Publikationsliste Dr. Michael Lehnert

• Originalarbeiten in wissenschaftlichen Fachzeitschriften
• Buchbeiträge
• Kurzbeiträge
• Vorträge
• Konferenzbeiträge

Originalarbeiten in wissenschaftlichen Fachzeitschriften

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2023

• S. Calabrese, A. M. Markl, M. Neugebauer, S. J. Krauth, N. Borst, F. von Stetten, M. Lehnert
  Reporter emission multiplexing in digital PCRs (REM-dPCRs): direct quantification of multiple target sequences per detection channel by population specific reporters
  2023 Analyst, Band: 148, Seiten: 5243 - 5254

2022

• E. Kipf, F. Schlenker, N. Borst, M. Fillies, R. Kirschner-Schwabe, R. Zengerle, C. Eckert, F. von Stetten, M. Lehnert
  Advanced minimal residual disease monitoring for acute lymphoblastic leukemia with multiplex mediator probe PCR
  2022 J. Mol. Diag, Band: 24, Nummer: 1, Seiten: 57 - 68
• M. Neugebauer, C. E. Grundmann, M. Lehnert, F. von Stetten, S. M. Früh, R. Süss
  Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse Transcription-qPCR
  2022 Pharmaceutics, Band: 2022, Nummer: 14, Seite: 1348
• Y.-T. Kao, S. Calabrese, N. Borst, M. Lehnert, Y.-K. Lai, F. Schlenker, P. Juelg, R. Zengerle, P. Garstecki, F. von Stetten
  Microfluidic One-Pot Digital Droplet FISH Using LNA/DNA Molecular Beacons for Bacteria Detection and Absolute Quantification
  2022 Biosensors, Band: 12, Nummer: 4, Seite: 237

2021

• F. Schlenker, E. Kipf, M. Deuter, I. Höffkes, M. Lehnert, R. Zengerle, F von Stetten, F. Scherer, J. Wehrle, N. von Bubnoff, P. Juelg, T. Hutzenlaub, N. Borst
  Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA
  2021 cancers, Band: 13, Nummer: 22, Seite: 5742
• P. Juelg, E. Kipf, M. Specht, M. Fillies, C. Eckert, N. Paust, R. Zengerle, M. Lehnert, T. Hutzenlaub
  The MRD disk: automated minimal residual disease monitoring by highly sensitive centrifugal microfluidic multiplex qPCR
  2021 Lab Chip, Band: 21, Seiten: 558 - 570
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  Kurzfassung

  We present a proof-of-principle study on automated, highly sensitive and multiplexed qPCR quantification by centrifugal microfluidics. The MRD disk can be used for standardisation of repetitive, longitudinal assays with high requirements on reproducibility and sensitivity, such as cancer monitoring. In contrast to high-throughput qPCR automation by bulky and expensive robotic workstations we employ a small centrifugal microfluidic instrument, addressing the need of low- to mid-throughput applications. As a potential application we demonstrate automated minimum residual disease (MRD) monitoring of prognostic markers in patients with acute lymphoblastic leukaemia (ALL). The disk-workflow covers all aspects of clinical gold standard MRD quantification: generation of standard curves, specificity controls, no template controls and quantification of the ALL patient sample. We integrated a highly sensitive, colorimetric 2-plex analysis of MRD targets, as well as a 2-plex analysis of reference genes, both in parallel and in a single LabDisk cartridge. For this purpose, a systematic procedure for crosstalk- and signal-to-noise-optimisation is introduced, providing a guideline for efficient multiplex readout inside microfluidic platforms. The qPCR standard curves (n = 12/12) generated on-disk reach clinically required linearity (R2 = 98.1% to R2 = 99.8%). In three consecutive MRD disk runs with an ALL patient sample containing the two representative MRD targets VH3D3D5JH3 and VkIkde, we observe high accordance between the on-disk quantifications (48 ± 6 copies/reaction and 69 ± 6 copies/reaction) and the expected concentrations (57 copies/reaction for both targets). In comparison to the clinical gold standard of manually pipetted, singleplex assays, the MRD disk yields comparable limit of quantification (1 × 10−4) in n = 6/6 analyses (vs. n = 4/4 in gold standard) and a limit of detection (1 × 10−5) in n = 6/6 analysis (vs. n = 2/4 in gold standard). The automation reduces the risk of manual liquid handling errors, making the MRD disk an attractive solution to assure reproducibility in moderate-throughput, longitudinal gene quantification applications.

