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Journal of Virological Methods 213 (2015) 65–67

Contents lists available at ScienceDirect

Journal of Virological Methods

journa l homepage: www.e lsev ier .com/ locate / jv i romet

hort communication

olecular detection of human adenovirus in sediment using a directetection method compared to the classical polyethylene glycolrecipitation

odrigo Staggemeier ∗, Marina Bortoluzzi, Tatiana Moraes da Silva Heck, Tiago da Silva,ernando Rosado Spilki, Sabrina Esteves de Matos Almeida

aboratory of Molecular Microbiology, Universidade Feevale, ERS 239, No. 2755, Novo Hamburgo, Rio Grande do Sul 93352-000, Brazil

rticle history:eceived 11 August 2014eceived in revised form0 November 2014ccepted 18 November 2014vailable online 6 December 2014

a b s t r a c t

Various effective methods have been developed to measure the concentration of viruses in sedimentsamples. However, there is need to standardize less laborious and simpler techniques. The objective ofthe present study was to compare two different methods to measure the concentration of viruses in soilsamples. The use of polyethylene glycol (PEG) was compared with a direct extraction of viral nucleicacids from the samples diluted in modified Eagle’s minimal essential medium (E-MEM). The presence ofadenovirus in the samples was detected by real-time quantitative polymerase chain reaction (qPCR). Only

eywords:irect methodnteric virusesediment

six samples (30%) were positive for adenovirus when PEG technique was used. The direct method showed16 (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 anattractive 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. How-ver, it is a suitable environment for many pathogens because of itshysicochemical characteristics (Santamaría and Toranzos, 2003).he presence of superficial sediments in bodies of water is causedy soil erosion. Sediments may also be important reservoirs of viralgents because they are transported by flowing water in rivers,akes, and ponds and are able to retain water (Alm et al., 2003).

Viral and bacterial loads are usually higher in soils than in waterodies, and it is estimated that there may be up to 109–1013 viralarticles per kilogram of sediment in soils (Staggemeier et al.,011). Despite the predominance of aquatic environments on thearth’s surface, microbial abundance and diversity in soil mayxceed that found in aquatic environments (Srinivasiah et al., 2008).iral particles in soil may be transported through this matrix byeans of successive adsorption–desorption cycles, thus reaching

urface and groundwater (Keeley et al., 2003).

Historically, the main focus of research about the presence of

iruses in soils has been fate and transport of viruses, as well astandardization of techniques of virus detection (Staggemeier et al.,

∗ Corresponding author at: Laboratório de Microbiologia, Universidade Feevale,ovo Hamburgo, Rio Grande do Sul, Brazil. Tel.: +55 51 3586 8800.

E-mail address: rstaggemeier@gmail.com (R. Staggemeier).

ttp://dx.doi.org/10.1016/j.jviromet.2014.11.019166-0934/© 2014 Elsevier B.V. All rights reserved.

2011). There are two common methods for detection of microor-ganisms in environmental samples: (1) cell culture infectivity assayand (2) molecular biology techniques for detection of viral genomes(Barardi et al., 2012). Using the viral concentration method beforenucleic acid extraction is often recommended for molecular detec-tion of viral agents in environmental samples (Katayama et al.,2002). For soil and sediment samples, methods based on acid pre-cipitation (Sobsey et al., 1978), organic flocculation (Katzenelsonet al., 1976), and polyethylene glycol precipitation (PEG) (Lewisand Metcalf, 1988) have been proposed and regularly used throughthe years. However, these methods have some drawbacks that mayhinder their usefulness and effectiveness in the recovery of viralparticles. The low pH used in these techniques may compromise theviability of some microorganisms, particularly RNA viruses (Keeleyet al., 2003). Flocculation and precipitation are often performed byadding purified proteins or other sources of organic matter to thesample, thus impairing the viral detection methods (Staggemeieret al., 2011). Since the analytical sensitivity of the molecular meth-ods used for amplification of viral genomes has highly increasedduring the last 20 years, it is now possible to investigate whether

these protocols are still necessary. Previous studies have reportedon the direct detection of viral particles using different elutionbuffers or pH ranging from 7.2 to 11.5 (Miura et al., 2011; Pradoet al., 2014).

