VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Summary

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

Abstract

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.

At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.

The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Introduction

During the first phases of any humanitarian emergency, access to clean water supplies, sanitation, and hygiene are critical for the survival of those affected. Therefore, monitoring water quality is a priority to prevent waterborne outbreaks. It is well-known that contaminated water is frequently the origin of diseases, but it is often difficult to determine the sources of viral outbreaks such as Hepatitis E virus (HEV), even with the availability of conventional laboratory methods. The control of water quality is based on the quantification of FIB^1,2,3,

Protocol

1. Preparation and packaging

NOTE: The materials/equipment to be packed is listed in Table 1. Use gloves to handle the reagents required for the process control, the concentration reagents, the nucleic acid extraction reagents and the detection reagents. Wear protective glasses to handle the reagents required for nucleic acid extraction.

1. Process control
   1. Prepare MS2 bacteriophage stock culture (American Type Culture Collection [ATCC] 15597-B1) containing 1 x 10¹⁰ PFU/mL in the laboratory by following the ISO procedure 10705-1:1995¹⁴.
   ....

Results

Method development

This procedure has been developed in the Laboratory of Viruses Contaminants of Water and Food with the collaboration of GenIUL and Oxfam Intermón. It comprises of three different steps. The first one, the viral particle concentration, is an adaptation of a skimmed milk flocculation method previously described^12,17,