2019

• P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, M. Keller, T. Hutzenlaub, F. von Stetten, R. Zengerle, N. Paust
  Automated serial dilutions for high-dynamic-range assays enabled by fill-level-coupled valving in centrifugal microfluidics
  2019 Lab Chip, Band: 19, Seiten: 2205 - 2219
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  Kurzfassung

  We introduce a new concept for centrifugal microfluidics that enables fully automated serial dilution generation without any additional means besides temperature control. Key feature is time-independent, serial valving of mixing chambers by fill-level-coupled temperature change rate (FLC-TCR) actuated valving. The automated dilution is realized under continuous rotation which enables reliable control of wetting liquids without the need of any additional fabrication steps such as hydrophobic coatings. All fluidic features are implemented in a monolithic fashion and disks are manufactured by foil thermoforming for scalable manufacturing. The new valving concept is demonstrated to reliably prevent valving if the diluted sample is not added to the mixing chamber (n = 30) and ensures valving if the dilution stage is completed (n = 15). Accuracy and precision of the automated serial dilution are verified by on-disk generation of qPCR standard curve dilutions and compared with manually generated reference dilutions. In a first step, the 5-log-stages standard curves are evaluated in a commercial qPCR thermocycler revealing a linearity of R² ≥ 99.92 % for the proposed LabDisk method vs. R² ≥ 99.67 % in manual reference dilutions. In a second step, the disk automated serial dilution is combined with on-disk qPCR thermocycling and readout, both inside a LabDisk Player. A 4-log-stages linearity of R² ≥ 99.81 % and a sensitivity of one leukemia associated ETV6-RUNX1 mutant DNA copy in a background of 100,000 wild-type DNA copies is achieved.

2018

• M. Lehnert, E. Kipf, F. Schlenker, N. Borst, R. Zengerle, F. von Stetten
  Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides
  2018 Anal Methods-uk
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  Kurzfassung

  In this study we optimised the fluorescence signal generation of contact quenched universal reporter oligonucleotides. These are used as secondary probes in real-time Mediator Probe PCR to detect the sequence-specific cleavage of label-free primary mediator probes. Since the fluorescence signal generation of a universal reporter is not influenced by the target DNA sequence, optimisation of the fluorescence signal-to-noise ratio will improve the performance of all Mediator Probe PCRs that are based on this type of universal reporter. To determine the critical factors influencing signal-to-noise optimisation, we systematically analysed four parameters. These parameters were type of fluorophore, type of quencher molecule, intramolecular orientation of both residuals, and the number of quencher labels. In total, more than 30 different fluorogenic universal reporter structures were analysed, covering the whole fluorescence spectrum from green to crimson. From our results, we deduced a novel set of guidelines for signal-to-noise optimisation in the design of contact quenched, fluorogenic universal reporter oligonucleotides. We confirmed these guidelines in a different thermocycler, and by designing a second set of universal reporters, which were used for multiplex real-time PCR quantification of acute lymphoblastic leukaemia marker sequences. This optimised biplex Mediator Probe PCR showed an improved performance under clinical conditions, with a 10 times higher resolution regarding the limit of quantification. In addition to Mediator Probe PCR, these guidelines may also prove useful in signal-to-noise optimisation of other fluorescence-based assays where contact quenched oligonucleotides or secondary reporter molecules are used.

