RPA has enabled researchers in Germany to develop an automated DNA amplification and chemiluminescence microarray platform that can simultaneously identify and quantify multiple viral and bacterial contaminants in water1.
The multiplex technology is an adaptation of a flow-through device called the MCR 3 (microarray chip reader, third generation), which has been developed by Dr. Michael Seidel at the Technical University of Munich’s (TUM) Institute for Hydrochemistry (Head Prof. Dr. Niessner)2. The researchers’ team originally designed the flow-based MCR 3 system to process automated immunoassays for food and water hygiene testing. The potential to combine RPA for isothermal DNA amplification with streptavidin-HRP labelling for chemiluminescence detection on the same chip opened up opportunities for developing a portable, field-based system for water testing.
The team reconstructed the device’s chip loading unit to incorporate a controllable thermoelectric heating module, and to integrate a hybridization chamber3. “The 37-40°C temperature at which RPA operates is ideal for flow-through microarrays,” Dr Seidel points out. “This is a key feature of RPA that allows us to carry out on-chip amplification. The higher temperatures required by other isothermal amplification techniques can cause water in the chip to evaporate, which will ruin the experiment or destroy the microarray. RPA is the most promising method that we know for working directly on DNA microarrays.”
The approach developed by the TUM team involves both solid- and solution-phase amplification. Spatially separated spots of unlabelled reverse primers specific to each pathogen are spotted directly onto the chip surface. Biotin-labelled forward primers, unlabelled reverse primers and RPA reagents are added to the solution phase above the microarray surface. Biotinylated amplification products are generated during the reaction both directly, by on-chip synthesis, and are also created in the bulk phase and hybridise to the immobilised primer. This dual amplification cycle enhances the assay sensitivity, the team claims. Reagent flow is one way, and a minimum number of fluidic components are required – theoretically just one valve and one syringe pump. Streptavidin-HRP binding to the biotin label allows detection by chemiluminescence.
The team tested the platform by first carrying out assays to detect single organisms, and then multiple organisms on the same chip, by spiking water samples with DNA templates for each or combinations of the water-borne bacterial and viral pathogens, human adenovirus 41 (HAdV 41), Enterococcus faecalis, and coliphage Phi X 174. The researchers also successfully used genomic DNA from culture extracts. The limits of detection (LOD) for HAdV 41, Phi X 174 and E. faecalis, were 35 genomic units/μL, 1 GU/μL and 5 x 103 GU/μL, respectively, which the authors say is comparable to the sensitivity reported for qPCR analysis, but with a much shorter assay time.
“Since the amplification takes place individually for one target pathogen per spot, a multiplexing of the developed assay is enabled,” the researchers conclude in their published paper in Analytical Chemistry. “The pathogen is identified by the spot position on the microarray, while the chemiluminescence intensity correlates with the amount of pathogen DNA in the sample.”
Since publication of the paper the TUM researchers have developed their platform into a portable microarry for detecting Legionella in concentrated water samples from cooling towers or air conditioning systems. The platform is undergoing validation testing. “The idea is that the technology can be used by people with minimal training,” Dr. Seidel stresses. “The process is automated and simple. In the future we hope to develop a broadly applicable water hygiene-monitoring platform that can simultaneously detect 10 or 15 different bacterial and viral contaminants in concentrated samples of industrial water and drinking water.”
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1 Kunze, A.; Dilcher, M.; Abd El Wahed, A.; Hufert, F.; Niessner, R. and Seidel, M., On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria. Analytical Chemistry 2016, 88, 898–905. DOI:10.1021/acs.analchem.5b03540.
2 Seidel, M. and Niessner, R., Chemiluminescence microarrays: a critical review. Analytical and Bioanalytical Chemistry 2014,406, 5589–5612, https://www.ncbi.nlm.nih.gov/pubmed/25002333
3 Lengger, S.; Otto, J.; Elsaesser, D.; Schneider, O.; Tiehm, A.; Fleischer, J.; Niessner, R.; Seidel, M., Oligonucleotide microarray chip for the quantification of MS2, PhiX174, and adenoviruses on the multiplex analysis platform MCR 3. Analytical Bioanalytical Chemistry 2014, 406, 3323-3334, link.springer.com/article/10.1007%2Fs00216-014-7641-y.