International Journal of Food Microbiology 201 (2015) 1–6
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Application of viability PCR to discriminate the infectivity of hepatitis A virus in food samples L. Moreno a, R. Aznar a,b, G. Sánchez a,b,⁎ a b
Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustín Escardino, 7, 46980 Paterna, Valencia, Spain
a r t i c l e
i n f o
Article history: Received 9 December 2014 Received in revised form 6 February 2015 Accepted 9 February 2015 Available online 16 February 2015 Keywords: Hepatitis A virus Quantitative PCR Viability dyes Propidium monoazide Ethidium monoazide
a b s t r a c t Transmitted through the fecal–oral route, the hepatitis A virus (HAV) is acquired primarily through close personal contact and foodborne transmission. HAV detection in food is mainly carried out by quantitative RT-PCR (RT-qPCR). The discrimination of infectious and inactivated viruses remains a key obstacle when using RTqPCR to quantify enteric viruses in food samples. Initially, viability dyes, propidium monoazide (PMA) and ethidium monoazide (EMA), were evaluated for the detection and quantification of infectious HAV in lettuce wash water. Results showed that PMA combined with 0.5% Triton X-100 (Triton) was the best pretreatment to assess HAV infectivity and completely eliminated the signal of thermally inactivated HAV in lettuce wash water. This procedure was further evaluated in artificially inoculated foods (at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50) including lettuce, parsley, spinach, cockles and coquina clams. The PMA–0.5% Triton pretreatment reduced the signal of thermally inactivated HAV between 0.5 and 2 logs, in lettuce and spinach concentrates. Moreover, this pretreatment reduced the signal of inactivated HAV by more than 1.5 logs, in parsley and ten-fold diluted shellfish samples inoculated at the lowest concentration. Overall, this pretreatment (50 μM PMA–0.5% Triton) significantly reduced the detection of thermally inactivated HAV, depending on the initial virus concentration and the food matrix. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The hepatitis A virus (HAV) is mainly transmitted by person-toperson contact, ingestion of contaminated food or water, as well as by contact with contaminated fomites. Hepatitis A has recently been considered as a re-emerging foodborne public health threat due to the increasing number of outbreaks reported in industrialized countries and associated with food imports from areas of high hepatitis A endemicity (Sprenger, 2014). Because of this increasing number of HAV outbreaks, it has become even more important to have reliable and widely applicable techniques for the detection and quantification of HAV in food samples (reviewed by Bosch et al., 2011; Sánchez et al., 2007). Moreover, since the infectious dose of enteric viruses is very low (10 to 100 viral particles) (Teunis et al., 2008; Yezli and Otter, 2011), sensitive methods are therefore needed when screening food products for viral pathogens. So far, the current ‘gold standard’ for the detection of enteric viruses is quantitative reverse transcription PCR (RT-qPCR) but this method cannot discriminate between infectious and inactivated viruses (reviewed by Knight et al., 2013). ⁎ Corresponding author at: Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Avda. Agustín Escardino, 7, Paterna, Valencia, Spain. Tel.: +34 96 3900022; fax: +34 96 3939301. E-mail address: [email protected] (G. Sánchez).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.02.012 0168-1605/© 2015 Elsevier B.V. All rights reserved.
The relationship between the number of infectious viruses and the number of genome copies detected by RT-qPCR is not a constant, mainly in food samples, since this ratio may vary depending on environmental conditions. Several studies have reported that the reduction in the number of infectious viruses does not correlate with the number of genomes detected by RT-qPCR (Baert et al., 2008; Butot et al., 2008, 2009; Hewitt and Greening, 2004). Several different approaches to assess virus infectivity using PCR have been evaluated (reviewed by Hamza et al., 2011; Knight et al., 2013; Rodriguez et al., 2009). The detection of the whole or specific region of a viral genome may indicate that the virus capsid is protecting the genome from degradation, hence measuring infectivity (Bhattacharya et al., 2004). Another alternative strategy to increase the likelihood of detecting intact and potentially infectious viruses is to pretreat them with nucleases and/or proteolytic enzymes prior to nucleic acid extraction, thereby eliminating the detection of free nucleic acids or nucleic acids associated with damaged or inactivated viruses. This approach was first introduced by Nuanualsuwan and Cliver (2003) and consists of an enzymatic pretreatment with RNase, in some instances combined with a proteinase K treatment. Nuanualsuwan and Cliver (2003) applied this pretreatment to differentiate successfully between intact viruses (HAV, poliovirus and feline calicivirus) and viruses inactivated by ultraviolet light, chlorine disinfection, and thermal treatment at 72 °C. Later on, this approach has been applied for assessing norovirus (NoV)
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(Lamhoujeb et al., 2008; Mormann et al., 2010; Nowak et al., 2011; Topping et al., 2009) and hepatitis E virus (HEV) infectivity (Schielke et al., 2011). A promising new strategy to assess viral infectivity relies on the use of nucleic acid intercalating dyes, or viability dyes, such as ethidium monoazide (EMA) or propidium monoazide (PMA) as a sample pretreatment before applying molecular techniques (reviewed by Elizaquível et al., 2014). Theoretically, these compounds cannot penetrate intact capsids but are able to penetrate damaged or destroyed capsids. Once penetrated, the dye intercalates covalently into RNA after exposure to strong visible light, interfering with PCR amplification. Until now, viability dyes combined with RT-qPCR have successfully been applied to virus suspensions in order to discriminate between infectious and inactivated viruses (Table 1). However, the effectiveness of viability dyes to discriminate between infectious and inactivated viruses in food samples has yet to be explored. In a previous publication, our group reported that PMA pretreatment combined with RT-qPCR was a promising alternative for assessing HAV infectivity (Sánchez et al., 2012a). Moreover, Coudray-Meunier et al. (2013) found that treatments combining viability dyes and surfactants were effective for differentiating infectious and thermally inactivated HAV suspensions. The purpose of this work was then to assess the applicability of viability dyes for the detection and quantification of infectious HAV by RT-qPCR in lettuce wash water, as an example of the fresh-cut industry water, as well as in artificially inoculated foods including vegetables and shellfish. 2. Materials and methods 2.1. Virus and cells The cytopathogenic HM-175 strain of HAV (ATCC VR-1402) was propagated and assayed in confluent FRhK-4 cells (courtesy of Prof. Albert Bosch, University of Barcelona). Semi-purified stocks were obtained from infected FRhK-4 cells by three freeze–thaw cycles after 8–11 days post-infection. Cell debris was pelleted at 660 × g for 30 min. Infectious viruses were quantified by determining the 50% tissue culture infectious dose (TCID50) with eight wells per dilution and 20 μl of inoculum per well (Pintó et al., 1994). 2.2. Reagents Viability dyes, propidium monoazide (PMA) and ethidium monoazide (EMA), were purchased from Geniul (Spain). Both reagents were dissolved in 20% dimethylsulfoxide (DMSO) at 2 mM and stored at
