Sample preparation prior to molecular amplification: Complexitiesand opportunitiesSophie Butot, Sophie Zuber and Leen Baert

Available online at www.sciencedirect.com

ScienceDirect

Molecular amplification using Reverse Transcription

quantitative Polymerase Chain Reaction (RT-qPCR) is currently

considered as the gold standard to detect enteric human

pathogenic viruses such as norovirus and hepatitis A virus in

food and water. However, the molecular-based detection

requires an adequate sampling strategy and a sample

preparation specific for viruses. Sampling for enteric human

viruses in water and food should not necessarily follow

bacterial sampling plans. The development of a reference

detection method including sample preparation as proposed in

ISO/TS 15216 represents a milestone to facilitate the evaluation

of the performance and eventually validation of future virus

detection methods. The potential viral infectivity linked to a

positive PCR result is a remaining issue and pretreatments

allowing the differentiation of infectious viruses would be useful

for future risk assessments.

Addresses

Food Safety and Quality Competence Pillar, Nestle Research Centre,

Vers-chez-les-Blanc, Box 44, 1000 Lausanne 26, Switzerland

Corresponding author: Butot, Sophie (sophie.butot@rdls.nestle.com)

Current Opinion in Virology 2014, 4:66–70

This review comes from a themed issue on Environmental virology

Edited by Lee-Ann Jaykus and John Scott Meschke

For a complete overview see the Issue and the Editorial

Available online 16th January 2014

1879-6257/$ – see front matter, # 2013 Elsevier B.V. All rights

reserved.

http://dx.doi.org/10.1016/j.coviro.2013.12.004

IntroductionMolecular amplification using Reverse Transcription

quantitative Polymerase Chain Reaction (RT-qPCR) is

currently considered as the gold standard to detect enteric

human pathogenic viruses such as norovirus (NoV) and

hepatitis A virus (HAV) in food and water. Viruses cannot

grow outside their specific host cells and therefore repli-

cation does not occur in the environment nor in foods. For

this reason, enrichment, which is typically used for bac-

terial propagation for analytical purposes, cannot be

applied to increase virus concentration [1,2]. Therefore,

an adequate sampling strategy including sample prep-

aration, specifically for viruses, is required. The food or

water sample needs to be subjected to virus extraction

and concentration steps followed by nucleic acid purifi-

cation prior to molecular amplification. The complexities

and the opportunities associated with the sampling, the

Current Opinion in Virology 2014, 4:66–70

sample preparation consisting of virus extraction and

concentration steps and the preparation of ready-to-use

nucleic acids will be discussed.

SamplingAs for any other microbial contaminant, detection of human

enteric viruses in water and food should start with a rational

sampling plan in which sampling points, number and

volumes of samples should be determined based on the

aim of the testing, the anticipated prevalence and the

desired accuracy [3]. Unfortunately, this can be challen-

ging. While there is information available on the prevalence

of NoV in oysters where outbreak- and non-outbreak-

related positive samples were compared, there is only

limited information on the prevalence of viruses in other

foods such as different fruits and vegetables [4,5,6��]. This

general lack of data may explain why there is no specific

mention of sampling for human enteric viruses in any of the

available standards from international bodies [7�].

Currently, sampling for viruses is based on sampling

strategies for bacteria such as published by the Inter-

national Commission for the Microbiological Specifica-

tions of Foods (ICMSF) [8]. As an example, for the

enhanced monitoring for NoV and HAV in frozen straw-

berries imported from China to the European Union, a

sampling plan is proposed requiring 5 samples to be taken

throughout a batch [9�,10]. This could prevent highly

contaminated berries from reaching the consumer. In a

similar way, monitoring of NoV in shellfish appears to be a

relevant approach to determine whether contamination is

present above a particular level, in order to prevent highly

contaminated shellfish batches reaching the market [6��].

Sampling for virological analyses of food should not

necessarily follow the bacterial approach since important

differences are evident, such as the generally low level of

viral contamination, the inability to enrich viruses and the

complexity and high cost of assays [7�]. Relevant and

economically viable new approaches are needed for the

food industry to incorporate sampling for enteric viruses,

and the future standards for microbial sampling should

include recommendations for enteric viruses.

