Abstract

Convergence of Nucleic Acid Quantification Methods in a Single Platform

Stefan Rödiger, Group Leader, BTU Cottbus Senftenberg

The precise quantification of nucleic acids, essential for diagnostic applications, is performed through widely used multiplex methods based on DNA micro(bead)array, isothermal amplification and PCR. In addition to this, related technologies, including digital PCR, are gaining an increasing momentum. However, most platforms are limited to a specific task, such as qPCR. Moreover, the multiplex-level of the technologies is often constrained by available fluorescence dye combination. For example, conventional qPCRs are limited to five-plex assays. To address the problem of overspecialization, we developed the highly versatile VideoScan platform and unique probe detection systems. VideoScan is a real-time image analysis platform, which can be used for the quantification of thousands of microscopic objects within a sample. Applications of this platform include multiplex qPCRs, quantitative istothermal amplification reactions, genotyping and melting curve analysis. We achieve highly multiplex (15-plex) qPCRs employing only three fluorescence dyes. The VideoScan platform has also been used as a readout platform for droplet digital PCRs from different commercial vendors. To complete the convergence we developed a family of open source software packages for the data analysis and report generation. Our software is able to preprocess data (e.g., removal/imputation of missing values, outlier recognition, detection of background signal, data transformations as smoothing /normalization). This allows users to analyze data of different formats and origins in a single workflow. The talk will take the perspective of a developer. It will describe how a single platform can make the transition from an endpoint quantification method to a highly multiplex real-time platform. Dr. Rödiger graduated as engineer in Pharmabiotechnology at the University of Applied Sciences Jena. Later, he worked at the University of Jena, Technical University of Dresden and Max Plank institute Dresden