− 20 °C in the dark. Surfactants, Triton X-100 (Triton) and Span 20, were purchased from Sigma-Aldrich. 2.3. Lettuce wash water and food concentrates Lettuce wash water with 500 mg/l chemical oxygen demand (COD) was prepared as described by López-Gálvez et al. (2011). Lettuce, spinach and parsley concentrates were prepared as previously described (Sánchez et al., 2012b). Briefly, vegetables were washed with Buffered Peptone Water using the Pulsifier equipment (Microgen Bioproducts) and concentrated by polyethylene glycol (PEG) precipitation. The pellet was immediately resuspended in 500 μl of PBS. Aliquots of 100 μl were treated with viability dyes as described below. Cockle and coquina clam concentrates were prepared as described in the technical specification ISO/TS 15216-1. Briefly, 2 g of digestive glands was treated with proteinase K, and 100 μl of supernatant was treated with viability dyes as described below. 2.4. Viability dye treatments One-hundred microliters of lettuce wash water or food concentrates was inoculated with infectious HAV and thermally inactivated (5 min at 99 °C) HAV suspensions (at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50), and added to PMA 50 μM and EMA 20 μM, with or without surfactants. All the experiments were performed in DNA LoBind 1.5 ml tubes (Eppendorf) in triplicate. After the addition of the viability dye, incubation in the dark at room temperature was performed for 10 min at 150 rpm to allow reagent penetration. Thereafter, samples were exposed to light for 15 min using a photo-activation system (Led-Active Blue, Geniul). After photo-induced cross-linking, RNA was extracted using the NucleoSpin® RNA virus kit (Macherey-Nagel GmbH & Co.) according to the manufacturer's instructions. Three types of controls were always included in the experiments; infectious viruses treated with viability dyes and infectious and thermally inactivated viruses without viability dye treatment. 2.5. HAV quantification The set of primers and probes used has been previously validated (Costafreda et al., 2006) and targets the 5′ non-coding region (5′NCR) of HAV corresponding to the genomic region 68–240 of HAV (pHM175 43c). RNA samples were analyzed in duplicate by RT-qPCR using the RNA UltraSense One-Step quantitative RT-PCR system (Invitrogen SA) and the LightCycler 2.0 instrument (Roche Diagnostics). The standard curve for HAV was generated by amplifying 10-fold
Table 1 Application of viability dyes for the detection of infectious viruses. Virus targeted
Inactivation process
Matrix
Dye used
Successful monitoring
References
Virus suspension Virus suspension Virus suspension
EMA PMA PMA/EMA
Norovirus
UV Heat treatment, hypochlorite Heat treatment High pressure processing Heat treatment
Virus suspension
PMA
Yes Yes Yes Partially Yes
Poliovirus
Heat treatment, hypochlorite, UV
Virus suspension, river water
PMA/EMA
Yes
Sangsanont et al. (2014) Parshionikar et al. (2010) Coudray-Meunier et al. (2013) Sánchez et al. (2012a) Parshionikar et al. (2010)a Escudero-Abarca et al. (2014) Parshionikar et al. (2010) Kim et al. (2011) Sangsanont et al. (2014)
Other viruses Avian influenza MS2 phage Murine norovirus
Stability in water Heat treatment Heat treatment
Landfill leachate Virus suspension Virus suspension
T4 phages
Heat treatment
EMA PMA PMA EMA PMA
No Yes No Yes No
Enteric viruses Adenovirus Coxsackievirus, echovirus HAV
a
Discrimination was only achieved by PMA-RT-PCR but not PMA-RT-qPCR.
Graiver et al. (2010) Kim and Ko (2012) Kim and Ko (2012) Kim et al. (2011) Fittipaldi et al. (2010)
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dilutions of the viral stock by RT-qPCR in triplicate. The crossing points (Cp) obtained from the assay of each dilution were used to plot a standard curve by assigning a value of 1 RT-PCR unit (PCRU) to the highest dilution showing a positive crossing point value and progressively 10-fold-higher values to the lower dilutions (y = −3.4x + 36.3; R2 = 0.99). 2.6. Statistical analyses Differences among the mean numbers of viruses determined after the various treatments were evaluated by Student's t-test, with a significance level of p b 0.05 (Microsoft Office Excel; Microsoft, Redmond, US). 3. Results 3.1. Efficacy of pretreatment combining viability dyes and surfactants for the selective detection of infectious HAV in lettuce wash water As a first step in exploring the potential of viability dyes to be applied in the fresh-cut industry, aliquots of thermally inactivated HAV suspensions (containing ca. 6 × 104 TCID50) were added in lettuce wash water and treated with 20 μM EMA or 50 μM PMA. Results showed that both viability dyes, PMA and EMA, reduced the signal of inactivated HAV by 3.17 and 2.17 logs, respectively (Table 2). When surfactants, i.e. Triton and Span 20, were combined with viability dyes (Table 3) only the combination of PMA and 0.5% Triton eliminated the signal of thermally inactivated HAV by more than 3.4 logs. The use of Span 20 rendered similar or even worse results than the use of the viability dyes alone. 3.2. Efficacy of PMA for the discrimination of inactivated HAV in vegetables To verify that PMA combined with 0.5% Triton was the best combination to selectively detect infectious HAV in vegetables, thermally inactivated HAV suspensions were inoculated at ca. 6 × 104 and 6 × 103 TCID50 and treated with 50 μM PMA with or without 0.5% Triton. Results showed that PMA–0.5% Triton pretreatment reduced the signal of inactivated HAV by 1.53 and 2.04 logs when inoculated at 6 × 104 and 6 × 103 TCID50, respectively, whereas PMA alone only reduced the signal by 1.09 and 1.56 logs, respectively (Table 4). The pretreatment with PMA and 0.5% Triton was further evaluated in lettuce, parsley and spinach artificially inoculated at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50, in triplicate. Results showed that the pretreatment was partially efficient in reducing the RT-qPCR signal between 0.5 and 2 logs (Table 5). The RT-qPCR signal was only completely eliminated in PMA–0.5% Triton treated concentrates corresponding to parsley inoculated at 6 × 102 TCID50. Table 2 Quantification by RT-qPCR and cell culture of infectious and thermally inactivated HAV suspensions inoculated in lettuce wash water after viability dye treatment. RT-qPCR
Infectious Inactivatedc Inactivated + PMA (50 μM) Inactivated + EMA (20 μM)
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Table 3 Log scale reduction of RT-qPCR titers of thermally inactivated HAV inoculated in lettuce wash water by viability dye treatments. Viability dyes
No surfactant 0.5% Triton X-100 0.1% Span 20 0.5% Span 20 1% Span 20
PMA (50 μM)
EMA (20 μM)
2.96 ± 0.24 N3.42 ± 0.48 2.96 ± 0.17 2.61 ± 0.39 2.18 ± 0.13
2.58 ± 0.14 2.39 ± 0.59 2.24 ± 0.33 2.11 ± 0.17 2.46 ± 0.18
3.3. Application of PMA–Triton pretreatment in shellfish samples The quantification levels of thermally inactivated HAV in shellfish samples with or without pretreatment with PMA–0.5% Triton are shown in Table 6. When shellfish concentrates were used undiluted, less than 0.5 log reduction was observed after PMA–0.5% Triton pretreatment indicating low performance of the pretreatment. Therefore, in order to assess the effect of the matrix, shellfish concentrates were ten-fold diluted and further inoculated with thermally inactivated HAV at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50. No detection of thermally inactivated HAV by the PMA–0.5% Triton pretreatment was observed at the lowest inoculum (i.e. 6 × 102 TCID50) whereas at higher levels, reductions of 1.10 to 1.95 logs were recorded (Table 6).