6 Virological Methods 213 (2015) 65–67

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

Source Sediment

Direct method PEG

P1 Spring 1 1.20E+04 2.35E+03Spring 2 Neg NegDam 1.97E+03 1.55E+03

P2 Spring 5.87E+04 NegStream 4.27E+03 2.91E+03

P3 Stream 1.96E+04 Neg

P4 Spring 1.26E+04 NegDam 2.36E+04 Neg

P5 Stream 8.20E+03 Neg

P6 Stream Neg NegSpring 1.41E+04 Neg

P7 Spring 7.51E+03 9.00E+02Dam2 Neg Neg

P8 Stream1 5.80E+04 NegStream2 6.96E+04 2.02E+03

P9 Spring 1.66E+04 NegP10 Stream 3.78E+04 Neg

P11 Dam Neg NegStream 5.34E+03 Neg

6 R. Staggemeier et al. / Journal of

The present study aims to compare the classical PEG technique,ommonly used for the recovery of viruses in soil and sedimentamples, with the direct extraction of viral nucleic acids in samplesiluted in modified Eagle’s minimal essential medium (E-MEM).implified methods using elution buffers for direct detection of viralarticles have been proposed (Miura et al., 2011; Staggemeier et al.,011; Prado et al., 2014). Nevertheless, a variation of these proto-ols using cell culture medium at pH 11.5 instead of other elutionuffers is demonstrated herein.

Superficial sediments were aseptically collected from 12 farmsocated in the state of Rio Grande do Sul, Brazil. Twenty (n = 20)amples of surface sediment from three different sources: dams,treams, and springs were collected. Each sample consisted of 100 gf sediment, which were stored in sterile glass vials under refrig-ration until use. The classical PEG protocol was performed asescribed by Lewis and Metcalf (1988). In the direct method, sam-les were only diluted in 10× (v/v) in E-MEM. In the PEG technique,ediment samples were mixed with beef extract 3% – 2 M NaNO3luant (pH 5.5) for 30 min and the solids were removed by centrifu-ation at 10,000 × g for 10 min. The pH was adjusted at 7.5 and PEG000 was added to a final concentration of 15% (w/v). The mix-ure was stirred for 1.5–2 h at 4 ◦C and centrifuged at 10,000 × gor 20 min. The PEG-containing supernatant was discarded and theellet was suspended in 0.15 M Na2HPO4 (pH 9), sonicated for0 s, shaken for 20 min at 250 rpm and centrifuged at 10,000 × gor 30 min. Next, the supernatant was adjusted to a pH of 7.4 andreated with antimicrobial agent. In order to detect viral particlessing the new method, 1 g of solid (sediment) and 1 ml of E-MEM,utricell, were mixed (pH = 11.5). The solution was homogenizedy vigorous agitation (vortex) for 1 min and then centrifuged at0,000 × g for 10 min. The supernatant was then used for the extrac-ion of DNA using the RTP® DNA/RNA Virus Mini Kit (Invitek, Berlin,ermany).

Before field sample testing, the two methods were standard-zed using experimentally contaminated samples. Cell cultivatedAdV-5 prototype strain Ad5 was spiked repeatedly onto ster-

le soil samples, after autoclaved and stored in 50-mL tubes.AdV-5 was inoculated using 10-fold serial dilutions, ranging

rom the 104.75TCID50/50 �L to less than 10 infective doses100.07TCID50/50 �L). The samples were then submitted to nucleiccid extraction as described above, and conventional PCR [primersTB2 HAdvC (Wolf et al., 2010)] was performed with annealing

emperature at 55 ◦C. Positive and negative controls were usedor all reactions, and the conventional GoTaq® Green Master Mix× (Promega, Madison, USA) was used according to the manufac-urer’s guidelines: 50 �L of reaction mixtures consisting of 25 �Lf GoTaq® Green Master Mix, 18 �L of nuclease-free water, 1 �Lf each primer (20 pM), and 5 �L of nucleic acid. Amplification ofhe target genomic fragments was performed using a thermal cyclerMultiGene®, Labnet International, Edison, USA). After reaction, theroduct was analyzed by electrophoresis in 2% agarose gel withthidium bromide for staining and subsequently visualized underV light. During these assays, both protocols showed the detectionf a minimum of 1.174 tissue culture infective doses (TCID50) per/g,r approximately 1300 equivalent genome copies.

Quantitative polymerase chain reactions (qPCR) were per-ormed for the field and experimentally contaminated samples byhe partial amplification of the hexon gene of HAdV, using the pre-iously described primers VTB2-R (5′-GATGAACCGCAGCGTCAA-3′)nd VTB2-F (5′-GAGACGTACTTCAGCCTGAAT-3′) (Wolf et al., 2010).he commercial SYBR® Green Platinun® qPCR Supermix-UDG kitLife TechnologiesTM Corporation, Carlsbad, CA 92008, USA) was

sed for qPCR in accordance with the manufacturer’s instructions.he qPCR reactions were optimized and carried out under the sameonditions, being used as controls for absolute quantification ofiral DNA from prototype samples of HAdV-5, in a thermal cycler

P12 Dam 7.67E+03 4.00E+03

Neg: negative.

