal reporters with improved signal-to-noise ratios was established by successive testing over 40 molecular structures, with different fluorophore-quencher labels and configurations.1 As a result, distinct guidelines exist, which enable fast adaption of new DNA targets and facilitate multiplex Mediator Probe PCR design. The capability of the technology was shown by highly sensitive, precise and specific multiplex Mediator Probe real-time PCRs in different areas of molecular diagnostics. These fields include monitoring of oncological disease, detection of pathogens or analysis of food samples.1,3
1. Lehnert M, Kipf E, Schlenker F, Borst N, Zengerle R, von Stetten F. Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides. Anal. Methods. 2018;10:190. doi: 10.1039/C8AY00812D
2. Wadle S, Lehnert M, Rubenwolf S, Zengerle R, von Stetten F. Real-time PCR probe optimization using design of experiments approach. Biomolecular detection and quantification. 2016;7:1–8. doi: 10.1016/j.bdq.2015.12.002
3. Wadle S, Lehnert M, Schuler F et al. Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters. BioTechniques. 2016;61(3):123–8. doi: 10.2144/000114443

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Directed Evolution of Enzymes for Streamlined and Reliable RT-qPCR and NGS Workflows

David Mark Schuster
Quantabio, United States of America

Reverse transcription remains an essential and sometimes problematic initial step in methods and workflows for the analysis of RNA by NGS or PCR-based amplification methods. Despite advancements in these technologies and the introduction of engineered reverse transcriptases, efficient conversion of RNAs that form stable secondary structures, and/or the presence of inhibitors in sample matrix can influence the efficacy of first-strand synthesis, introducing bias in RNA sequence coverage or transcript enumeration. This talk will describe novel thermostable, inhibitor tolerant RNA directed DNA polymerases obtained through our molecular screening and directed evolution program and their application to streamlined workflows for RT-PCR and NGS methods. Collectively, the properties of these new enzymes and associated reagent systems offer the promise to simplify, accelerate and improve the reliability and flexibility of detection and analysis of mRNA, noncoding RNA and viral targets.

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Quality Standards in quantitative PCR; Specification, Validation, Controls and Standards

Peter Rossmanith
Vetmeduni Vienna, Vienna, Austria

Introduction: The implementation of molecular methods such as real-time PCR for food pathogen detection is desired and reasonable. Nevertheless the obstacles of precise specification and meaningful validation are not yet overcome and therefore broad range use in food testing is not yet accomplished. Specification is generally based on the determination of the detection limit, the overall efficiency of the reaction and exclusivity and inclusiveness of the assay respectively. These parameters do not provide sufficient information about the real performance of the underlying enzymatic reaction. The validation according to ISO 16140, the validation of alternative methods, has many drawbacks based on its original sense and purpose, the comparison of microbiological methods. Evaluation of real-time PCR is therefore not significant with this process due to the different nature of molecular biological methods.
Purpose: Establishment of a significant specification and validation approach in consideration of the inherent qualities of real-time PCR. Methods: A validation system including testing algorithms derived from software engineering; per se specification of the enzymatic reaction, controls covering all necessary steps and the investigation of surrounding parameters was designed. The whole approach is based on fundamental principles of systems theory and cybernetics. This alternative strategy includes every necessary detail thus leading to a maximum performance of the assay and most precise specification and validation of the whole analytical chain. Results: We present the practical application of this new approach by example of an analytical chain for the detection of L. monocytogenes, including sample preparation, DNA isolation/purification and real-time PCR detection.
Significance: New approaches for the significant specification and validation of molecular biological methods are necessary to gain confidence in such methods and furthermore support widespread implementation. The whole system approach presented herein is an equivalent attempt, which effectively supports the standard validation method ISO 16140.

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