4. Discussion In the last five years, several outbreaks of hepatitis A associated with foods of foreign origin have been reported in western countries (Collier et al., 2014; Guzman-Herrado et al., 2014; Wenzel et al., 2014). Despite advances in the development of standardized methods (i.e. ISO/TS 15216), the food virology field still presents many difficulties at the analytical level. For instance, molecular detection methodologies need approaches to better assess the infectivity of the samples. In this sense, the use of photoactivatable dyes has been shown as an innovative technology to selectively detect infectious viruses by RT-qPCR (Table 1). Recently, the utility of viability RT-qPCR has been expanded by modifications to this basic strategy. Surfactant cotreatment was reported to facilitate PMA and EMA penetration in thermally inactivated HAV and rotavirus, thereby improving the ability to discern their viability (Coudray-Meunier et al., 2013). However, reports on the application of this methodology in environmental samples are somewhat limited. Until now, only Parshionikar et al. (2010) have successfully applied PMA treatment in water samples for poliovirus detection, showing that the matrix did not interfere with this pretreatment. The aim of this study was to evaluate the previously outlined procedure (Sánchez et al., 2012a) for its application in routine food analysis. First, the pretreatment for the discrimination of infectious HAV was
Cell culture
Quantification (log PCRU)a
Reduction Quantification (log TCID50)b
3.83 ± 0.25 3.79 ± 0.07 0.42 ± 0.06 1.12 ± 0.35
0.04 3.17d 2.47d
4.77 ± 0.12 b1.32 ± 0.24 NA NA
Reduction
N3.45
NA: not applicable. a Each condition was replicated three times, and HAV titers were obtained by RT-qPCR using a standard curve made by means of HAV PCRU. b Each condition was replicated three times, and HAV titers were obtained by determining the 50% tissue culture infectious dose (TCID50) with eight wells per dilution and 20 μl of inoculum per well. c Inactivated HAV suspensions were obtained by heat treatment at 99 °C for 5 min. d Reduction in titers obtained between inactivated viruses before and after viability dye treatment.
Table 4 Quantification of thermally inactivated HAV suspensions inoculated in lettuce concentrates by RT-qPCR. Pretreatment
Levels of HAV 6 × 104 TCID50
PMA (50 μ M)
Triton (0.5%)
Quantification (log PCRU)a
− + +
− − +
4.14 ± 0.03 3.05 ± 0.28 2.61 ± 0.80
6 × 103 TCID50 Reduction
1.09 1.53
b
3.32 ± 0.12 1.76 ± 0.26 1.28 ± 0.90
1.56 2.04
a Each condition was replicated three times, and HAV titers were obtained by RT-qPCR using a standard curve made by means of HAV PCRU. b Reduction in titers between thermally inactivated viruses before and after pretreatment.
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Table 5 Quantification of thermally inactivated HAV suspensions inoculated in vegetable concentrates by PMA–0.5% Triton pretreatment and RT-qPCR. PMA–0.5% Triton
Levels of HAV 6 × 104 TCID50 Quantification (log PCRU)a
Lettuce Spinach Parsley
− + − + − +
4.04 ± 0.01 2.40 ± 0.10 2.87 ± 0.06 1.72 ± 0.08 3.24 ± 0.05 2.18 ± 0.20
6 × 103 TCID50 Reductionb
Quantification (log PCRU)a 3.07 ± 0.12 1.27 ± 0.08 1.51 ± 0.02 0.80 ± 0.13 2.31 ± 0.21 0.87 ± 0.27
1.64 1.15 1.06
6 × 102 TCID50 Reductionb
2.00 0.71 1.44
Quantification (log PCRU)a 1.32 ± 0.06 0.77 ± 0.12 1.19 ± 0.03 0.33 ± 0.23 1.55 ± 0.20 ND
Reductionb
0.55 0.86 N1.55
ND: non-detected. a Each condition was replicated three times, and HAV titers were obtained by RT-qPCR using a standard curve made by means of HAV PCRU. b Reduction in titers between thermally inactivated viruses before and after pretreatment.
optimized by employing DNA LoBind tubes (to avoid PMA interaction with the plastic surface of the tubes) and extending to 10 min the incubation time with the viability dye under agitation (data not shown). When applied in lettuce wash water, as a model of water susceptible to be analyzed for virus presence, 50 μM PMA treatment was significantly (p b 0.05) more effective than 20 μM EMA treatment. The efficacy of viability dyes has also recently been assessed on HAV suspensions diluted in PBS inactivated at 80 °C for 10 min; Coudray-Meunier et al. (2013) reported 1.06 and 1.75 log reduction after 50 μM PMA and 20 μM EMA treatment, respectively. Deviations from the results reported here may be due to matrix differences, the length of PCR product and viability dye treatment conditions, since Coudray-Meunier et al. (2013) incubated the viability dyes for 2 h at 4 °C whereas in this study the incubation was maintained for 10 min at room temperature. Following, procedure improvement was approached by testing the addition of surfactants, i.e. Triton and Span 20. The combination of 50 μM PMA and 0.5% Triton was highly useful to completely reduce the signal of inactivated HAV in lettuce wash water. These results are in line with the successful application of PMA-RT-PCR in environmental water concentrates to distinguish between infectious and noninfectious polioviruses (Parshionikar et al., 2010). The incidence of viral foodborne diseases conveyed by fresh-cut vegetables is on the rise (EFSA, 2014). In salad vegetables from European countries 1.32, 3.42, 2 and 2.95% of samples were positive for HAV, HEV, NoV GI and NoV GII, respectively (Kokkinos et al., 2012). In samples from Mexico, HAV was found in 28.2% of samples, NoV in 32.6% and rotavirus in 13.0% (Felix-Valenzuela et al., 2012). Moreover, NoV genomes have frequently been detected in salad vegetables (28.2, 33.3 and 50% of samples from Canada, Belgium and France, respectively); however, sequence confirmation was not successful for the majority of the samples tested. All the mentioned reports used RT-qPCR for virus detection; therefore, the infectivity of the samples could not be
assessed, and thereby the impact of these results on the public health is under discussion (Baert et al., 2011). This highlights the importance of implementing new methodologies to assess infectivity in food samples. So far, viability PCR has successfully been applied for bacterial detection in several types of food (reviewed by Elizaquível et al., 2014) but not for enteric viruses. This study shows for the first time the potential of PMA to discriminate between thermally inactivated HAV and infectious HAV in food applications. In contrast to the results obtained for bacteria (Elizaquível et al., 2012), this study shows that viability RTqPCR cannot completely prevent PCR amplification from thermally inactivated HAV in food samples. From results on vegetables and shellfish (Table 5 and 6), signal reductions of inactivated HAV are influenced by food matrix and virus concentration. Several authors have reported that the detection of some bacteria (reviewed by Elizaquível et al., 2014) and viruses (Sánchez et al., 2012a) by viability PCR is limited when high concentrations of microorganisms are present. Additionally, Escudero-Abarca et al. (2014) reported that successful discrimination of infectious noroviruses was only achieved when applying it to monodispersed viruses. This may partially explain the differences on the efficacy of PMA pretreatment, since not only the concentration of the virus may play a role but also the extent of virus aggregates. Therefore, in-depth assessment of the influence of virus concentration and presence of virus aggregates will be an essential part for optimizing viability PCR for virus testing in food. In this study PMA–0.5% Triton pretreatment was quite effective in vegetable concentrates, but not in shellfish concentrates. This result indicates that PMA is capable of binding the RNA of the thermally inactivated HAV present in vegetable samples and it partially prevents amplification without interferences of the food matrix. For shellfish concentrates, the high turbidity of the sample may avoid the action of PMA pretreatment and this effect was reverted by diluting shellfish concentrates. The interferences of turbidity on viability PCR has also been
Table 6 Quantification of thermally inactivated HAV suspensions inoculated in shellfish concentrates by PMA–0.5% Triton pretreatment and RT-qPCR. PMA–0.5% Triton
Levels of HAV 6 × 104 TCID50 Quantification (log PCRU)a
Cockles Diluted cockles Coquina clams Diluted coquina clams
− + − + − + − +
2.53 ± 0.02 2.52 ± 0.10 3.16 ± 0.04 1.80 ± 0.16 3.60 ± 0.06 3.33 ± 0.12 4.03 ± 0.64 2.08 ± 0.11
6 × 103 TCID50 Reductionb
0.01 1.36 0.27 1.95
Quantification (log PCRU)a 1.92 ± 0.09 1.69 ± 0.11 2.36 ± 0.09 0.83 ± 0.33 2.66 ± 0.14 2.36 ± 0.05 2.83 ± 0.13 1.73 ± 0.11
6 × 102 TCID50 Reductionb
0.23 1.53 0.30 1.10
ND: non-detected. a Each condition was replicated three times, and HAV titers were obtained by RT-qPCR using a standard curve made by means of HAV PCRU. b Reduction in titers between inactivated viruses before and after pretreatment.