Discussion

The VirWaTest method enables the concentration of viruses and nucleic acid extraction from water samples at the point of use by non-experienced users. It is an affordable, rapid, and simple protocol. The concentration is based on the principle of organic flocculation using skimmed milk, by which the low pH and high conductivity conditions make skimmed milk proteins aggregate into flocs the viruses adsorb to. When the flocs sediment, it is easy to collect them, making it possible to concentrate 10 L of water, whereas trad.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5x HOT FIREPol Probe qPCR Mix Plus (ROX)	Solis BioDyne	08-14-00001	Includes Solis Biodyne's 5x HOT FIREPol Probe qPCR Mix Plus (qPCR Mix), 50 Reactions
8-Microtube Strips with Caps	dD Biolab	840637	Low Profile, Thin Walls, Adapted for Quantitative and Qualitative PCR
Batteries and Power Adapters for Magnetic Stirrer	GenIUL	900011674	Includes 12V car power adapter
Bucket Support	GenIUL	900011648	Aluminium support
Bucket, 10 L	Cater4You	10LTR	Polypropilene, Tamperproof, Clear color
Centrifuge Tube, 50 mL	LabBox	CTSP-E50-050	Polypropylene, Sterile, Graduated, With Skirt
Citric Acid 1-Hydrate, 500 g	PanReac AppliChem	1410181211	Pure, Pharma Grade, 1 Kilogram
Clear PET Bottle	LabBox	FPET-500-088	Clear Color, PET, Cap Not Included
Difco Skim Milk, 500 g	Becton Dickinson	232100	Dehydrated
DNA/RNA Shield, 250 mL	Zymo Research	R1100-250	DNA/RNA Preservation Medium, 250 mL
Easy9 Pipette Controller	LabBox	EAS9-001-001	0.3 μm filter, Pipettes from 0.1 to 100 mL, Autoclavable silicone pipette holder
Eppendorf Tube, 0.5 mL	Eppendorf	0030121023	Polypropilene, Safe-Lock
Eppendorf Tube, 2 mL	Eppendorf	0030120094	Polypropilene, Safe-Lock
Eppendorf Tube, 5 mL	dD Biolab	999542	Polypropylene, Sterile, Graduated
Ethanol 96% V/V, 1 L	Panreac AppliChem	361085-1611	For UV, IR  and HPLC
Laboratory Tweezers	LabBox	FORS-007-002	Thin, Curved End, L= 120 mm
Magnetic Stirrer	GenIUL	900017000	Battery-powered
Marker	dD Biolab	929203	Black, Extra fine Tip, Water Resistant, Fast Drying, For Plastic and Glassware
Micro Rota-Rack for Microtubes	dD Biolab	37782	4 Modules, L x W x H= 208 x 100 x 100 mm
Mini8 Real-Time PCR Cycler	Coyote Biosciences, China	Mini-8	Portable, Works with 12V Power Supplies or External Batteries, Two channels, Capacity for 8 Tubes
NucliSens Lysis Buffer	Biomerieux	200292	Reagents for up to 48 Isolations, Store at Ambient Temperature
Open Tip Serological Pipette, 10 mL	Deltalab	900136N	Sterile, Individually Wrapped (Paper/Plastic)
PE Screw Cap PP28	LabBox	TP28-004-020	For PET Bottles
pH Indicator Strip	LabBox	WSPH-001-001	Range pH 2.8 to pH 4.4, 50 Strips per Pack
Plastic Test Tube	Quimikals	300913	Includes Cap
Polyethylene Pasteur Pipette	LabBox	PIPP-003-500	Graduated, 7 mL Overall Volume, Non-Sterile
Polypropylene Screw Flask With Screw Cap, 150 mL	Deltalab	409726	Screw cap, Sterile, graduated up to 100 mL
Polypropylene Screw Flask With Screw Cap, 60 mL	Deltalab	409526G	Screw cap, Sterile, Graduated up to 50 mL
Powder Powder Detergent	-	-	Regular Powder Soap for washing clothes
Power Cables for Magnetic Stirrer	GenIUL	900011692	Connection between batteries and magnetic stirrers
QuickPick Magnetic Tool	BioNobile	24001	Hand-held tool for magnetic particles
QuickPick Tips in Box	BioNobile	24296	RNase-Free, Autoclaved, 96 Units
QuickPick XL gDNA Magnetic Particles	BioNobile	SN51100	3.2 mL
Sea Salts	Sigma-Aldrich	S9883-500G	An artificial salt mixture closely resembling the composition of the dissolved salts of ocean water
Silicone Tubing	LabBox	SILT-006-005	Roll of 5 Meters, Inner  ø x Outer  ø= 6 x 10 mm
Sodium Hydroxide Pellets, 98.5 - 100.5%	VWR Chemicals	28244295	Pellets, 1 Kg
Solar Rotary Platform	SOL-EXPERT Group	70020	Acrylic Plate, 10 RPM, Supports up to 300 Grams
SOLIScript 1-step Probe Kit	Solis BioDyne	08-57-00250	Includes Solis Biodyne's 5x One-Step Probe Mix (qPCR Mix) and 40x One-Step SOLIScript Mix (Reverse Transcriptase Enzyme), 250 Reactions
SPEEDTOOLS RNA Virus Extraction Kit	BioTools	21.141-4197	Includes BioTools's BAW Buffer (Washing Buffer 1), BAV3 Buffer (Washing Buffer 2 and 3) and BRE Buffer (Elution Buffer).
SpinBar Octhaedral Stirring Magnet	dD Biolab	045926	Pivot Ring, L x  ø = 38 x 8 mm, Blue
Tape-End Serological Pipette, 10 mL	Deltalab	PN10E1	Sterile, Individually Wrapped (Paper/Plastic)
Tape-End Serological Pipette, 50 mL	Deltalab	900043	Sterile, Individually Wrapped (Paper/Plastic)
Termi-DNA-Tor - Nucleic Acid Remover	BioTools	22001-4291	Remover of nucleic acids, bacteria, fungi and mycoplasma from material and surfaces, 450 mL
Water Molecular Biology Reagent, 1L	Sigma-Aldrich	W4502-1L	Nuclease and Protease Free, 0.1 μm Filtered
Whirl-Pak Bag, 540 mL	Deltalab	200361	Stable bottom
Zip Lock Plain Bag	LabBox	BZIP-080-100	Polyethylene, L x W= 120 x 80 mm