Systeme für die Chip-basierte Nukleinsäure-Analytik mit integrierter DNA-Amplifikation und Detektion

Die vorgelegte Arbeit beschäftigt sich mit der Entwicklung von Lab-on-a-Chip Systemen für die Bioanalyik. Die Zielstellung bestand in der Übertragung molekularbiologischer Nachweismethoden zur spezifischen Amplifikation und Untersuchung von Nukleinsäuresequenzen in den miniaturisierten Maßstab. Dabei wurde ein System zur on-Chip Real-Time PCR (Polymerase Kettenreaktion, engl. Polymerase Chain Reaction) umgesetzt, wobei die Reaktionslösung als Tropfen mit einem Volumen von wenigen Mikrolitern auf der Oberfläche eines Probenträgers appliziert wurde. Die Oberfläche dieses Probenträgers wurde zu diesem Zweck mit Repel-Silan sowie Teflon beschichtet und strukturiert, wodurch die in Mineralöl eingebettete Reaktionslösung einen halbkugelförmigen Tropfen ausbildet und somit vor Verdunstung geschützt wird. Ein zweites System zur Verknüpfung der DNA-Amplifikation mit einem Mikroarray- basierten Nachweis wurde entwickelt. An diesem System wurde speziell der Nachweis verschiedener Arten des Pflanzenpathogens Phytophthora etabliert. Die Funktionsfähigkeit der beiden Systeme konnte anhand unterschiedlicher Anwendungsbeispiele demonstriert werden. Mit Hilfe der on-Chip Real-Time PCR konnten Genabschnitte detektiert werden, darunter der Tumorsupressor p53. Neben DNA und RNA wurden auch Zellen bzw. Sporen direkt als Ausgangsmaterial bei der Chip-PCR eingesetzt, wodurch auf eine aufwändige Extraktion der Nukleinsäuren verzichtet werden konnte. Hinsichtlich der Sensitivität konnte gezeigt werden, dass einzelne DNA-Moleküle sowie einzelne Zellen bzw. wenige Sporen nachweisbar sind. Die hohe Spezifität der Nachweismethoden konnte ebenfalls demonstriert werden, vor allen bei der Verwendung Sequenz-spezifischer Oligonukleotide, beispielsweise als Fluoreszenz-markierte Sonden bei der Real-Time PCR oder in Form von Fänger-Molekülen auf dem DNA-Mikroarray. Mittels Sequenz-spezifischer Hybridisierung konnten fünf nah verwandte Phytophthora-Arten eindeutig voneinander unterschieden werden

Standoff laser induced fluorescence of living and inactivated bacteria

Biological hazards, such as bacteria, represent a non-assessable threat in case of an accident or a terroristic attack. Rapid detection and highly sensitive identification of released, suspicious substances at low false alarm rates are challenging requirements which one single technology cannot cope with. It has been shown that standoff detection using laser-induced fluorescence (LIF) can provide information on the class of bioorganic substances in real-time1. In combination with traditional, highly sensitive, but non-standoff methods, the time for identification of the threat can be optimized. This work is aimed at the selectivity of LIF technology for different bacterial strains. A second important aspect examines how to deal with inactivated bacteria and how their fluorescence signature changes after deactivation. LIF spectra of closely and more distantly related bacterial strains are presented as well as spectra of bacteria treated by different inactivation methods

Standoff detection: distinction of bacteria by hyperspectral laser induced fluorescence

Sensitive detection and rapid identification of hazardous bioorganic material with high sensitivity and specificity are essential topics for defense and security. A single method can hardly cover these requirements. While point sensors allow a highly specific identification, they only provide localized information and are comparatively slow. Laser based standoff systems allow almost real-time detection and classification of potentially hazardous material in a wide area and can provide information on how the aerosol may spread. The coupling of both methods may be a promising solution to optimize the acquisition and identification of hazardous substances. The capability of the outdoor LIF system at DLR Lampoldshausen test facility as an online classification tool has already been demonstrated. Here, we present promising data for further differentiation among bacteria. Bacteria species can express unique fluorescence spectra after excitation at 280 nm and 355 nm. Upon deactivation, the spectral features change depending on the deactivation method

Evaluation of a microfluidic chip system for preparation of bacterial DNA from swabs, air, and surface water samples

AbstractThe detection of bacterial pathogens from complex sample matrices by PCR requires efficient DNA extraction. In this study, a protocol for extraction and purification of DNA from swabs, air, and water samples using a microfluidic chip system was established. The optimized protocol includes a combination of thermal, chemical and enzymatic lysis followed by chip-based DNA purification using magnetic particles. The procedure was tested using Gram-positive Bacillus thuringiensis Berliner var. kurstaki as a model organism for Bacillus anthracis and the attenuated live vaccine strain of Francisella tularensis subsp. holarctica as Gram-negative bacterium. The detection limits corresponded to 103 genome equivalents per milliliter (GE/ml) for surface water samples spiked with F. tularensis and 102 GE/ml for B. thuringiensis. In air, 10 GE of F. tularensis per 10 L and 1 GE of B. thuringiensis per 10 L were detectable. For swab samples obtained from artificially contaminated surfaces the detection limits were 4 × 103 GE/cm2 for F. tularensis and 4 × 102 GE/cm2 for B. thuringiensis. Suitability of the chip-assisted procedure for DNA preparation of real samples was demonstrated using livestock samples. The presence of thermophilic Campylobacter spp. DNA could be confirmed in air samples collected on pig and broiler farms

Electrochemiluminescence DNA sensor array for multiplex detection of biowarfare agents

Development of a fully automated electrochemiluminescence (ECL) DNA assay for multiplex detection of six biowarfare agents is described. Aminated-DNA capture probes were covalently immobilised on activated-carbon electrodes and subsequently hybridised to target strands. Detection was achieved via a sandwich-type assay after Ru(bpy)<inf>3</inf>2+-labelled reporter probes were hybridised to the formed probetarget complexes. The assay was performed in an automated microsystem in a custom-designed ECL detection box with integrated fluidics, electronics, and movable photomultiplier detector. The obtained limits of detection were 0.61.2 nmol L1 for six targets ranging from 50 to 122 base pairs in size, with linear range 115 nmol L1. Non-specific adsorption and cross-reactivity were very low. Detection of six targets on a single chip was achieved with subnanomolar detection limits

Hare-to-Human Transmission of Francisella tularensis subsp. holarctica, Germany

In November 2012, a group of 7 persons who participated in a hare hunt in North Rhine-Westphalia, Germany, acquired tularemia. Two F. tularensis subsp. holarctica isolates were cultivated from human and hare biopsy material. Both isolates belonged to the FTN002–00 genetic subclade (derived for single nucleotide polymorphisms B.10 and B.18), thus indicating likely hare-to-human transmission