Quantification (log PCRU)a 0.89 ± 0.44 0.88 ± 0.22 1.52 ± 0.10 ND 1.94 ± 0.04 1.47 ± 0.07 2.14 ± 0.19 ND
Reductionb
0.01 N1.52 0.47 N2.14
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described for Vibrio parahaemolyticus in raw seafood. This study showed that PMA treatment with turbidities greater than 10 NTU did not adequately inhibit the amplification of DNA (Zhu et al., 2012). Turbid samples containing suspended solids lowered the effective dye concentration by chemical adsorption with organic and inorganic compounds, resulting in decreased effectiveness of the viability treatment (Fittipaldi et al., 2012). Overall, the application of PMA–0.5% Triton pretreatment for the detection of HAV in food products greatly helps generating more meaningful data, since 1 to 2 logs of the PCR signal can be eliminated by a simple pretreatment. This pretreatment can be applied straightforward for routine analyses, since it lasts 30 min only. Furthermore it can be easily incorporated to the ISO norm for virus detection (ISO/TS 15216), as the RT-qPCR assay suggested there is the same as the one used in this study. However, this pretreatment faces some challenges that need to be tackled in the future. One of the most evident challenges is the fact that, depending on the complexity of the food sample, suppression of inactivated virus signals is not complete, leading to an overestimation of infectious viruses. Additional strategies to increase PMA treatment efficiency include incubation with other surfactants, repeated dye exposure, amplification of longer RNA sequences or modification of the incubation temperature (Fittipaldi et al., 2012; Nkuipou-Kenfack et al., 2013). Acknowledgments This study was supported by grants AGL2009-08603 from the Spanish Ministry of Science and Innovation, and ACOMP/2010/279 and ACOMP/2012/199 from the Generalitat Valenciana. G. Sánchez was supported by the “Ramón y Cajal” Young Investigator program of the Spanish Ministry of Economy and Competitiveness RYC-2012-09950. References Baert, L., Wobus, C.E., Van Coillie, E., Thackray, L.B., Debevere, J., Uyttendaele, M., 2008. Detection of murine norovirus 1 by using plaque assay, transfection assay, and realtime reverse transcription-PCR before and after heat exposure. Appl. Environ. Microbiol. 74, 543–546. Baert, L., Mattison, K., Loisy-Hamon, F., Harlow, J., Martyres, A., Lebeau, B., Stals, A., Van Coillie, E., Herman, L., Uyttendaele, M., 2011. Review: norovirus prevalence in Belgian, Canadian and French fresh produce: a threat to human health. Int. J. Food Microbiol. 15, 261–269. Bhattacharya, S.S., Kulka, M., Lampel, K.A., Cebula, T.A., Goswami, B.B., 2004. Use of reverse transcription and PCR to discriminate between infectious and non-infectious hepatitis A virus. J. Virol. Methods 116, 181–187. Bosch, A., Sánchez, G., Abbaszadegan, M., Carducci, A., Guix, S., Le Guyader, F., Netshikweta, R., Pinto, R.M., van der Poel, W., Rutjes, S.A., Sano, D., Taylor, M., van Zyl, W., Rodríguez-Lázaro, D., Kovac, K., Sellwood, J., 2011. Analytical methods for virus detection in water and food. Food Anal. Methods 4 (1), 4–12. Butot, S., Putallaz, T., Sánchez, G., 2008. Effects of sanitation, freezing and frozen storage on enteric viruses in berries and herbs. Int. J. Food Microbiol. 126, 30–35. Butot, S., Putallaz, T., Amoroso, R., Sánchez, G., 2009. Inactivation of enteric viruses in minimally processed berries and herbs. Appl. Environ. Microbiol. 75, 4155–4161. Collier, M.G., Khudyakov, Y.E., Selvage, D., Adams-Cameron, M., Epson, E., Cronquist, A., Jervis, R.H., Lamba, K., Kimura, A.C., Sowadsky, R., Hassan, R., Park, S.Y., Garza, E., Elliott, A.J., Rotstein, D.S., Beal, J., Kuntz, T., Lance, S.E., Dreisch, R., Wise, M.E., Nelson, N.P., Suryaprasad, A., Drobeniuc, J., Holmberg, S.D., Xu, F., for the Hepatitis A Outbreak investigation Team, 2014. Outbreak of hepatitis A in the USA associated with frozen pomegranate arils imported from Turkey: an epidemiological case study. Lancet Infect. Dis. 14, 976–981. Costafreda, M.I., Bosch, A., Pintó, R.M., 2006. Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples. Appl. Environ. Microbiol. 72, 3846–3855. Coudray-Meunier, C., Fraisse, A., Martin-Latil, S., Guiller, L. Perelle S., 2013. Discrimination of infectious hepatitis A virus and rotavirus by combining dyes and surfactants with RT-qPCR. BMC Microbiol. 13, 216–231. EFSA, 2014. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA J. 12 (2), 3547 (312 pp.). Elizaquível, P., Sanchez, G., Aznar, R., 2012. Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR. Food Control 25, 704–708. Elizaquível, P., Aznar, R., Sánchez, G., 2014. Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field. J. Appl. Microbiol. 116, 1–13.
