This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/μL, 1 GU/μL, and 5 × 10(3) GU/μL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.
The state-of-the-art monitoring of drinking water hygiene is based on the cultivation and enumeration of indicator bacteria. Despite its proven reliability, this approach has the disadvantages of being (a) relatively slow and (b) limited to a small number of indicator bacteria. Ideally, alternative methods would be less time-consuming while providing information about a larger set of hygienically relevant microorganisms including viruses. In this paper, we present insights into the design of a modular concentration and detection system for bacteria, bacteriophages and viruses. Following further validation, this or similar techniques have the potential to extend and speed up the monitoring of raw and drinking water hygiene in the future. The system consists of different modules for the concentration of microorganisms, an amplification and detection unit that includes a module for the differentiation between live and dead microorganisms, and an automated system for decision support and self-diagnosis. The ongoing testing under controlled laboratory conditions and real-life conditions in the water supply industry yields further system improvements. Moreover, the increased sensitivity and broader range of microbiological parameters emphasize the need for a reconsideration of the currently used criteria for the assessment of (drinking) water hygiene
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