(iQ5TM Bio-Rad, BioradTM, Hercules, CA, USA). For each 25 �L reac-tion, the following was used: 12.5 �L of the mix, 1 �L of eachprimer (20 pM), 5.5 �L of DNAse/RNAse free sterile water, and5.0 �L of nucleic acid extracted from each sample. Each reactionwas composed of a denaturation cycle at 95 ◦C for 10 min, followedby 40 cycles composed of one step at 95 ◦C for 20 s, and a com-bined annealing/extension step at 55 ◦C for 1 min. Fluorescencedata were collected during the annealing/extension step. To gen-erate standard curves, 10-fold serial dilutions of standard controlsfrom 10−1 to 10−5 were prepared, starting at 6.01 × 107 genomecopies per reaction (HAdV-5). All standard controls and sampleswere run in duplicates. Both “no template controls” (NTC) and neg-ative controls were used in each run to confirm that there wasnot contamination in the assay. Melting curve analysis was doneusing high resolution melting curve (HRM) to verify PCR productspecificity (melting step between 55 and 95 ◦C), after completionof the amplification steps. Typical HAdV amplicons had a specifictemperature of 86 ◦C in this protocol.

The results obtained by qPCR using the technique of precipita-tion with PEG for the 20 field samples of superficial sediments wereas follows: 30% of samples positive for HAdV (6/20), and viral con-centrations ranging between 9.00 × 102 gc/g and 4.00 × 103 gc/g. Incontrast, positivity rates of 80% HAdV (16/20) were found usingthe direct method, and viral loads ranging from 1.97 × 103 gc/g to6.96 × 104 gc/g (Table 1).

Direct dilution of sediments made it possible to find higherrates of positivity and detect higher viral loads than those foundusing the method proposed by Lewis and Metcalf (1988). A recov-ery 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% (13–28%). Monpoeho et al. (2001)also compared methods of viral recovery for enteroviruses using

the methodologies proposed by other authors and the recoveryefficiency varied from approximately 30% (Tartera and Jofre, 1987;Jofre et al., 1989; Albert and Schwartzbrod, 1991; Grabow et al.,

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991; Alouini and Sobsey, 1995) to 45% (Ahmed and Sorensen,995; Schlindwein et al., 2009). Schlindwein et al. (2009) reportedrecovery efficiency of 90% and 20% of RV and adenovirus, respec-

ively, using the PEG protocol. In the present study, besides HAdV,he samples were analyzed for the presence of RV (data not shown),esulting in 6 positive samples according to the direct method andll negative samples based on the PEG method, also demonstratinghe ability to recovery of RNA viruses.

In general, although viruses may be concentrated using differentethods, some viral species are more susceptible to pH changes

nd other organic components that may be present during samplerocessing, which may hamper the recovery of these viral particlesWyn Jones and Selwood, 2001; Queiroz et al., 2001). A possiblexplanation for the variation in the number of positive samplesetected by the two techniques may be associated with the amountf viral particles in the samples (Silva et al., 2010).

The isoelectric point (IEP) of approximately 150 different virusesas determined, with a mean value of 5 ± 1.3, meaning thatost viruses are negatively charged under natural pH conditions

Michen and Graule, 2010). Viruses may be considered colloidalarticles, their adsorption is significantly influenced by a numberf parameters, such as the type of virus, soil type, salt concen-ration, pH, virus load, hydrogen bonding, electrostatic attractionnd repulsion, van der Waals forces, hydrophobicity, and cova-ent ionic interactions (Staggemeier et al., 2011). This electrostaticharge provides mobility to soft particles in an electric field andhus regulates their colloidal behavior, which plays an impor-ant role in virus adsorption (Michen and Graule, 2010). It haseen demonstrated that even sharp increases in the pH maynhance the detachment from soil matrices (Keeley et al., 2003).hat is why the direct method and those techniques previouslyeported by other authors require an alkaline buffer or mediumpH = 11.5).

The advantage of PEG concentration is to obtain a precip-tate at neutral pH or at high ionic concentrations with thebsence of ionic compounds (Lewis and Metcalf, 1988). How-ver, precipitation with PEG may not be as effective for somenvironmental samples. This precipitate may cause a concentra-ion of PCR inhibitors (Schvoerer et al., 2000). The PEG techniqueonsists of several steps during which the samples need to beentrifuged, recentrifuged, stirred, and sonicated. It also requireshe addition of eluants and numerous other reagents such as PEGtself and beef extract. Some eluants may inhibit the methodssed to concentrate and detect viruses (Schvoerer et al., 2000).urthermore, effective flocculation and precipitation of virusesequire the presence of proteins or organic matter. However,hese waste materials may compromise the subsequent steps inhe methods of viral detection (Staggemeier et al., 2011), thusirectly influencing the results of the samples. Also, in this sense,he time elapsed during the procedure may interfere with theuantity/viability of viral particles in the sample (Wyn Jones andelwood, 2001).

The method described in the present study proved to be reli-ble for the detection of HAdV in sediment samples. The reagentssed are commonly found in virology laboratories and the method

s very easy to perform. According to Mehnert (2003), the methodssed to perform virus concentration in sediments have low effi-iency and sensitivity, in addition to being costly and cumbersome.abutti (2000) also reported that there is a need for developmentnd standardization of new techniques.

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