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Microbiol. 55, 182–188. Kim, K., Katayama, H., Kitajima, M., Tohya, Y., Ohgaki, S., 2011. Development of a realtime RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure. Water Sci. Technol. 63, 502–507. Knight, A., Li, D., Uyttendaele, M., Jaykus, L.A., 2013. A critical review of methods for detecting human noroviruses and predicting their infectivity. Crit. Rev. Microbiol. 39, 295–309. Kokkinos, P., Kozyra, I., Lazic, S., Bouwknegt, M., Rutjes, S., Willems, K., Moloney, R., de Roda Husman, A., Kaupke, A., Legaki, E., D'Agostino, M., Cook, N., Rzeżutka, A., Petrovic, T., Vantarakis, A., 2012. Harmonised investigation of the occurrence of human enteric viruses in the leafy green vegetable supply chain in three European countries. Food Environ. Virol. 4 (4), 179–191. Lamhoujeb, S., Fliss, I., Ngazoa, S.E., Jean, J., 2008. 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Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Application of viability PCR to discriminate the infectivity of hepatitis A virus in food samples L. Moreno a, R. Aznar a,b, G. Sánchez a,b,⁎ a b
Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustín Escardino, 7, 46980 Paterna, Valencia, Spain
a r t i c l e
i n f o
Article history: Received 9 December 2014 Received in revised form 6 February 2015 Accepted 9 February 2015 Available online 16 February 2015 Keywords: Hepatitis A virus Quantitative PCR Viability dyes Propidium monoazide Ethidium monoazide
a b s t r a c t Transmitted through the fecal–oral route, the hepatitis A virus (HAV) is acquired primarily through close personal contact and foodborne transmission. HAV detection in food is mainly carried out by quantitative RT-PCR (RT-qPCR). The discrimination of infectious and inactivated viruses remains a key obstacle when using RTqPCR to quantify enteric viruses in food samples. Initially, viability dyes, propidium monoazide (PMA) and ethidium monoazide (EMA), were evaluated for the detection and quantification of infectious HAV in lettuce wash water. Results showed that PMA combined with 0.5% Triton X-100 (Triton) was the best pretreatment to assess HAV infectivity and completely eliminated the signal of thermally inactivated HAV in lettuce wash water. This procedure was further evaluated in artificially inoculated foods (at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50) including lettuce, parsley, spinach, cockles and coquina clams. The PMA–0.5% Triton pretreatment reduced the signal of thermally inactivated HAV between 0.5 and 2 logs, in lettuce and spinach concentrates. Moreover, this pretreatment reduced the signal of inactivated HAV by more than 1.5 logs, in parsley and ten-fold diluted shellfish samples inoculated at the lowest concentration. Overall, this pretreatment (50 μM PMA–0.5% Triton) significantly reduced the detection of thermally inactivated HAV, depending on the initial virus concentration and the food matrix. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The hepatitis A virus (HAV) is mainly transmitted by person-toperson contact, ingestion of contaminated food or water, as well as by contact with contaminated fomites. Hepatitis A has recently been considered as a re-emerging foodborne public health threat due to the increasing number of outbreaks reported in industrialized countries and associated with food imports from areas of high hepatitis A endemicity (Sprenger, 2014). Because of this increasing number of HAV outbreaks, it has become even more important to have reliable and widely applicable techniques for the detection and quantification of HAV in food samples (reviewed by Bosch et al., 2011; Sánchez et al., 2007). Moreover, since the infectious dose of enteric viruses is very low (10 to 100 viral particles) (Teunis et al., 2008; Yezli and Otter, 2011), sensitive methods are therefore needed when screening food products for viral pathogens. So far, the current ‘gold standard’ for the detection of enteric viruses is quantitative reverse transcription PCR (RT-qPCR) but this method cannot discriminate between infectious and inactivated viruses (reviewed by Knight et al., 2013). ⁎ Corresponding author at: Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Avda. Agustín Escardino, 7, Paterna, Valencia, Spain. Tel.: +34 96 3900022; fax: +34 96 3939301. E-mail address: [email protected] (G. Sánchez).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.02.012 0168-1605/© 2015 Elsevier B.V. All rights reserved.
The relationship between the number of infectious viruses and the number of genome copies detected by RT-qPCR is not a constant, mainly in food samples, since this ratio may vary depending on environmental conditions. Several studies have reported that the reduction in the number of infectious viruses does not correlate with the number of genomes detected by RT-qPCR (Baert et al., 2008; Butot et al., 2008, 2009; Hewitt and Greening, 2004). Several different approaches to assess virus infectivity using PCR have been evaluated (reviewed by Hamza et al., 2011; Knight et al., 2013; Rodriguez et al., 2009). The detection of the whole or specific region of a viral genome may indicate that the virus capsid is protecting the genome from degradation, hence measuring infectivity (Bhattacharya et al., 2004). Another alternative strategy to increase the likelihood of detecting intact and potentially infectious viruses is to pretreat them with nucleases and/or proteolytic enzymes prior to nucleic acid extraction, thereby eliminating the detection of free nucleic acids or nucleic acids associated with damaged or inactivated viruses. This approach was first introduced by Nuanualsuwan and Cliver (2003) and consists of an enzymatic pretreatment with RNase, in some instances combined with a proteinase K treatment. Nuanualsuwan and Cliver (2003) applied this pretreatment to differentiate successfully between intact viruses (HAV, poliovirus and feline calicivirus) and viruses inactivated by ultraviolet light, chlorine disinfection, and thermal treatment at 72 °C. Later on, this approach has been applied for assessing norovirus (NoV)
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(Lamhoujeb et al., 2008; Mormann et al., 2010; Nowak et al., 2011; Topping et al., 2009) and hepatitis E virus (HEV) infectivity (Schielke et al., 2011). A promising new strategy to assess viral infectivity relies on the use of nucleic acid intercalating dyes, or viability dyes, such as ethidium monoazide (EMA) or propidium monoazide (PMA) as a sample pretreatment before applying molecular techniques (reviewed by Elizaquível et al., 2014). Theoretically, these compounds cannot penetrate intact capsids but are able to penetrate damaged or destroyed capsids. Once penetrated, the dye intercalates covalently into RNA after exposure to strong visible light, interfering with PCR amplification. Until now, viability dyes combined with RT-qPCR have successfully been applied to virus suspensions in order to discriminate between infectious and inactivated viruses (Table 1). However, the effectiveness of viability dyes to discriminate between infectious and inactivated viruses in food samples has yet to be explored. In a previous publication, our group reported that PMA pretreatment combined with RT-qPCR was a promising alternative for assessing HAV infectivity (Sánchez et al., 2012a). Moreover, Coudray-Meunier et al. (2013) found that treatments combining viability dyes and surfactants were effective for differentiating infectious and thermally inactivated HAV suspensions. The purpose of this work was then to assess the applicability of viability dyes for the detection and quantification of infectious HAV by RT-qPCR in lettuce wash water, as an example of the fresh-cut industry water, as well as in artificially inoculated foods including vegetables and shellfish. 2. Materials and methods 2.1. Virus and cells The cytopathogenic HM-175 strain of HAV (ATCC VR-1402) was propagated and assayed in confluent FRhK-4 cells (courtesy of Prof. Albert Bosch, University of Barcelona). Semi-purified stocks were obtained from infected FRhK-4 cells by three freeze–thaw cycles after 8–11 days post-infection. Cell debris was pelleted at 660 × g for 30 min. Infectious viruses were quantified by determining the 50% tissue culture infectious dose (TCID50) with eight wells per dilution and 20 μl of inoculum per well (Pintó et al., 1994). 2.2. Reagents Viability dyes, propidium monoazide (PMA) and ethidium monoazide (EMA), were purchased from Geniul (Spain). Both reagents were dissolved in 20% dimethylsulfoxide (DMSO) at 2 mM and stored at
