Versatile, automated sample preparation and detection of contaminants and biological materials

Abstract

Contamination of food, water, medicine and ingestible household products is a public health hazard that episodically causes outbreaks worldwide. Existing laboratory methods are often expensive, require a laboratory environment and/or trained staff to perform manual steps. The aim of this PhD thesis was to create and test methods and instruments for affordable diagnostic tests for contaminants and pathogens. To achieve this goal, the LabReader was introduced, which employs a LED-based detection scheme for four simultaneous fluorescence- and UV-measurements. Assays were developed to detect (di-)ethylene glycol in consumables ≥0.1wt% and alcohols ≥1ppb. Pathogens in water, foods and blood were detected at ≥104 CFU/ml using nonspecific intercalating dyes. To gain sensitivity and specificity for cell-based analysis, biochemical amplification methods had to be incorporated. To be deployable outside a laboratory, sample preparation needed to be automated. Automation was achieved by combining the LabReader with the already developed LabTube, a disposable platform for automated DNA extraction inside a standard centrifuge. Performing DNA amplification/readout in an external optical reader, made the LabSystem broadly deployable and flexible. DNA extraction of food bacteria (E.coli and Alicyclobacillus) was optimized inside the LabTube for 102-109 inserted DNA copies. The extracted DNA was amplified using the qualitative isothermal LAMP method and semi-quantitative, real-time PCR inside the LabReader. The combined extraction and amplification detection limit of the LAMP-LabSystem and the quantitation limit of the PCR-LabSystem were as low as 102 copies. Performing extraction and amplification inside the centrifuge/LabTube was also outlined, which may be preferable when contamination risks are high. After theoretically evaluating heating methods, a battery-driven heated LabTube was designed, in which 102-108 DNA copies of VTEC E.coli were extracted, LAMP-amplified and visually readout within 1.5 hrs. The major contribution of this thesis is the full system integration of versatile, automated sample preparation and detection systems. They offer great flexibility as they may be used with each other or in combination with other analytic methods, depending on the application. At the same time, they are frugal and deployable at low-to-medium throughput - even outside a traditional laboratory. Whilst the focus was put on food safety, the systems were also used for medical, environmental or consumer product quality applications, hence demonstrating their broad applicability.