2016

• S. Wadle, M. Lehnert, S. Rubenwolf, R. Zengerle, F. von Stetten
  Real-time PCR probe optimization using design of experiments approach
  2016 Biomolecular Detection and Quantification, Band: 7, Seiten: 1 - 8
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  Kurzfassung

  Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3–14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7–11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times.
• S. Wadle, M. Lehnert, F. Schuler, R. Köppel, A. Serr, R. Zengerle, F. von Stetten
  Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters
  2016 BioTechniques, Band: 61, Seiten: 123 - 128
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  Kurzfassung

  Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2–5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength.

Buchbeiträge

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2014

• S. Wadle, S. Rubenwolf, M. Lehnert, B. Faltin, M. Weidmann, F. Hufert, R. Zengerle, F. von Stetten
  Mediator Probe PCR: Detection of Real-Time PCR by Label-Free Probes and a Universal Fluorogenic Reporter
  In: Quantitative Real-Time PCR: Methods and Protocols
  2014, Springer, R. Biassoni, A. Raso, HUMANA PRESS INC., Seiten: 55 - 73, R. Biassoni, A. Raso, HUMANA PRESS INC.,
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  Kurzfassung

  Mediator probe PCR (MP PCR) is a novel detection format for real-time nucleic acid analysis. Label-free mediator probes (MP) and fluorogenic universal reporter (UR) oligonucleotides are combined to accomplish signal generation. Compared to conventional hydrolysis probe PCRs costs can thus be saved by using the same fluorogenic UR for signal generation in different assays. This tutorial provides a practical guideline to MP and UR design. MP design rules are very similar to those of hydrolysis probes. The major difference is in the replacement of the fluorophore and quencher by one UR-specific sequence tag, the mediator. Further protocols for the setup of reactions, to detect either DNA or RNA targets with clinical diagnostic target detection as models, are explained. Ready to use designs for URs are suggested and guidelines for their de novo design are provided as well, including a protocol for UR signal generation characterization.

Kurzbeiträge

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2012

• B. Faltin, S. Wadle, G. Roth, S. Rubenwolf, M. Lehnert, R. Zengerle, F. von Stetten
  Neuer Echtzeit-Nachweis von Nukleinsäuren
  2012 Laborwelt, Band: 4, Seiten: 10 - 11

Vorträge

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2024

• E. Kipf, P. Jülg, F. Schlenker, M. Fillies, C. Kranig, M. Specht, S. Groeneveld-Krentz, S. Neumann, R. Zengerle, F. von Stetten, N. Borst, R. Kirschner-Schwabe, T. Hutzenlaub, M. Lehnert, C. Eckert
  Messung des individuellen Therapieansprechens von akuten lymphoblastischen Leukämien (IRMA-4-ALL)
  2024 Translationale Forschung in der Personalisierten Medizin – Chancen und Hürden, Netzwerkveranstaltung für geförderte Projekte der „Innovationen für die individualisierte Medizin“ und der „Translationsp

2013

• Lehnert M, Wadle S, Stetten F, Zengerle R
  Mediator probe PCR as tool for personalized medicine
  2013 BmbF

Konferenzbeiträge

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2025

• J. Sum, T. Truong, C. Mikloska, M. Oberländer, N. von Bubnoff, L. Christiansen, S. Tornow, S. Derer, F. Janke, H. Sültmann, S. Zeissig, M. Linnebacher, C. Schafmayer, I. Nazarenko, F. Scherer, H. Becker, M. Kalweit, M. Lehnert, T. Hutzenlaub, R. Zengerle, N. Paust, P. Juelg
  Liquid Biopsy using Methylation Analysis-coupled digital PCR of Biomarkers to Detect Early-Onset Colorectal Cancer
  2025 Kongress "Prevention of eoCRC" HARMONIZE des OUTLIVE Verbundes, Lübeck, 24. – 26.03.2025