− 20 °C in the dark. Surfactants, Triton X-100 (Triton) and Span 20, were purchased from Sigma-Aldrich. 2.3. Lettuce wash water and food concentrates Lettuce wash water with 500 mg/l chemical oxygen demand (COD) was prepared as described by López-Gálvez et al. (2011). Lettuce, spinach and parsley concentrates were prepared as previously described (Sánchez et al., 2012b). Briefly, vegetables were washed with Buffered Peptone Water using the Pulsifier equipment (Microgen Bioproducts) and concentrated by polyethylene glycol (PEG) precipitation. The pellet was immediately resuspended in 500 μl of PBS. Aliquots of 100 μl were treated with viability dyes as described below. Cockle and coquina clam concentrates were prepared as described in the technical specification ISO/TS 15216-1. Briefly, 2 g of digestive glands was treated with proteinase K, and 100 μl of supernatant was treated with viability dyes as described below. 2.4. Viability dye treatments One-hundred microliters of lettuce wash water or food concentrates was inoculated with infectious HAV and thermally inactivated (5 min at 99 °C) HAV suspensions (at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50), and added to PMA 50 μM and EMA 20 μM, with or without surfactants. All the experiments were performed in DNA LoBind 1.5 ml tubes (Eppendorf) in triplicate. After the addition of the viability dye, incubation in the dark at room temperature was performed for 10 min at 150 rpm to allow reagent penetration. Thereafter, samples were exposed to light for 15 min using a photo-activation system (Led-Active Blue, Geniul). After photo-induced cross-linking, RNA was extracted using the NucleoSpin® RNA virus kit (Macherey-Nagel GmbH & Co.) according to the manufacturer's instructions. Three types of controls were always included in the experiments; infectious viruses treated with viability dyes and infectious and thermally inactivated viruses without viability dye treatment. 2.5. HAV quantification The set of primers and probes used has been previously validated (Costafreda et al., 2006) and targets the 5′ non-coding region (5′NCR) of HAV corresponding to the genomic region 68–240 of HAV (pHM175 43c). RNA samples were analyzed in duplicate by RT-qPCR using the RNA UltraSense One-Step quantitative RT-PCR system (Invitrogen SA) and the LightCycler 2.0 instrument (Roche Diagnostics). The standard curve for HAV was generated by amplifying 10-fold
Table 1 Application of viability dyes for the detection of infectious viruses. Virus targeted
Inactivation process
Matrix
Dye used
Successful monitoring
References
Virus suspension Virus suspension Virus suspension
EMA PMA PMA/EMA
Norovirus
UV Heat treatment, hypochlorite Heat treatment High pressure processing Heat treatment
Virus suspension
PMA
Yes Yes Yes Partially Yes
Poliovirus
Heat treatment, hypochlorite, UV
Virus suspension, river water
PMA/EMA
Yes
Sangsanont et al. (2014) Parshionikar et al. (2010) Coudray-Meunier et al. (2013) Sánchez et al. (2012a) Parshionikar et al. (2010)a Escudero-Abarca et al. (2014) Parshionikar et al. (2010) Kim et al. (2011) Sangsanont et al. (2014)
Other viruses Avian influenza MS2 phage Murine norovirus
Stability in water Heat treatment Heat treatment
Landfill leachate Virus suspension Virus suspension
T4 phages
Heat treatment
EMA PMA PMA EMA PMA
No Yes No Yes No
Enteric viruses Adenovirus Coxsackievirus, echovirus HAV
a
Discrimination was only achieved by PMA-RT-PCR but not PMA-RT-qPCR.
Graiver et al. (2010) Kim and Ko (2012) Kim and Ko (2012) Kim et al. (2011) Fittipaldi et al. (2010)
L. Moreno et al. / International Journal of Food Microbiology 201 (2015) 1–6
dilutions of the viral stock by RT-qPCR in triplicate. The crossing points (Cp) obtained from the assay of each dilution were used to plot a standard curve by assigning a value of 1 RT-PCR unit (PCRU) to the highest dilution showing a positive crossing point value and progressively 10-fold-higher values to the lower dilutions (y = −3.4x + 36.3; R2 = 0.99). 2.6. Statistical analyses Differences among the mean numbers of viruses determined after the various treatments were evaluated by Student's t-test, with a significance level of p b 0.05 (Microsoft Office Excel; Microsoft, Redmond, US). 3. Results 3.1. Efficacy of pretreatment combining viability dyes and surfactants for the selective detection of infectious HAV in lettuce wash water As a first step in exploring the potential of viability dyes to be applied in the fresh-cut industry, aliquots of thermally inactivated HAV suspensions (containing ca. 6 × 104 TCID50) were added in lettuce wash water and treated with 20 μM EMA or 50 μM PMA. Results showed that both viability dyes, PMA and EMA, reduced the signal of inactivated HAV by 3.17 and 2.17 logs, respectively (Table 2). When surfactants, i.e. Triton and Span 20, were combined with viability dyes (Table 3) only the combination of PMA and 0.5% Triton eliminated the signal of thermally inactivated HAV by more than 3.4 logs. The use of Span 20 rendered similar or even worse results than the use of the viability dyes alone. 3.2. Efficacy of PMA for the discrimination of inactivated HAV in vegetables To verify that PMA combined with 0.5% Triton was the best combination to selectively detect infectious HAV in vegetables, thermally inactivated HAV suspensions were inoculated at ca. 6 × 104 and 6 × 103 TCID50 and treated with 50 μM PMA with or without 0.5% Triton. Results showed that PMA–0.5% Triton pretreatment reduced the signal of inactivated HAV by 1.53 and 2.04 logs when inoculated at 6 × 104 and 6 × 103 TCID50, respectively, whereas PMA alone only reduced the signal by 1.09 and 1.56 logs, respectively (Table 4). The pretreatment with PMA and 0.5% Triton was further evaluated in lettuce, parsley and spinach artificially inoculated at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50, in triplicate. Results showed that the pretreatment was partially efficient in reducing the RT-qPCR signal between 0.5 and 2 logs (Table 5). The RT-qPCR signal was only completely eliminated in PMA–0.5% Triton treated concentrates corresponding to parsley inoculated at 6 × 102 TCID50. Table 2 Quantification by RT-qPCR and cell culture of infectious and thermally inactivated HAV suspensions inoculated in lettuce wash water after viability dye treatment. RT-qPCR
Infectious Inactivatedc Inactivated + PMA (50 μM) Inactivated + EMA (20 μM)
3
Table 3 Log scale reduction of RT-qPCR titers of thermally inactivated HAV inoculated in lettuce wash water by viability dye treatments. Viability dyes
No surfactant 0.5% Triton X-100 0.1% Span 20 0.5% Span 20 1% Span 20
PMA (50 μM)
EMA (20 μM)
2.96 ± 0.24 N3.42 ± 0.48 2.96 ± 0.17 2.61 ± 0.39 2.18 ± 0.13
2.58 ± 0.14 2.39 ± 0.59 2.24 ± 0.33 2.11 ± 0.17 2.46 ± 0.18
3.3. Application of PMA–Triton pretreatment in shellfish samples The quantification levels of thermally inactivated HAV in shellfish samples with or without pretreatment with PMA–0.5% Triton are shown in Table 6. When shellfish concentrates were used undiluted, less than 0.5 log reduction was observed after PMA–0.5% Triton pretreatment indicating low performance of the pretreatment. Therefore, in order to assess the effect of the matrix, shellfish concentrates were ten-fold diluted and further inoculated with thermally inactivated HAV at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50. No detection of thermally inactivated HAV by the PMA–0.5% Triton pretreatment was observed at the lowest inoculum (i.e. 6 × 102 TCID50) whereas at higher levels, reductions of 1.10 to 1.95 logs were recorded (Table 6).
