High performance concentration method for viruses in drinking water

Kunze, Andreas; Pei, Lu; Elsäßer, Dennis; Nießner, Reinhard; Seidel, Michael

doi:10.1016/j.jviromet.2015.06.007

Cited by 22 publications

(21 citation statements)

References 30 publications

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“…The principle of the MAF adsorption/elution method was based on the procedure as previously described [18]. Monolithic discs, diameter 3.86 cm and height 1.0 cm, were synthesized by polymerization of polyglycerol-3-glycidyl ether (Ipox chemicals, Laupheim, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…To overcome these biases, different methods to concentrate viruses from water samples have been developed, including: polyethylene glycol precipitation (PEG) [8], FeCl 3 precipitation [13], skimmed milk flocculation (SMF) [14], glass wool filtration (GW) [15] or monolithic adsorption filtration (MAF) [16]. The influence of concentration method on viral recovery has been evaluated on sea water [17], spiked tap water [15,18] and raw sewage [19], cautioning of method associated biases. To our knowledge, no major comparison studies using metagenomics have been performed with sewage water.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing

et al. 2017

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Viral sewage metagenomics is a novel field of study used for surveillance, epidemiological studies, and evaluation of waste water treatment efficiency. In raw sewage human waste is mixed with household, industrial and drainage water, and virus particles are, therefore, only found in low concentrations. This necessitates a step of sample concentration to allow for sensitive virus detection. Additionally, viruses harbor a large diversity of both surface and genome structures, which makes universal viral genomic extraction difficult. Current studies have tackled these challenges in many different ways employing a wide range of viral concentration and extraction procedures. However, there is limited knowledge of the efficacy and inherent biases associated with these methods in respect to viral sewage metagenomics, hampering the development of this field. By the use of next generation sequencing this study aimed to evaluate the efficiency of four commonly applied viral concentrations techniques (precipitation with polyethylene glycol, organic flocculation with skim milk, monolithic adsorption filtration and glass wool filtration) and extraction methods (Nucleospin RNA XS, QIAamp Viral RNA Mini Kit, NucliSENS® miniMAG®, or PowerViral® Environmental RNA/DNA Isolation Kit) to determine the viriome in a sewage sample. We found a significant influence of concentration and extraction protocols on the detected viriome. The viral richness was largest in samples extracted with QIAamp Viral RNA Mini Kit or PowerViral® Environmental RNA/DNA Isolation Kit. Highest viral specificity were found in samples concentrated by precipitation with polyethylene glycol or extracted with Nucleospin RNA XS. Detection of viral pathogens depended on the method used. These results contribute to the understanding of method associated biases, within the field of viral sewage metagenomics, making evaluation of the current literature easier and helping with the design of future studies.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing

et al. 2017

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“…This method can also be applied in other experiments that require detection of a “neutralized” phage, which is merely a phage that is not active against bacteria. Notably, qPCR has already been applied for phage detection in other (than phage neutralizing) conditions: in phage cultures ( Edelman and Barletta, 2003 ; Clokie, 2009 ; Anderson et al, 2011 ; Refardt, 2012 ; Dieterle et al, 2016 ), food ( Imamovic and Muniesa, 2011 ; Flannery et al, 2014 ; Perrin et al, 2015 ; Parente et al, 2016 ; Hartard et al, 2017 ; Muhammed et al, 2017 ), environmental samples ( Farkas et al, 2015 ; Kunze et al, 2015 ; Unnithan et al, 2015 ; Mankiewicz-Boczek et al, 2016 ), and in feces ( Imamovic et al, 2010 ; Chehoud et al, 2016 ), where some interference from antibodies cannot be excluded ( Majewska et al, 2015 ).…”