2024

• M. Neugebauer, S. Calabrese, A. Markl, L. Schumacher, T. Wolper, T-T. Truong, P. Juelg, N. Borst, T. Hutzenlaub, F. Scherer, F. von Stetten, M. Lehnert
  Increasing digital PCR performance in liquid biopsy using reporter emission multiplexing
  2024 13th CNAPS International Symposium on Circulating Nucleic Acids in Plasma and Serum, Graz/Austria, 4-6 March 2024
• H. Gmoser, T. Groß, M. Ballweg, P. Sánchez-Martín, M. Lehnert, C. Jeney, A. Lux, P. Juelg, F. v. Stetten, N. Borst
  Isothermal PICO Assay for One-Step Library Prep and Nanopore Readout
  2024 nanodiag bw Innovative Retreat, Hohenfels, Bodensee, 30 September - 3 October 2024
• S. Calabrese, A. Markl, M. Neugebauer, L. Schumacher, N. Borst, F. Scherer, F. von Stetten, M. Lehnert
  Reporter emission multiplexing (REM) Increases detection capacities of digital PCR devices leading to sensitive, precise and specific liquid biopsy assays
  2024 EUDIP 2024 - European Digital PCR Symposium 2024, Gent/Belgium, 6-8 February 2024

2023

• H. Gmoser, T. Groß, M. Ballweg, A. Lux, C. Jeney, M. Lehnert, F. v. Stetten, N. Borst
  Isothermal Protein Interaction Coupling (PICO) Assay towards One-Step Library Prep and Nanopore Read-out
  2023 Black Forest Nanopore Meeting, Freiburg, 06.-09.11.2023

2021

• P. Juelg, E. Kipf, M. Specht, J. Menges, M. Fillies, C. Eckert, N. Paust, R. Zengerle, M. Lehnert, T. Hutzenlaub
  Die MRD Disk: Automatisiertes Monitoring Minimaler Resterkrankung durch hochsensitive, zentrifugal-mikrofluidische Multiplex-qPCR / The MRD disk: automated minimal residual disease monitoring by highly sensitive centrifugal microfluidic multiplex qPCR
  2021 MST
• Y.-T. Kao, S. Calabrese, N. Borst, M. Lehnert, Y.-K. Lai, F. Schlenker, R. Zengerle, P. Garstecki, F. von Stetten
  One-step amplification-free bacteria detection by optimizes LNA/DNA molecular beacons in droplets
  2021 MicroTAS 2021, Palm Springs/USA, 10.-14.10.2021, online

2019

• M. Lehnert, E. Kipf, F. Schlenker, R. Zengerle, N. Borst, F. von Stetten
  Multiplex Mediator Probe Real-Time PCR - Optimization and Guideline Development through Systematic Characterisation of Label Free Mediator Probes and Fluorogenic Universal Reporters
  2019 9th Gene Quantification Event qPCR dPCR & NGS 2019, 18 - 22 March 2019, in Freising-Weihenstephan, Germany
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  Kurzfassung

  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 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
• P. Juelg, M. Specht, M. Meyer, E. Kipf, F. Schlenker, F. Baensch, S. Neumann, F. von Stetten, R. Zengerle, N. Paust, M. Fillies, R. Kirschner-Schwabe, S. Groeneveld-Krentz, M. Lehnert, C. Eckert, T. Hutzenlaub
  Individual Response Monitoring Assay (IRMA) – Standardization Of Personalized Multiplex Biomarker Quantification
  2019 EACR-ESMO Joint Conference on Liquid Biopsies, Bergamo/Italien, 15. – 17.05. 2019
• P. Juelg, M. Specht, M. Meyer, E. Kipf, F. Schlenker, F. Bänsch, S. Neumann, F. von Stetten, R. Zengerle, N. Paust, M. Fillies, R. Kirschner-Schwabe, S. Groeneveld-Krentz, M. Lehnert, C. Eckert, T. Hutzenlaub
  Standardization of Personalized Multiplex Biomarker Quantification: Individual Response Monitoring Assay (IRMA)
  2019 MST-Kongress, 28. - 30.Oktober 2019, Berlin