4. Discussion In the last five years, several outbreaks of hepatitis A associated with foods of foreign origin have been reported in western countries (Collier et al., 2014; Guzman-Herrado et al., 2014; Wenzel et al., 2014). Despite advances in the development of standardized methods (i.e. ISO/TS 15216), the food virology field still presents many difficulties at the analytical level. For instance, molecular detection methodologies need approaches to better assess the infectivity of the samples. In this sense, the use of photoactivatable dyes has been shown as an innovative technology to selectively detect infectious viruses by RT-qPCR (Table 1). Recently, the utility of viability RT-qPCR has been expanded by modifications to this basic strategy. Surfactant cotreatment was reported to facilitate PMA and EMA penetration in thermally inactivated HAV and rotavirus, thereby improving the ability to discern their viability (Coudray-Meunier et al., 2013). However, reports on the application of this methodology in environmental samples are somewhat limited. Until now, only Parshionikar et al. (2010) have successfully applied PMA treatment in water samples for poliovirus detection, showing that the matrix did not interfere with this pretreatment. The aim of this study was to evaluate the previously outlined procedure (Sánchez et al., 2012a) for its application in routine food analysis. First, the pretreatment for the discrimination of infectious HAV was
Cell culture
Quantification (log PCRU)a
Reduction Quantification (log TCID50)b
3.83 ± 0.25 3.79 ± 0.07 0.42 ± 0.06 1.12 ± 0.35
0.04 3.17d 2.47d
4.77 ± 0.12 b1.32 ± 0.24 NA NA
Reduction
N3.45
NA: not applicable. a Each condition was replicated three times, and HAV titers were obtained by RT-qPCR using a standard curve made by means of HAV PCRU. b Each condition was replicated three times, and HAV titers were obtained by determining the 50% tissue culture infectious dose (TCID50) with eight wells per dilution and 20 μl of inoculum per well. c Inactivated HAV suspensions were obtained by heat treatment at 99 °C for 5 min. d Reduction in titers obtained between inactivated viruses before and after viability dye treatment.
Table 4 Quantification of thermally inactivated HAV suspensions inoculated in lettuce concentrates by RT-qPCR. Pretreatment
Levels of HAV 6 × 104 TCID50
PMA (50 μ M)
Triton (0.5%)
Quantification (log PCRU)a
− + +
− − +
4.14 ± 0.03 3.05 ± 0.28 2.61 ± 0.80
6 × 103 TCID50 Reduction
1.09 1.53
b
3.32 ± 0.12 1.76 ± 0.26 1.28 ± 0.90
1.56 2.04
a Each condition was replicated three times, and HAV titers were obtained by RT-qPCR using a standard curve made by means of HAV PCRU. b Reduction in titers between thermally inactivated viruses before and after pretreatment.
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Table 5 Quantification of thermally inactivated HAV suspensions inoculated in vegetable concentrates by PMA–0.5% Triton pretreatment and RT-qPCR. PMA–0.5% Triton
Levels of HAV 6 × 104 TCID50 Quantification (log PCRU)a
Lettuce Spinach Parsley
− + − + − +
4.04 ± 0.01 2.40 ± 0.10 2.87 ± 0.06 1.72 ± 0.08 3.24 ± 0.05 2.18 ± 0.20
6 × 103 TCID50 Reductionb
Quantification (log PCRU)a 3.07 ± 0.12 1.27 ± 0.08 1.51 ± 0.02 0.80 ± 0.13 2.31 ± 0.21 0.87 ± 0.27
1.64 1.15 1.06
6 × 102 TCID50 Reductionb
2.00 0.71 1.44
Quantification (log PCRU)a 1.32 ± 0.06 0.77 ± 0.12 1.19 ± 0.03 0.33 ± 0.23 1.55 ± 0.20 ND
Reductionb
0.55 0.86 N1.55
ND: non-detected. a Each condition was replicated three times, and HAV titers were obtained by RT-qPCR using a standard curve made by means of HAV PCRU. b Reduction in titers between thermally inactivated viruses before and after pretreatment.
optimized by employing DNA LoBind tubes (to avoid PMA interaction with the plastic surface of the tubes) and extending to 10 min the incubation time with the viability dye under agitation (data not shown). When applied in lettuce wash water, as a model of water susceptible to be analyzed for virus presence, 50 μM PMA treatment was significantly (p b 0.05) more effective than 20 μM EMA treatment. The efficacy of viability dyes has also recently been assessed on HAV suspensions diluted in PBS inactivated at 80 °C for 10 min; Coudray-Meunier et al. (2013) reported 1.06 and 1.75 log reduction after 50 μM PMA and 20 μM EMA treatment, respectively. Deviations from the results reported here may be due to matrix differences, the length of PCR product and viability dye treatment conditions, since Coudray-Meunier et al. (2013) incubated the viability dyes for 2 h at 4 °C whereas in this study the incubation was maintained for 10 min at room temperature. Following, procedure improvement was approached by testing the addition of surfactants, i.e. Triton and Span 20. The combination of 50 μM PMA and 0.5% Triton was highly useful to completely reduce the signal of inactivated HAV in lettuce wash water. These results are in line with the successful application of PMA-RT-PCR in environmental water concentrates to distinguish between infectious and noninfectious polioviruses (Parshionikar et al., 2010). The incidence of viral foodborne diseases conveyed by fresh-cut vegetables is on the rise (EFSA, 2014). In salad vegetables from European countries 1.32, 3.42, 2 and 2.95% of samples were positive for HAV, HEV, NoV GI and NoV GII, respectively (Kokkinos et al., 2012). In samples from Mexico, HAV was found in 28.2% of samples, NoV in 32.6% and rotavirus in 13.0% (Felix-Valenzuela et al., 2012). Moreover, NoV genomes have frequently been detected in salad vegetables (28.2, 33.3 and 50% of samples from Canada, Belgium and France, respectively); however, sequence confirmation was not successful for the majority of the samples tested. All the mentioned reports used RT-qPCR for virus detection; therefore, the infectivity of the samples could not be
assessed, and thereby the impact of these results on the public health is under discussion (Baert et al., 2011). This highlights the importance of implementing new methodologies to assess infectivity in food samples. So far, viability PCR has successfully been applied for bacterial detection in several types of food (reviewed by Elizaquível et al., 2014) but not for enteric viruses. This study shows for the first time the potential of PMA to discriminate between thermally inactivated HAV and infectious HAV in food applications. In contrast to the results obtained for bacteria (Elizaquível et al., 2012), this study shows that viability RTqPCR cannot completely prevent PCR amplification from thermally inactivated HAV in food samples. From results on vegetables and shellfish (Table 5 and 6), signal reductions of inactivated HAV are influenced by food matrix and virus concentration. Several authors have reported that the detection of some bacteria (reviewed by Elizaquível et al., 2014) and viruses (Sánchez et al., 2012a) by viability PCR is limited when high concentrations of microorganisms are present. Additionally, Escudero-Abarca et al. (2014) reported that successful discrimination of infectious noroviruses was only achieved when applying it to monodispersed viruses. This may partially explain the differences on the efficacy of PMA pretreatment, since not only the concentration of the virus may play a role but also the extent of virus aggregates. Therefore, in-depth assessment of the influence of virus concentration and presence of virus aggregates will be an essential part for optimizing viability PCR for virus testing in food. In this study PMA–0.5% Triton pretreatment was quite effective in vegetable concentrates, but not in shellfish concentrates. This result indicates that PMA is capable of binding the RNA of the thermally inactivated HAV present in vegetable samples and it partially prevents amplification without interferences of the food matrix. For shellfish concentrates, the high turbidity of the sample may avoid the action of PMA pretreatment and this effect was reverted by diluting shellfish concentrates. The interferences of turbidity on viability PCR has also been
Table 6 Quantification of thermally inactivated HAV suspensions inoculated in shellfish concentrates by PMA–0.5% Triton pretreatment and RT-qPCR. PMA–0.5% Triton
Levels of HAV 6 × 104 TCID50 Quantification (log PCRU)a
Cockles Diluted cockles Coquina clams Diluted coquina clams
− + − + − + − +
2.53 ± 0.02 2.52 ± 0.10 3.16 ± 0.04 1.80 ± 0.16 3.60 ± 0.06 3.33 ± 0.12 4.03 ± 0.64 2.08 ± 0.11
6 × 103 TCID50 Reductionb
0.01 1.36 0.27 1.95
Quantification (log PCRU)a 1.92 ± 0.09 1.69 ± 0.11 2.36 ± 0.09 0.83 ± 0.33 2.66 ± 0.14 2.36 ± 0.05 2.83 ± 0.13 1.73 ± 0.11
6 × 102 TCID50 Reductionb
0.23 1.53 0.30 1.10
ND: non-detected. a Each condition was replicated three times, and HAV titers were obtained by RT-qPCR using a standard curve made by means of HAV PCRU. b Reduction in titers between inactivated viruses before and after pretreatment.
