title: On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.
abstract: 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.
authors.affiliation: Institute of Hydrochemistry, Chair of Analytical Chemistry, Technical University of Munich , Marchioninistrasse 17, D-81377 Munich, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Kunze
authors.affiliation: Department of Virology, University Medical Center, Georg-August-University Göttingen , Kreuzbergring 57, D-37075 Göttingen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Dilcher
authors.affiliation: Division of Microbiology and Animal Health, Georg-August-University Göttingen , Burckhardtweg 2, D-37077 Göttingen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Abd El Wahed
authors.affiliation: Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane , Grossenhainer Strasse 57, D-01968 Senftenberg, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Hufert
authors.affiliation: Institute of Hydrochemistry, Chair of Analytical Chemistry, Technical University of Munich , Marchioninistrasse 17, D-81377 Munich, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Niessner
authors.affiliation: Institute of Hydrochemistry, Chair of Analytical Chemistry, Technical University of Munich , Marchioninistrasse 17, D-81377 Munich, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Seidel
mesh_terms: D000260:Adenoviruses, Human / Q000302:isolation & purification*; D010584:Bacteriophage phi X 174 / Q000302:isolation & purification*; D013293:Enterococcus faecalis / Q000302:isolation & purification*; D008163:Luminescent Measurements / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D013696:Temperature