Combining ultrafiltration, immunoseparation and analytical microarrays for the monitoring of viruses from large drinking water samples with a volume of 30,000 L
Zusammenfassung der Projektergebnisse
According to the risk assessment of the WHO, pathogenic viruses should not be present in large-volume (up to 30 m3 (WHO, 2004) and up to 90 m3 (WHO, 2011)) drinking water samples. In contrast, analysis methods for viruses are only operable in small volumes. Primary and secondary concentration methods have to be combined efficiently to achieve high recoveries in a shortest possible time for the analysis of viruses in such large-volume water samples. A proof-of-principle study was executed to show the route for concentrating viruses from 90 m3 tap water to a final 1-mL sample which is ready for the analyses with qRT-PCR or cell culture assay. The instrumentation for ultrafiltration of large-volume drinking water samples was built up for primary concentration by either dead-end or cross-flow ultrafiltration (UF). Filtration flow rates of up to 1.5 m3/h were achieved by a hollow fiber multibore UF module with a membrane area of 6 m2 and a pore size of about 20 nm. First recovery experiments were conducted by concentration of bacteriophage MS2 as model viruses. They were spiked directly in tap water during the ultrafiltration by injection in the water tube before the ultrafiltration unit. 1 - 30 m3 of tap water was ultrafiltrated in dead-end and cross-flow mode. For processing 1 m3 in DE-UF mode the recovery of MS2 was comparable using plaque assay, or qPCR as quantification method (around 20%). Increased recoveries were determined for CF-UF. The result for qPCR (45 (±23)%) has shown higher recoveries than with plaque assay (30 (±6)%). We could conclude that more MS2 are inactivated for plaque assay by using the CF-UF mode. Processing 30 m3 of tap water besides of MS2 bacteriophages particles and agglomerated phages will be concentrated resulting in a reduced recovery quantified by plaque assay. The quantified recovery of 8 (± 7)% by plaque assay and 39 (± 30)% by qRT-PCR has suggested inactivation processes. In summary, dead-end UF was the promising method for low turbidity water in respect of total recovery and time-efficiency. A new MAF method was established as secondary concentration method which is able to process rapidly (20 L in 20 min) the UF-eluate. The MAF column contained the macroporous activated polyepoxide supports in form of disks with a diameter of 3.86 cm and a height of 1.0 cm. For a principle study acidified 10-L tap water samples (pH 3.0) spiked with MS2 in concentrations ranging from 2.2 (± 0.5) x 103 to 1.9 (± 0.2) x 108 PFU were tested. Almost all seeded MS2 were recovered (recovery rates of 102 (± 23)%, n = 10, m = 3) in 15 min. For tap water the binding capacity of viruses on MAF-disks was not a limiting factor. In cooperation with UBA a mixture of bacteriophage MS2 and φX174, murine noroviruses and human adenoviruses were concentrated in tap water. 106% and 40 (± 17)% of bacteriophage MS2 and φX174, respectively were recovered by plaque assay analysis. Human adenoviruses and murine noroviruses were determined by qPCR. Recoveries of 67 (± 59)% and 12 (± 6)%, respectively, were found. The results prove the efficiency of this secondary concentration method and the possibility to simultaneously concentrate various viruses by MAF-disk columns. In summary, a secondary concentration method was found which is fast, highly effective and the production costs of MAF-disk columns (< 2 Euro) are low. At the end of the project the combined concentration methods were applied to tap water samples of 1 to 90 m³. The concentration method CF-UF/DE-UF & MAF-disks & centrifugal UF has achieved a concentration factor up to 108 and a total recovery of up to 13%. This method was also applied at UBA for 35-m3 groundwater spiked with waste water. There, it could be shown that MAF-disks concentration is more efficient than glass wool filtration. In cooperation with Prof. Drosten we could show that the virus concentration of 30 m3 groundwater by DE-UF & MAF-disks & CeUF was suitable to identify viruses by next generation sequencing without a prior enrichment by cultivation. 4 different bacteriophages were identified. With this project we achieved the first combined concentration system that is able to concentrate viruses from large-volume water samples (up to 90.000 L) to small volumes (1 mL) applicable for molecular biological detection methods.
Projektbezogene Publikationen (Auswahl)
- Combination of crossflow ultrafiltration, monolithic affinity filtration, and quantitative reverse transcriptase PCR for rapid concentration and quantification of model viruses in water. Environmental Science & Technology 2012, 46, 10073-10080
Pei, L., Rieger, M., Lengger, S., Ott, S., Zawadsky, C., Hartmann, N.M., Selinka, H.C., Tiehm, A., Niessner, R., Seidel, M.
(Siehe online unter https://doi.org/10.1021/es302304t) - Pathogens in Water - enrich and verify. Nachrichten aus der Chemie 2012, 60, 1208-1212
Lengger, S., Niessner, R., Seidel, M.
