Search Results
A manual and an automatic TERS based virus discrimination
2015, Olschewski, Konstanze, Kämmer, Evelyn, Stöckel, Stephan, Bocklitz, Thomas, Deckert-Gaudig, Tanja, Zell, Roland, Cialla-May, Dana, Weber, Karina, Deckert, Volker, Popp, Jürgen
Rapid techniques for virus identification are more relevant today than ever. Conventional virus detection and identification strategies generally rest upon various microbiological methods and genomic approaches, which are not suited for the analysis of single virus particles. In contrast, the highly sensitive spectroscopic technique tip-enhanced Raman spectroscopy (TERS) allows the characterisation of biological nano-structures like virions on a single-particle level. In this study, the feasibility of TERS in combination with chemometrics to discriminate two pathogenic viruses, Varicella-zoster virus (VZV) and Porcine teschovirus (PTV), was investigated. In a first step, chemometric methods transformed the spectral data in such a way that a rapid visual discrimination of the two examined viruses was enabled. In a further step, these methods were utilised to perform an automatic quality rating of the measured spectra. Spectra that passed this test were eventually used to calculate a classification model, through which a successful discrimination of the two viral species based on TERS spectra of single virus particles was also realised with a classification accuracy of 91%.
Non-instrumented DNA isolation, amplification and microarray-based hybridization for a rapid on-site detection of devastating Phytophthora kernoviae
2015, Schwenkbier, Lydia, Pollok, Sibyll, Rudloff, Anne, Sailer, Sebastian, Cialla-May, Dana, Weber, Karina, Popp, Jürgen
A rapid and simple instrument-free detection system was developed for the identification of the plant pathogen Phytophthora kernoviae (P. kernoviae). The on-site operable analysis steps include magnetic particle based DNA isolation, helicase-dependent amplification (HDA) and chip-based DNA hybridization. The isothermal approach enabled the convenient amplification of the yeast GTP-binding protein (Ypt1) target gene in a miniaturized HDA-zeolite-heater (HZH) by an exothermic reaction. The amplicon detection on the chip was performed under room temperature conditions – either by successive hybridization and enzyme binding or by a combined step. A positive signal is displayed by enzymatically generated silver nanoparticle deposits, which serve as robust endpoint signals allowing an immediate visual readout. The hybridization assay enabled the reliable detection of 10 pg μL−1 target DNA. This is the first report of an entirely electricity-free, field applicable detection approach for devastating Phytophthora species, exemplarily shown for P. kernoviae.
Label-free detection of Phytophthora ramorum using surface-enhanced Raman spectroscopy
2015, Yüksel, Sezin, Schwenkbier, Lydia, Pollok, Sibyll, Weber, Karina, Cialla-May, Dana, Popp, Jürgen
In this study, we report on a novel approach for the label-free and species-specific detection of the plant pathogen Phytophthora ramorum from real samples using surface enhanced Raman scattering (SERS). In this context, we consider the entire analysis chain including sample preparation, DNA isolation, amplification and hybridization on SERS substrate-immobilized adenine-free capture probes. Thus, the SERS-based detection of target DNA is verified by the strong spectral feature of adenine which indicates the presence of hybridized target DNA. This property was realized by replacing adenine moieties in the species-specific capture probes with 2-aminopurine. In the case of the matching capture and target sequence, the characteristic adenine peak serves as an indicator for specific DNA hybridization. Altogether, this is the first assay demonstrating the detection of a plant pathogen from an infected plant material by label-free SERS employing DNA hybridization on planar SERS substrates consisting of silver nanoparticles.