Oligonucleotide microarray chip for the quantification of MS2, ΦX174, and adenoviruses on the multiplex analysis platform MCR 3.

Pathogenic viruses are emerging contaminants in water which should be analyzed for water safety to preserve public health. A strategy was developed to quantify RNA and DNA viruses in parallel on chemiluminescence flow-through oligonucleotide microarrays. In order to show the proof of principle, bacteriophage MS2, ΦX174, and the human pathogenic adenovirus type 2 (hAdV2) were analyzed in spiked tap water samples on the analysis platform MCR 3. The chemiluminescence microarray imaging unit was equipped with a Peltier heater for a controlled heating of the flow cell. The efficiency and selectivity of DNA hybridization could be increased resulting in higher signal intensities and lower cross-reactivities of polymerase chain reaction (PCR) products from other viruses. The total analysis time for DNA/RNA extraction, cDNA synthesis for RNA viruses, polymerase chain reaction, single-strand separation, and oligonucleotide microarray analysis was performed in 4-4.5 h. The parallel quantification was possible in a concentration range of 9.6 × 10(5)-1.4 × 10(10) genomic units (GU)/mL for bacteriophage MS2, 1.4 × 10(5)-3.7 × 10(8) GU/mL for bacteriophage ΦX174, and 6.5 × 10(3)-1.2 × 10(5) for hAdV2, respectively, by using a measuring temperature of 40 °C. Detection limits could be calculated to 6.6 × 10(5) GU/mL for MS2, 5.3 × 10(3) GU/mL for ΦX174, and 1.5 × 10(2) GU/mL for hAdV2, respectively. Real samples of surface water and treated wastewater were tested. Generally, found concentrations of hAdV2, bacteriophage MS2, and ΦX174 were at the detection limit. Nevertheless, bacteriophages could be identified with similar results by means of quantitative PCR and oligonucleotide microarray analysis on the MCR 3.

Combination of crossflow ultrafiltration, monolithic affinity filtration, and quantitative reverse transcriptase PCR for rapid concentration and quantification of model viruses in water.

We present a rapid and effective adsorption-elution method based on monolithic affinity filtration (MAF) for the concentration and purification of waterborne viruses. The MAF column consists of a hydrolyzed macroporous epoxy-based polymer. High recoveries were achieved by columns for the bacterial virus (bacteriophage) MS2 110 (±19)%, as model organism, as well as for human adenoviruses 42.4 (±3.4)% and murine noroviruses 42.6 (±1.9)%. This new concentration and purification method was combined with crossflow ultrafiltration (CUF). Because of the adsorption of the examined viruses to the macroporous surface of the MAF column at pH 3, concentrated matrix components by CUF can be removed. Bacteriophages MS2 were spiked in tap water and concentrated with the new CUF-MAF concentration method by a volumetric factor of 10(4) within 33 min. Furthermore, the detection limit for quantification of bacteriophage MS2 by quantitative reverse transcriptase PCR (qRT-PCR) could be improved from 79.47 to 0.0056 GU mL(-1) by a factor of 1.4 × 10(4). In a first study, we have shown that this method could also be applied for river water containing naturally MS2 and MS2-like phages.