Journal of Virological Methods 213 (2015) 6567

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Journal of Virological Methods

journal homepage: www.elsevier.com/locate/jviromet

Short communication

Molecular detection of human adenovirus in sediment using a direct

detection method compared to the classical polyethylene glycol
precipitation
Rodrigo Staggemeier , Marina Bortoluzzi, Tatiana Moraes da Silva Heck, Tiago da Silva,
Fernando Rosado Spilki, Sabrina Esteves de Matos Almeida
Laboratory of Molecular Microbiology, Universidade Feevale, ERS 239, No. 2755, Novo Hamburgo, Rio Grande do Sul 93352-000, Brazil

a b s t r a c t
Article history:
Received 11 August 2014
Received in revised form
10 November 2014
Accepted 18 November 2014
Available online 6 December 2014
Keywords:
Direct method
Enteric viruses
Sediment

Various effective methods have been developed to measure the concentration of viruses in sediment
samples. However, there is need to standardize less laborious and simpler techniques. The objective of
the present study was to compare two different methods to measure the concentration of viruses in soil
samples. The use of polyethylene glycol (PEG) was compared with a direct extraction of viral nucleic
acids from the samples diluted in modied Eagles minimal essential medium (E-MEM). The presence of
adenovirus in the samples was detected by real-time quantitative polymerase chain reaction (qPCR). Only
six samples (30%) were positive for adenovirus when PEG technique was used. The direct method showed
16 (80%) samples positive for adenovirus. Therefore, direct detection (i.e. without previous concentration)
demonstrated a higher rate of detection, better effectiveness, and shorter execution time. Furthermore,
direct detection uses reagents that are often readily available in virology laboratories. Thus, it is an
attractive alternative to other methods of detection of virus particles in sediments.
2014 Elsevier B.V. All rights reserved.

The soil is as an important reservoir of natural resources. However, it is a suitable environment for many pathogens because of its
physicochemical characteristics (Santamara and Toranzos, 2003).
The presence of supercial sediments in bodies of water is caused
by soil erosion. Sediments may also be important reservoirs of viral
agents because they are transported by owing water in rivers,
lakes, and ponds and are able to retain water (Alm et al., 2003).
Viral and bacterial loads are usually higher in soils than in water
bodies, and it is estimated that there may be up to 109 1013 viral
particles per kilogram of sediment in soils (Staggemeier et al.,
2011). Despite the predominance of aquatic environments on the
Earths surface, microbial abundance and diversity in soil may
exceed that found in aquatic environments (Srinivasiah et al., 2008).
Viral particles in soil may be transported through this matrix by
means of successive adsorptiondesorption cycles, thus reaching
surface and groundwater (Keeley et al., 2003).
Historically, the main focus of research about the presence of
viruses in soils has been fate and transport of viruses, as well as
standardization of techniques of virus detection (Staggemeier et al.,

Corresponding author at: Laboratrio de Microbiologia, Universidade Feevale,

Novo Hamburgo, Rio Grande do Sul, Brazil. Tel.: +55 51 3586 8800.
E-mail address: rstaggemeier@gmail.com (R. Staggemeier).
http://dx.doi.org/10.1016/j.jviromet.2014.11.019
0166-0934/ 2014 Elsevier B.V. All rights reserved.

2011). There are two common methods for detection of microorganisms in environmental samples: (1) cell culture infectivity assay
and (2) molecular biology techniques for detection of viral genomes
(Barardi et al., 2012). Using the viral concentration method before
nucleic acid extraction is often recommended for molecular detection of viral agents in environmental samples (Katayama et al.,
2002). For soil and sediment samples, methods based on acid precipitation (Sobsey et al., 1978), organic occulation (Katzenelson
et al., 1976), and polyethylene glycol precipitation (PEG) (Lewis
and Metcalf, 1988) have been proposed and regularly used through
the years. However, these methods have some drawbacks that may
hinder their usefulness and effectiveness in the recovery of viral
particles. The low pH used in these techniques may compromise the
viability of some microorganisms, particularly RNA viruses (Keeley
et al., 2003). Flocculation and precipitation are often performed by
adding puried proteins or other sources of organic matter to the
sample, thus impairing the viral detection methods (Staggemeier
et al., 2011). Since the analytical sensitivity of the molecular methods used for amplication of viral genomes has highly increased
during the last 20 years, it is now possible to investigate whether
these protocols are still necessary. Previous studies have reported
on the direct detection of viral particles using different elution
buffers or pH ranging from 7.2 to 11.5 (Miura et al., 2011; Prado
et al., 2014).

