Werner Lehmann

A Highly Versatile Microscope Imaging Technology Platform for the Multiplex Real-Time Detection of Biomolecules and Autoimmune Antibodies

The analysis of different biomolecules is of prime importance for life science research and medical diagnostics. Due to the discovery of new molecules and new emerging bioanalytical problems, there is an ongoing demand for a technology platform that provides a broad range of assays with a user-friendly flexibility and rapid adaptability to new applications. Here we describe a highly versatile microscopy platform, VideoScan, for the rapid and simultaneous analysis of various assay formats based on fluorescence microscopic detection. The technological design is equally suitable for assays in solution, microbead-based assays and cell pattern recognition. The multiplex real-time capability for tracking of changes under dynamic heating conditions makes it a useful tool for PCR applications and nucleic acid hybridization, enabling kinetic data acquisition impossible to obtain by other technologies using endpoint detection. The paper discusses the technological principle of the platform regarding data acquisition and processing. Microbead-based and solution applications for the detection of diverse biomolecules, including antigens, antibodies, peptides, oligonucleotides and amplicons in small reaction volumes, are presented together with a high-content detection of autoimmune antibodies using a HEp-2 cell assay. Its adaptiveness and versatility gives VideoScan a competitive edge over other bioanalytical technologies.

Nucleic acid detection based on the use of microbeads: a review

AbstractMicrobead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. Contains 211 references. Figureᅟ

Adhesion of human and animal escherichia coli strains in association with their virulence-associated genes and phylogenetic origins

ABSTRACT Intestinal colonization is influenced by the ability of the bacterium to inhabit a niche, which is based on the expression of colonization factors. Escherichia coli carries a broad range of virulence-associated genes (VAGs) which contribute to intestinal (inVAGs) and extraintestinal (exVAGs) infection. Moreover, initial evidence indicates that inVAGs and exVAGs support intestinal colonization. We developed new screening tools to genotypically and phenotypically characterize E. coli isolates originating in humans, domestic pigs, and 17 wild mammal and avian species. We analyzed 317 isolates for the occurrence of 44 VAGs using a novel multiplex PCR microbead assay (MPMA) and for adhesion to four epithelial cell lines using a new adhesion assay. We correlated data for the definition of new adhesion genes. inVAGs were identified only sporadically, particularly in roe deer (Capreolus capreolus) and the European hedgehog ( Erinaceus europaeus). The prevalence of exVAGs depended on isolation from a specific host. Human uropathogenic E. coli isolates carried exVAGs with the highest prevalence, followed by badger (Meles meles) and roe deer isolates. Adhesion was found to be very diverse. Adhesion was specific to cells, host, and tissue, though it was also unspecific. Occurrence of the following VAGs was associated with a higher rate of adhesion to one or more cell lines: afa-dra, daaD, tsh, vat, ibeA, fyuA, mat, sfa-foc, malX, pic, irp2, and papC. In summary, we established new screening methods which enabled us to characterize large numbers of E. coli isolates. We defined reservoirs for potential pathogenic E. coli. We also identified a very broad range of colonization strategies and defined potential new adhesion genes.

Mikropartikelsysteme für die Nukleinsäurediagnostik

PCR is a simplistic and robust laboratory technology for nucleic acid detection. However, for research and diagnostics processing multiple targets within one reaction in an automatic fashion is a demanded feature. Combining two multiplex read out technologies, such as microarray and microbeads, the VideoScan platform was designed. This microscope imaging technology enables an automatable high throughput multiplex measurement of genetic material from biological and patient samples.

Algorithms for automated detection of hook effect-bearing amplification curves

Amplification curves from quantitative Real-Time PCR experiments typically exhibit a sigmoidal shape. They can roughly be divided into a ground or baseline phase, an exponential amplification phase, a linear phase and finally a plateau phase, where in the latter, the PCR product concentration no longer increases. Nevertheless, in some cases the plateau phase displays a negative trend, e.g. in hydrolysis probe assays. This cycle-to-cycle fluorescence decrease is commonly referred to in the literature as the hook effect. Other detection chemistries also exhibit this negative trend, however the underlying molecular mechanisms are different. In this study we present two approaches to automatically detect hook effect-like curvatures based on linear (hookreg) and nonlinear regression (hookregNL). As the hook effect is typical for qPCR data, both algorithms can be employed for the automated identification of regular structured qPCR curves. Therefore, our algorithms streamline quality control, but can also be used for assay optimization or machine learning.

Digitale PCR in der Labordiagnostik

The need for new highly sensitive, cost-efficient, fast and robust nucleic acid detection and quantification technologies is a driving force. PCR, especially quantitative PCR (qPCR), is the method of choice in diagnostics and life-sciences. The digital PCR (dPCR) provides a new technology to measure absolute quantities of nucleic acids without the need for calibration curves. This review gives details on the dPCR technology and available platforms. We discuss the platform differences, common features as well as their advantages and disadvantages.