Section: Discussionmentioning

confidence: 99%

“…Bacteriophages have been quantitatively analyzed and discriminated by real-time qPCR directly in microbiological cultures, and the authors found this method to be a good alternative to the plaque assay ( Edelman and Barletta, 2003 ; Clokie, 2009 ; Anderson et al, 2011 ; Refardt, 2012 ; Dieterle et al, 2016 ). Real-time PCR has been further demonstrated as applicable for a rapid screening allowing phage detection in food (milk, fruits, vegetables, seafood, meat) ( Imamovic and Muniesa, 2011 ; Flannery et al, 2014 ; Perrin et al, 2015 ; Parente et al, 2016 ; Hartard et al, 2017 ) and water samples ( Farkas et al, 2015 ; Kunze et al, 2015 ; Unnithan et al, 2015 ; Mankiewicz-Boczek et al, 2016 ) or in feces ( Imamovic et al, 2010 ; Chehoud et al, 2016 ). However, potential applicability of real-time qPCR for detection of inactivated (non-infective) but still biologically active (e.g., immunoreactive) phage has never been investigated.…”

Section: Introductionmentioning

confidence: 99%

Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles

et al. 2017

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The most common method for phage quantitation is the plaque assay, which relies on phage ability to infect bacteria. However, non-infective phage particles may preserve other biological properties; specifically, they may enter interactions with the immune system of animals and humans. Here, we demonstrate real-time quantitative polymerase chain reaction (qPCR) detection of bacteriophages as an alternative to the plaque assay. The closely related staphylococcal bacteriophages A3R and 676Z and the coliphage T4 were used as model phages. They were tested in vivo in mice, ex vivo in human sera, and on plastic surfaces designed for ELISAs. T4 phage was injected intravenously into pre-immunized mice. The phage was completely neutralized by specific antibodies within 5 h (0 pfu/ml of serum, as determined by the plaque assay), but it was still detected by qPCR in the amount of approximately 107 pfu/ml of serum. This demonstrates a substantial timelapse between “microbiological disappearance” and true clearance of phage particles from the circulation. In human sera ex vivo, qPCR was also able to detect neutralized phage particles that were not detected by the standard plaque assay. The investigated bacteriophages differed considerably in their ability to immobilize on plastic surfaces: this difference was greater than one order of magnitude, as shown by qPCR of phage recovered from plastic plates. The ELISA did not detect differences in phage binding to plates. Major limitations of qPCR are possible inhibitors of the PCR reaction or free phage DNA, which need to be considered in procedures of phage sample preparation for qPCR testing. We propose that phage pharmacokinetic and pharmacodynamic studies should not rely merely on detection of antibacterial activity of a phage. Real-time qPCR can be an alternative for phage detection, especially in immunological studies of bacteriophages. It can also be useful for studies of phage-based drug nanocarriers or biosensors.

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“…Where analytes are present in the matrix at a too low concentration several enrichment procedures have been proposed, e.g., desalting; size exclusion; ion exchange [ 83 ]; filter enrichment as for instance ultrafiltration and monolithic filtration [ 84 ]; magnetic particles; and MIPs.…”

Section: Sample Preparationmentioning

confidence: 99%

Analytical Protein Microarrays: Advancements Towards Clinical Applications

Sauer

2017

Sensors

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Protein microarrays represent a powerful technology with the potential to serve as tools for the detection of a broad range of analytes in numerous applications such as diagnostics, drug development, food safety, and environmental monitoring. Key features of analytical protein microarrays include high throughput and relatively low costs due to minimal reagent consumption, multiplexing, fast kinetics and hence measurements, and the possibility of functional integration. So far, especially fundamental studies in molecular and cell biology have been conducted using protein microarrays, while the potential for clinical, notably point-of-care applications is not yet fully utilized. The question arises what features have to be implemented and what improvements have to be made in order to fully exploit the technology. In the past we have identified various obstacles that have to be overcome in order to promote protein microarray technology in the diagnostic field. Issues that need significant improvement to make the technology more attractive for the diagnostic market are for instance: too low sensitivity and deficiency in reproducibility, inadequate analysis time, lack of high-quality antibodies and validated reagents, lack of automation and portable instruments, and cost of instruments necessary for chip production and read-out. The scope of the paper at hand is to review approaches to solve these problems.

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High performance concentration method for viruses in drinking water

Cited by 22 publications

References 30 publications

Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing

Evaluation of Methods for the Concentration and Extraction of Viruses from Sewage in the Context of Metagenomic Sequencing

Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles

Analytical Protein Microarrays: Advancements Towards Clinical Applications

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