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R. Staggemeier et al. / Journal of Virological Methods 213 (2015) 6567

The present study aims to compare the classical PEG technique,

commonly used for the recovery of viruses in soil and sediment
samples, with the direct extraction of viral nucleic acids in samples
diluted in modied Eagles minimal essential medium (E-MEM).
Simplied methods using elution buffers for direct detection of viral
particles have been proposed (Miura et al., 2011; Staggemeier et al.,
2011; Prado et al., 2014). Nevertheless, a variation of these protocols using cell culture medium at pH 11.5 instead of other elution
buffers is demonstrated herein.
Supercial sediments were aseptically collected from 12 farms
located in the state of Rio Grande do Sul, Brazil. Twenty (n = 20)
samples of surface sediment from three different sources: dams,
streams, and springs were collected. Each sample consisted of 100 g
of sediment, which were stored in sterile glass vials under refrigeration until use. The classical PEG protocol was performed as
described by Lewis and Metcalf (1988). In the direct method, samples were only diluted in 10 (v/v) in E-MEM. In the PEG technique,
sediment samples were mixed with beef extract 3% 2 M NaNO3
eluant (pH 5.5) for 30 min and the solids were removed by centrifugation at 10,000 g for 10 min. The pH was adjusted at 7.5 and PEG
6000 was added to a nal concentration of 15% (w/v). The mixture was stirred for 1.52 h at 4 C and centrifuged at 10,000 g
for 20 min. The PEG-containing supernatant was discarded and the
pellet was suspended in 0.15 M Na2 HPO4 (pH 9), sonicated for
30 s, shaken for 20 min at 250 rpm and centrifuged at 10,000 g
for 30 min. Next, the supernatant was adjusted to a pH of 7.4 and
treated with antimicrobial agent. In order to detect viral particles
using the new method, 1 g of solid (sediment) and 1 ml of E-MEM,
Nutricell, were mixed (pH = 11.5). The solution was homogenized
by vigorous agitation (vortex) for 1 min and then centrifuged at
10,000 g for 10 min. The supernatant was then used for the extraction of DNA using the RTP DNA/RNA Virus Mini Kit (Invitek, Berlin,
Germany).
Before eld sample testing, the two methods were standardized using experimentally contaminated samples. Cell cultivated
HAdV-5 prototype strain Ad5 was spiked repeatedly onto sterile soil samples, after autoclaved and stored in 50-mL tubes.
HAdV-5 was inoculated using 10-fold serial dilutions, ranging
from the 104.75 TCID50 /50 L to less than 10 infective doses
(100.07 TCID50 /50 L). The samples were then submitted to nucleic
acid extraction as described above, and conventional PCR [primers
VTB2 HAdvC (Wolf et al., 2010)] was performed with annealing
temperature at 55 C. Positive and negative controls were used
for all reactions, and the conventional GoTaq Green Master Mix
2 (Promega, Madison, USA) was used according to the manufacturers guidelines: 50 L of reaction mixtures consisting of 25 L
of GoTaq Green Master Mix, 18 L of nuclease-free water, 1 L
of each primer (20 pM), and 5 L of nucleic acid. Amplication of
the target genomic fragments was performed using a thermal cycler
(MultiGene, Labnet International, Edison, USA). After reaction, the
product was analyzed by electrophoresis in 2% agarose gel with
ethidium bromide for staining and subsequently visualized under
UV light. During these assays, both protocols showed the detection
of a minimum of 1.174 tissue culture infective doses (TCID50 ) per/g,
or approximately 1300 equivalent genome copies.
Quantitative polymerase chain reactions (qPCR) were performed for the eld and experimentally contaminated samples by
the partial amplication of the hexon gene of HAdV, using the previously described primers VTB2-R (5 -GATGAACCGCAGCGTCAA-3 )
and VTB2-F (5 -GAGACGTACTTCAGCCTGAAT-3 ) (Wolf et al., 2010).
The commercial SYBR Green Platinun qPCR Supermix-UDG kit
(Life TechnologiesTM Corporation, Carlsbad, CA 92008, USA) was
used for qPCR in accordance with the manufacturers instructions.
The qPCR reactions were optimized and carried out under the same
conditions, being used as controls for absolute quantication of
viral DNA from prototype samples of HAdV-5, in a thermal cycler

Table 1
Adenovirus in sediment samples diluted in E-MEM or concentrated by polyethylene
glycol 6000 (PEG). Results are expressed in (qPCR) genome copies per gram (gc/g).
Source

Sediment
Direct method

PEG

P1

Spring 1
Spring 2
Dam

1.20E+04
Neg
1.97E+03

2.35E+03
Neg
1.55E+03

P2

Spring
Stream

5.87E+04
4.27E+03

Neg
2.91E+03

P3

Stream

1.96E+04

Neg

P4

Spring
Dam

1.26E+04
2.36E+04

Neg
Neg

P5

Stream

8.20E+03

Neg

P6

Stream
Spring

Neg
1.41E+04

Neg
Neg

P7

Spring
Dam2

7.51E+03
Neg

9.00E+02
Neg

P8

Stream1
Stream2

5.80E+04
6.96E+04

Neg
2.02E+03

P9
P10

Spring
Stream

1.66E+04
3.78E+04

Neg
Neg

P11

Dam
Stream

Neg
5.34E+03

Neg
Neg

P12

Dam

7.67E+03

4.00E+03

Neg: negative.