2018

• P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, N. Paust, F. von Stetten, R. Zengerle, T. Hutzenlaub
  Automation of qPCR based Minimal Residual Disease Monitoring by Centrifugal Microfluidics
  2018 11th Symposium on minimal residual cancer, 03. – 05.05.2018, Montpellier, Frankreich
• P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, N. Paust, R. Zengerle, T. Hutzenlaub
  LabDisk for fully automated quantification of two leukemia associated gene targets
  2018 MicroTAS 2018, 11. -15. November 2018, Kaohsiung / Taiwan
• P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, N. Paust, F. von Stetten, R. Zengerle, T. Hutzenlaub
  Towards Standardization of Molecular Diagnostic Workflows: Centrifugal Microfluidic Automation of qPCR for Cancer Monitoring
  2018 6th International Molecular Diagnostics Europe, Lissabon / Portugal, 22. - 24. 05.2018

2017

• P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, S. Wadle, M. Keller, N. Paust, R. Zengerle, T. Hutzenlaub
  Centrifugal microfluidic automation of dilution series for high dynamic range assays demonstrated for decadal dilutions up to 1:100,000
  2017 MicroTAS 2017, Savannah /USA, 22.-26.10.2017
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  Kurzfassung

  For the first time we present the centrifugal microfluidic automation of an extensive dilution series that can cover all biochemically relevant dilution ranges. In contrast to existing methods, the new concept is based on iteration loops of individual dilution steps that can be easily combined in a theoretically unlimited fashion. Thus, any combination and range of dilutions can be implemented. The concept was demonstrated by generating a qPCR standard curve for cancer biomarker ETV6-RUNX1 up to 1:100,000. We achieved the following analytical performance characteristics: A high coefficient of determination of R²=98.9 %, equidistant quantification cycles of ΔCq=3.4±0.3 and low replicate variation coefficients of CV<3.5 %.

2015

• S. Wadle, M. Lehnert, F. Schuler, R. Zengerle, F. von Stetten
  Universelle Reporter der Mediatorsonden PCR als Zielsequenz-unabhängige Biosensoren zur Detektion fünf verschiedener RNA- und DNA-Sequenzen
  2015 9. Deutsches BioSensor Symposium 11.–13. März 2015, München
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  Kurzfassung

  In der Molekulardiagnostik werden meist sogenannte Hydrolysesonden (hydrolysis probe, HP) zur Echtzeitdetektion von Nukleinsäuren verwendet. Hierbei ist für jede zu detektierende Zielsequenz die Synthese einer spezifischen dual-markierten HP notwendig. Deren Anschaffung ist sehr kostenintensiv, vor allem beim Bedarf nur geringer Bestellmengen. Zudem müssen HP bei jeder neu zu detektierenden Zielsequenz individuell bezüglich der Signalgenerierungseffizienz optimiert werden. Ein von uns publiziertes neuartiges Verfahren, die Mediatorsonden PCR (mediator probe PCR, MP PCR) [1, Abb.1] löst diese Probleme durch Verwendung eines dual-markierten Universellen Reporter Oligonukleotids (UR). Dieser Biosensor erzeugt unabhängig der zu detektierenden Zielsequenz ein Signal, initiiert durch die Interaktion mit nicht-markierten und somit kostengünstigen sequenzspezifischen Mediatorsonden. Im Vergleich zu [1] wurde die Fluoreszenz-Quenchingeffizienz am UR und die Reaktionszusammensetzung der MP (RT-)PCR Assays verbessert, um fünf verschiedene DNA und auch RNA Sequenzen von Atemwegsinfektion-verursachenden Viren zu detekieren. Die Detektionsreaktionen wurden mit HP-basierten Assays als Referenz verglichen, bei denen fünf verschiedene dual-markierte Sonden benötigt werden.

