Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Summary

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Abstract

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.

Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.

In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.

The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

Protocol

1. Concentration of the viral particles present in water samples

1. Collection and conditioning of water samples
   1. Collect a minimum of 2 replicates of 10 L per sample in plastic containers with flat bottom and one extra sample as a process control. This last sample will be spiked with a known amount of viral particles and used as a control.
      Note: It is recommended to have special separate material (bottles, tube, etc.) for spiked samples.
   2. Check the calibration of the conductimeter and recalibrate if necessary. Prepare a negative control by using tap water previously adjusted to the adequate conductivity (see below 1.6) in one extr....

Discussion

The procedure described would fulfill the conditions for a fitting method for routine environmental and public health laboratories: reproducible, reliable, straightforward and cost-effective. The protocol is simple; however it must be followed carefully. Low conductivity in the samples without adding the requested concentration of artificial seawater salts would dramatically reduce the recovery of viruses as would be the case if the stirring time for flocculation is significantly reduced (less than 5 hours for example).<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
High speed centrifuge (8,000xg)	Berckman Coulter	Avanti J-20XP
pH-meter, thermometer and conductimeter	Afora	LPPC3003
Plastic tubes 100-200 cm length	Deltalab	350059
Sterile graduated disposable pipettes	Labclinics	PN10E1
Sterile plastic tubes of 1.5 and 10-15 mL (Eppendorf, Falcons, etc.)	Afora	KA298/00
Centrifuge pots (500 mL)	Fisher Scientific	SE5753512
Magnetic stirrers and magnets (one per sample)	Fisher Scientific	10510
Glass or plastic containers having flat bottoms to allow the use of magnetic stirrers	Deltalab	191642
A peristaltic pump for removing the supernatant (or a water-jet vacuum pump)	Watson-Marlow Pumps Group	323E/D
Timer to switch-off the stirring after 8-10 hours	Deltalab	900400
Hydrochloric acid (1N and 0.1N)	Panreac	141020.1611
Sodium hydroxide (1N)	Panreac	131687.1211
Artificial seawater sea salts	Sigma-Aldrich	S9883
Skimmed milk (SM)	Difco Laboratories	232100
Phosphate buffer pH 7,5			1:2 v/v of sterile Na2HPO4 0,2M and NaH2PO4 0,2M at pH 7.5
Thiosulphate	Panreac	121879.1209	Make a 10% solution in water
QIAamp Viral RNA Mini Kit	Qiagen	52904
96-well optical reaction plate (500 units)	Applied Biosystems	43426659
Optical adhesive covers (100 units)	Applied Biosystems	4311971
TaqMan Environmental PCR Master Mix 2x	Applied Biosystems	4396838