Quantification (log PCRU)a 0.89 ± 0.44 0.88 ± 0.22 1.52 ± 0.10 ND 1.94 ± 0.04 1.47 ± 0.07 2.14 ± 0.19 ND
Reductionb
0.01 N1.52 0.47 N2.14
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described for Vibrio parahaemolyticus in raw seafood. This study showed that PMA treatment with turbidities greater than 10 NTU did not adequately inhibit the amplification of DNA (Zhu et al., 2012). Turbid samples containing suspended solids lowered the effective dye concentration by chemical adsorption with organic and inorganic compounds, resulting in decreased effectiveness of the viability treatment (Fittipaldi et al., 2012). Overall, the application of PMA–0.5% Triton pretreatment for the detection of HAV in food products greatly helps generating more meaningful data, since 1 to 2 logs of the PCR signal can be eliminated by a simple pretreatment. This pretreatment can be applied straightforward for routine analyses, since it lasts 30 min only. Furthermore it can be easily incorporated to the ISO norm for virus detection (ISO/TS 15216), as the RT-qPCR assay suggested there is the same as the one used in this study. However, this pretreatment faces some challenges that need to be tackled in the future. One of the most evident challenges is the fact that, depending on the complexity of the food sample, suppression of inactivated virus signals is not complete, leading to an overestimation of infectious viruses. Additional strategies to increase PMA treatment efficiency include incubation with other surfactants, repeated dye exposure, amplification of longer RNA sequences or modification of the incubation temperature (Fittipaldi et al., 2012; Nkuipou-Kenfack et al., 2013). Acknowledgments This study was supported by grants AGL2009-08603 from the Spanish Ministry of Science and Innovation, and ACOMP/2010/279 and ACOMP/2012/199 from the Generalitat Valenciana. G. Sánchez was supported by the “Ramón y Cajal” Young Investigator program of the Spanish Ministry of Economy and Competitiveness RYC-2012-09950. References Baert, L., Wobus, C.E., Van Coillie, E., Thackray, L.B., Debevere, J., Uyttendaele, M., 2008. Detection of murine norovirus 1 by using plaque assay, transfection assay, and realtime reverse transcription-PCR before and after heat exposure. Appl. Environ. Microbiol. 74, 543–546. Baert, L., Mattison, K., Loisy-Hamon, F., Harlow, J., Martyres, A., Lebeau, B., Stals, A., Van Coillie, E., Herman, L., Uyttendaele, M., 2011. Review: norovirus prevalence in Belgian, Canadian and French fresh produce: a threat to human health. Int. J. Food Microbiol. 15, 261–269. Bhattacharya, S.S., Kulka, M., Lampel, K.A., Cebula, T.A., Goswami, B.B., 2004. Use of reverse transcription and PCR to discriminate between infectious and non-infectious hepatitis A virus. J. Virol. Methods 116, 181–187. Bosch, A., Sánchez, G., Abbaszadegan, M., Carducci, A., Guix, S., Le Guyader, F., Netshikweta, R., Pinto, R.M., van der Poel, W., Rutjes, S.A., Sano, D., Taylor, M., van Zyl, W., Rodríguez-Lázaro, D., Kovac, K., Sellwood, J., 2011. Analytical methods for virus detection in water and food. Food Anal. Methods 4 (1), 4–12. Butot, S., Putallaz, T., Sánchez, G., 2008. Effects of sanitation, freezing and frozen storage on enteric viruses in berries and herbs. Int. J. Food Microbiol. 126, 30–35. Butot, S., Putallaz, T., Amoroso, R., Sánchez, G., 2009. Inactivation of enteric viruses in minimally processed berries and herbs. Appl. Environ. Microbiol. 75, 4155–4161. Collier, M.G., Khudyakov, Y.E., Selvage, D., Adams-Cameron, M., Epson, E., Cronquist, A., Jervis, R.H., Lamba, K., Kimura, A.C., Sowadsky, R., Hassan, R., Park, S.Y., Garza, E., Elliott, A.J., Rotstein, D.S., Beal, J., Kuntz, T., Lance, S.E., Dreisch, R., Wise, M.E., Nelson, N.P., Suryaprasad, A., Drobeniuc, J., Holmberg, S.D., Xu, F., for the Hepatitis A Outbreak investigation Team, 2014. Outbreak of hepatitis A in the USA associated with frozen pomegranate arils imported from Turkey: an epidemiological case study. Lancet Infect. Dis. 14, 976–981. Costafreda, M.I., Bosch, A., Pintó, R.M., 2006. Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples. Appl. Environ. Microbiol. 72, 3846–3855. Coudray-Meunier, C., Fraisse, A., Martin-Latil, S., Guiller, L. Perelle S., 2013. Discrimination of infectious hepatitis A virus and rotavirus by combining dyes and surfactants with RT-qPCR. BMC Microbiol. 13, 216–231. EFSA, 2014. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA J. 12 (2), 3547 (312 pp.). Elizaquível, P., Sanchez, G., Aznar, R., 2012. Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR. Food Control 25, 704–708. Elizaquível, P., Aznar, R., Sánchez, G., 2014. Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field. J. Appl. Microbiol. 116, 1–13.
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