(iQ5TM Bio-Rad, BioradTM , Hercules, CA, USA). For each 25 L reaction, the following was used: 12.5 L of the mix, 1 L of each
primer (20 pM), 5.5 L of DNAse/RNAse free sterile water, and
5.0 L of nucleic acid extracted from each sample. Each reaction
was composed of a denaturation cycle at 95 C for 10 min, followed
by 40 cycles composed of one step at 95 C for 20 s, and a combined annealing/extension step at 55 C for 1 min. Fluorescence
data were collected during the annealing/extension step. To generate standard curves, 10-fold serial dilutions of standard controls
from 101 to 105 were prepared, starting at 6.01 107 genome
copies per reaction (HAdV-5). All standard controls and samples
were run in duplicates. Both no template controls (NTC) and negative controls were used in each run to conrm that there was
not contamination in the assay. Melting curve analysis was done
using high resolution melting curve (HRM) to verify PCR product
specicity (melting step between 55 and 95 C), after completion
of the amplication steps. Typical HAdV amplicons had a specic
temperature of 86 C in this protocol.
The results obtained by qPCR using the technique of precipitation with PEG for the 20 eld samples of supercial sediments were
as follows: 30% of samples positive for HAdV (6/20), and viral concentrations ranging between 9.00 102 gc/g and 4.00 103 gc/g. In
contrast, positivity rates of 80% HAdV (16/20) were found using
the direct method, and viral loads ranging from 1.97 103 gc/g to
6.96 104 gc/g (Table 1).
Direct dilution of sediments made it possible to nd higher
rates of positivity and detect higher viral loads than those found
using the method proposed by Lewis and Metcalf (1988). A recovery of approximately 70% of hepatitis A virus (HAV) and rotavirus
(RV) was reported using the PEG-based protocol (Green and Lewis,
1999). However, the results are variable. Colombert et al. (2007)
reported a recovery rate of 23% (1328%). Monpoeho et al. (2001)
also compared methods of viral recovery for enteroviruses using
the methodologies proposed by other authors and the recovery
efciency varied from approximately 30% (Tartera and Jofre, 1987;
Jofre et al., 1989; Albert and Schwartzbrod, 1991; Grabow et al.,

R. Staggemeier et al. / Journal of Virological Methods 213 (2015) 6567

1991; Alouini and Sobsey, 1995) to 45% (Ahmed and Sorensen,

1995; Schlindwein et al., 2009). Schlindwein et al. (2009) reported
a recovery efciency of 90% and 20% of RV and adenovirus, respectively, using the PEG protocol. In the present study, besides HAdV,
the samples were analyzed for the presence of RV (data not shown),
resulting in 6 positive samples according to the direct method and
all negative samples based on the PEG method, also demonstrating
the ability to recovery of RNA viruses.
In general, although viruses may be concentrated using different
methods, some viral species are more susceptible to pH changes
and other organic components that may be present during sample
processing, which may hamper the recovery of these viral particles
(Wyn Jones and Selwood, 2001; Queiroz et al., 2001). A possible
explanation for the variation in the number of positive samples
detected by the two techniques may be associated with the amount
of viral particles in the samples (Silva et al., 2010).
The isoelectric point (IEP) of approximately 150 different viruses
was determined, with a mean value of 5 1.3, meaning that
most viruses are negatively charged under natural pH conditions
(Michen and Graule, 2010). Viruses may be considered colloidal
particles, their adsorption is signicantly inuenced by a number
of parameters, such as the type of virus, soil type, salt concentration, pH, virus load, hydrogen bonding, electrostatic attraction
and repulsion, van der Waals forces, hydrophobicity, and covalent ionic interactions (Staggemeier et al., 2011). This electrostatic
charge provides mobility to soft particles in an electric eld and
thus regulates their colloidal behavior, which plays an important role in virus adsorption (Michen and Graule, 2010). It has
been demonstrated that even sharp increases in the pH may
enhance the detachment from soil matrices (Keeley et al., 2003).
That is why the direct method and those techniques previously
reported by other authors require an alkaline buffer or medium
(pH = 11.5).
The advantage of PEG concentration is to obtain a precipitate at neutral pH or at high ionic concentrations with the
absence of ionic compounds (Lewis and Metcalf, 1988). However, precipitation with PEG may not be as effective for some
environmental samples. This precipitate may cause a concentration of PCR inhibitors (Schvoerer et al., 2000). The PEG technique
consists of several steps during which the samples need to be
centrifuged, recentrifuged, stirred, and sonicated. It also requires
the addition of eluants and numerous other reagents such as PEG
itself and beef extract. Some eluants may inhibit the methods
used to concentrate and detect viruses (Schvoerer et al., 2000).
Furthermore, effective occulation and precipitation of viruses
require the presence of proteins or organic matter. However,
these waste materials may compromise the subsequent steps in
the methods of viral detection (Staggemeier et al., 2011), thus
directly inuencing the results of the samples. Also, in this sense,
the time elapsed during the procedure may interfere with the
quantity/viability of viral particles in the sample (Wyn Jones and
Selwood, 2001).
The method described in the present study proved to be reliable for the detection of HAdV in sediment samples. The reagents
used are commonly found in virology laboratories and the method
is very easy to perform. According to Mehnert (2003), the methods
used to perform virus concentration in sediments have low efciency and sensitivity, in addition to being costly and cumbersome.
Gabutti (2000) also reported that there is a need for development
and standardization of new techniques.
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