Simon Wadle
University of Freiburg
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Publication
Featured researches published by Simon Wadle.
BioTechniques | 2014
Markus Rombach; D. Kosse; Bernd Faltin; Simon Wadle; Günter Roth; Roland Zengerle; von Stetten F
A method for conserving primers and differently labeled fluorogenic hydrolysis (i.e., TaqMan) probes at ambient conditions is presented. Primers and hydrolysis probes with four different fluorophore-quencher combinations (6- FAM-BHQ1, HEX-BHQ1, ROX-BHQ650, and Cy5-BHQ2) were mixed with trehalose and xanthan at final concentrations of 56 mM and 2.78 mM, respectively. Mixtures were air-dried at 23°C for 30 min on strips composed of cyclo olefin polymer (COP), a material widely used in the manufacturing of in vitro diagnostic (IVD) test carriers. After one year of storage, the functionality of the primers and fluorophore-quencher combinations was validated by real-time polymerase chain reaction (real-time PCR), confirming their stability when stored in the presence of stabilizers, with the best results achieved using trehalose. This approach could be of great benefit for manufacturing IVD systems, for example, for genotyping applications based on multiplexing using different fluorescent dyes.
Biomolecular Detection and Quantification | 2016
Simon Wadle; Michael Lehnert; Stefanie Rubenwolf; Roland Zengerle; F. von Stetten
Graphical abstract
RSC Advances | 2015
M. Keller; Simon Wadle; Nils Paust; L. Dreesen; C. Nuese; Oliver Strohmeier; Roland Zengerle; F. von Stetten
We introduce microfluidic automation of geometrically multiplexed real-time PCR to off-the-shelf Rotor-Gene Q thermocyclers (RGQ, QIAGEN GmbH, Hilden, Germany). For centrifugal fluid control the RGQ provides low and constant rotation of 400 rpm, only. Compatibility to this very limited flexibility of centrifugal actuation is achieved by using thermal gas compression and expansion for valving and aliquoting. In contrast to existing thermo-pneumatic actuation, centrifugo-thermopneumatic (CTP) fluid control employs the induced change of partial vapor pressure by global temperature control as actuation parameter for two new unit operations: CTP siphon valving and CTP two-stage aliquoting. CTP siphon valving was demonstrated to reliably transfer sample liquid in all cases (n = 35) and CTP two-step aliquoting transfers metered aliquots of 18.2 ± 1.2 μl (CV 6.7%, n = 8) into reaction cavities within 5 s (n = 24). Thermal characteristics of CTP two-stage aliquoting were found to be in good agreement with an introduced analytical model (R2 = 0.9876, n = 3). A microfluidic disk segment comprising both new unit operations was used for automation of real-time PCR amplification of Escherichia coli DNA. Required primers and probes were pre-stored in the reaction cavities and a comparison with reference reactions in conventional PCR tubes yielded the same PCR efficiency, repeatability, and reproducibility.
Analytical Methods | 2016
Friedrich Schuler; Clara Siber; Sebastian Hin; Simon Wadle; Nils Paust; Roland Zengerle; Felix von Stetten
Digital nucleic acid amplification methods are a growing research field that allows for absolute quantification of DNA making the need of standard curves redundant. However, most of the existing digital amplification systems require specialized laboratory devices and costly investments. The required disposable cartridges are device specific and not interchangeable. Here, we present digital droplet loop-mediated isothermal amplification (ddLAMP) as a microfluidic app on standard laboratory devices. ddLAMP is implemented on a disposable polymer chip (DropChip) in the format of a standard microscope slide. After off-chip DNA denaturation, the reaction mix is emulsified in the DropChip in a mini centrifuge for 6 minutes. The DropChip is transferred to an in situ thermal cycler for 1 hour of incubation. Afterwards, a fluorescence scan in a microarray scanner is performed. The DropChip allows for absolute quantification with a dynamic range of 15–1500 DNA copies per μl. Assay conditions were optimized for ddLAMP and comparison of ddPCR and ddLAMP for genomic E. coli DNA reveals very good concordance.
Methods of Molecular Biology | 2014
Simon Wadle; Stefanie Rubenwolf; Michael Lehnert; Bernd Faltin; Manfred Weidmann; Frank T. Hufert; Roland Zengerle; Felix von Stetten
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.
BioTechniques | 2016
Simon Wadle; Michael Lehnert; Friedrich Schuler; René Köppel; Annerose Serr; Roland Zengerle; Felix von Stetten
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.
Labmedicine | 2017
Nadine Borst; Friedrich Schuler; Simon Wadle; Martin Schulz; Mara Specht; Jia Li; Lisa Becherer; Martin Trotter; Ana Belén Rodríguez-Martínez; Nils Paust; Roland Zengerle; Felix von Stetten
Abstract The combination of digital amplification and centrifugal microfluidics can enable quantitative and fast diagnostics at the point of care (PoC). The new unit operation of centrifugal step emulsification allows high throughput droplet generation. Different methods for digital nucleic acid analysis, including PCR, recombinase polymerase amplification (RPA) and loop mediated isothermal amplification (LAMP), have already been demonstrated. Our novel approach of integrated sample-to-answer analysis is introduced, and examples for the detection of HIV and single cell analysis of antibiotic resistant bacteria are presented. Next to these LabDisk based systems, a microfluidic cartridge termed DropChip allows for digital amplification using only commercially available laboratory devices.
Lab on a Chip | 2010
Maximilian Focke; Fabian Stumpf; Bernd Faltin; Patrick Reith; Dylan Bamarni; Simon Wadle; Claas Müller; Holger Reinecke; Jacques Schrenzel; Patrice Francois; Daniel Mark; Günter Roth; Roland Zengerle; Felix von Stetten
Lab on a Chip | 2015
Gregor Czilwik; Thorsten Messinger; Oliver Strohmeier; Simon Wadle; F. von Stetten; Nils Paust; Günter Roth; Roland Zengerle; P. Saarinen; J. Niittymäki; K. McAllister; Orla Sheils; John J. O'Leary; Daniel Mark
Lab on a Chip | 2015
Friedrich Schuler; Frank Schwemmer; Martin Trotter; Simon Wadle; Roland Zengerle; Felix von Stetten; Nils Paust