2014

• S. Wadle, M. Follo, M. Lehnert, F. Schuler, N. von Bubnoff, R. Zengerle, F. von Stetten
  Specific SNP detection by mediator probe digital droplet PCR
  2014 Advances in dPCR & qPCR, Barcelona, Spain, 14. – 15. Mai 2014
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  Kurzfassung

  Mediator probe digital droplet PCR (MP ddPCR) was used to detect single nucleotide polymorphisms (SNP) at the cKit V559D gene locus, a gastrointestinal cancer biomarker [1]. In comparison to conventional PCR with dual-labelled hydrolysis probes (HP) the MP PCR combines label-free mediator probes with universal fluorogenic reporters [2]. In the presented experiment, specificity of the MP-based SNP detection was as high as SNP detection using HPs with locked-nucleic acid (LNA) modified nucleotides.
• S. Wadle, M. Lehnert, F. Schuler, R. Zengerle, F. von Stetten
  Universal reporters of mediator probe PCR as target-independent biosensors
  2014 IFCC WorldLab Istanbul, 22.-26.06.2014, Istanbul, Türkei
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  Kurzfassung

  Molecular diagnostics often uses hydrolysis probes (HP) for real-time nucleic acid sensing. However, each target sequence requires synthesis of specific dual-labelled HPs, which are expensive, especially when used at low batch sizes. Also, HPs must be individually optimized for signal generation efficiencies for each target sequence to be detected. We have published a novel approach, the mediator probe PCR (MP PCR) [B. Faltin et al.: Clin Chem, vol. 58, pp. 1546-1556, 2012] which overcomes these issues by using a labelled but universal reporter oligonucleotide (UR) as a biosensor for target-independent signal generation. It is triggered by unlabelled and thus cost-effective sequence-specific mediator probes. Compared to [Faltin 2012] we improved UR quenching efficiencies and reaction setup of MP PCRs to detect 5 different DNA and also RNA target sequences of viruses causing respiratory tract infections. HP based assays, which required 5 different dual-labelled probes were run as references. MPs and the UR designs were adapted from [Faltin 2012] with the sequence-specific MP section equal to corresponding HP sequences. Nucleic acid standards from human adenovirus (hAdV), influenza virus A&B (InfA & B), human metapneumovirus (hMPV), and respiratory syncytial virus (RSV) were serially diluted enabling efficiency calculation and detection limit determination. Reaction efficiencies and the correlation of input- with back-calculated output concentrations were better for MP RT-PCRs than for HP RT-PCRs. 95 % detection limits were: hAdV 7 / 7 copies per reaction (MP / HP (RT-)PCR), InfA 4 / 18, InfB 10 / 14, hMPV 11 / 29, RSV 14 / 22. These correspond well to commercially available assays [L. Van Wesenbeeck et al.: J. Clin Microbiol., vol. 51, pp. 2977-2985, 2013]. As conclusion, one UR was used for sensing 5 different DNA and RNA targets by MP (RT-) PCR. Even higher reaction efficiencies and lower detection limits as with the more expensive HP (RT-) PCRs could be reached. The method is especially recommended if many different target-specific probes are required at low batch sizes. In future, multiplexing degrees shall be increased using UR-microarrays.
• S. Wadle, M. Lehnert, R. Zengerle, F. von Stetten
  Universal reporters of mediator probe PCR as target-independent biosensors for detection of five different RNA and DNA sequences
  2014 Biosensors 2014, Melbourne, Australia, 27.05.2014 – 30.05.2014

2013

• S. Wadle, S. Rubenwolf, M. Lehnert, B. Faltin, R. Zengerle, F. von Stetten
  Cost-effective geometric multiplex mediator probe PCR
  2013 7. Senftenberger Innovationsforum Multiparameteranalytik, 18. - 19.04.2013
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  Kurzfassung

  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).
• S. Wadle, S. Rubenwolf, M. Lehnert, B. Faltin, R. Zengerle, F. von Stetten
  Cost-effective real-time analysis by mediator probe (RT-)PCR
  2013 qPCR & NGS 2013 Symposium, 18th -20th March 2013, Technical University of Munich, Freising, Germany