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Concentration of viruses

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Alma Pérez-Méndez

on 16 January 2014

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Transcript of Concentration of viruses

Resin-based method for concentration
of viruses from water samples
Alma Pérez-Méndez

Introduction
Food borne diseases
Viruses
Pathogens
Indicators
The challenge
Concentration Methods
Particle size
Adsorption/elution
Hypothesis
- Positively charged polymers binds negatively charged
viruses.
- Nucleic acids may be extracted with no
elution/concentration steps

Anion exchange resin
Methods
Results
Concentration method
Detection
Gastrointestinal infections kill around 2.2 million people every year
In USA more than 9.4 million episodes of food borne diseases / yr.
Norovirus
Rotavirus *
Poliovirus
Hepatits A virus *
Hepatitis E virus
Adenovirus *
Astrovirus
Coronavirus
Coxsackievirus
Echovirus
Parvovirus
Torovirus
Fecal contamination of: drinking water,
irrigation water, washing water, recreational water or water used for shellfish production
Indicators of fecal contamination
and for source tracking
Similar structure, resistance
behavior to enteric viruses
F-RNA bacteriophages
WHO 2013
Scallan 2011, Emerg Infect Dis 17(1)
Low concentration in water samples
Enrichment or culture is not always possible or desirable
Ultracentrifugation

Ultrafiltration
Elution
Problems:
- Clogging
- Low elution recovery
- Large elution volume
- Co-concentration of
inhibitors
Objective
To develop a new virus concentration method:
- Suitable for large volume and high turbidity waters
- Small elution volume
- Fast, inexpensive, easy, field based
The resin
RNA isolation and real time RT-PCR
Bosh 1998, Internatl Microbiol 1(191)
Water sample
mixing at room temperature
Direct RNA isolation
Spiked samples
Naturally contaminated samples
Discussion
Grabow (2001), Water SA 27(2)
Wyn-Jones (2001), J. App. Microbiol 91(6)
Wyn-Jones (2001), J. App. Microbiol 91(6)
The resin-based method is useful for concentration of F-RNA coliphages and enteric viruses as Rotavirus, Adenovirus and Hepatitis A virus, providing comparable results to filter based methods at much lower cost and simplicity
Ikner (2012), Food Environ Virol 4
Resin-based method performed well in a wide variety of samples
Thank you!
January 16th, 2014
Environmental matrixes are challenging
Colloidal nature
Tap water
F-RNA coliphages, 50ml
Enteric viruses, 10L
Field evaluation
Spike F-RNA
coliphages
RNA isolation and RT-PCR
Detection
Inhibition
www.lookfordiagnosis.com/levivirus
Flocculation

Hydroextraction
Wyn-Jones (2001), J. App. Microbiol 91(6)
There is a lot of room for improvement: a more efficient extraction of nucleic acids from the resin and better systems to lower the amount of PCR inhibitors present in the final sample
The fluid nature of the resin offers several advantages over filter systems such as no clogging, very small volume of "eluent" and adaptability to flowing systems.
This was only one type of resin tested. If needed, it is possible to combine different resins in a single set to make broader the adsorption capacity of the system
Conclusions
Different performance of the four RT-PCR assays was found in different samples.
IAC did not reflect accurately the inhibition on detection of the target
Spiked samples allowed the assessment of the method performance for each sample
The different frequency of detection of the different genogroups may introduce certain bias towards GII and GIV detection. Caution is needed in source tracking
Spiked samples by genogroup
Spiked samples by water source
Spiked samples by sample type
Spiked samples, individual results
Internal amplification control
Inhibition of the target and the IAC detection
Degree of inhibition by genogroup
Naturally contaminated samples, individual results
Naturally contaminated samples, consecutive sampling days
Full transcript