For instance, viral sampling of fresh produce presents

many difficulties and limitations, including especially low

numbers of heterogeneously distributed viruses in com-

bination with the high cost of the analysis. In such

circumstances, testing environmental samples, for

example irrigation or washing water at a production or

www.sciencedirect.com

Sample preparation prior to molecular amplification Butot, Zuber and Baert 67

a processing stage, may prove more relevant than testing

the produce directly [11��]. In comparison to food

samples, sampling volumes and procedures for enteric

virus detection in water are more comprehensively docu-

mented and related to water quality [3]. Following the

same logic, it could be more useful to undertake virus

screening on swabs from the hands of harvesting person-

nel or other environmental surfaces than to sample the

produce or food directly [11��]. Such indirect evidence of

virus contamination could be added to certain raw

material specifications to ensure greater traceability and

enhance the confidence in critical raw materials such as

berries.

Alternatively, rather than attempting to monitor the pre-

sence of viral pathogens, sampling for a more prevalent

index virus could be used to indicate the potential pre-

sence of human pathogenic viruses in water coming into

contact with food or even the food itself. Candidate

viruses would be those which are largely carried by

healthy people and eliminated via the fecal route. Atten-

tion has focused on human Adenovirus (hAdV) which are

excreted in large quantities by the populations of widely

divergent geographical areas and are more resistant to

environmental degradation than many other enteric

viruses [12,13]. Other virus types such as polyomavirus

and more recently, pepper mild mottle virus, have also

been proposed [14,15]. The use of such ‘indicator’ viruses

might represent a more reliable approach in terms of risk

management and could be used by the food industry to

build trust in certain supply chains.

Sample preparationSample preparation for the detection of viruses in food

and water requires two steps: (i) virus extraction and

concentration from the sample and (ii) nucleic acid

extraction and purification. The latter step no longer

represents a bottleneck as reliable and reproducible com-

mercial kits are available [7�,16]. However, the former

step can be restrictive due to the high variability of virus

recoveries and the low extraction efficiency [17–19].

Furthermore, the large spectrum of matrices and the

broad diversity of existing virus extraction and concen-

tration approaches add complexity [17]. Indeed, a great

number of protocols with the aim of detecting viruses in

foods are published. In fact, these protocols represent

numerous variants on each other and can be grouped.

Two distinct approaches are applied: (i) elution–concen-

tration of virus particles or (ii) the direct viral RNA

extraction from food [17]. Similarly, for water, several

technologies are described to concentrate viruses; the

number of protocols can be categorized mainly as (i)

various adsorption–elution methods using electronega-

tive or electropositive filters and (ii) ultrafiltration-based

methods [20�]. The comparison of method performance

for those available methods is lacking and the limited

number of viral extraction studies that are available for

www.sciencedirect.com

any given combination of virus, water or food type are

limited.

To monitor the efficiency of a virus extraction method, a

process virus control needs to be included at the begin-

ning of the virus extraction step. This control should be a

virus with similar morphological and physicochemical

proprieties and environmental persistence to the target

virus [21]. ISO/TS 15216 [22��,23��], a reference method

for high risk food categories (bivalve molluscan shellfish,

soft fruits and salad vegetables, food surfaces and bottled

water) developed by the CEN/TC 275-Food analysis,

Horizontal Methods; Working Group 6, Technical Advi-

sory Group 4 (CEN TAG4), proposed the use of a

genetically modified mengovirus. Other candidates are

reported in the literature, such as feline calicivirus [24],

MS2 bacteriophage [19], and murine norovirus 1 (MNV-

1) [21]. It is important to point out that MNV-1 is not

easily accessible to private companies.

A meta-analysis comparing the recovery of the process

virus control can be used to evaluate the performance of

methods. Cashdollar [20�] carried out this type of analysis

for water, but the compilation of different process control

viruses resulted in a wide range of recoveries. It was not

evident if the divergence in recoveries was due to the

process virus control or the method itself. It was

suggested that the virus itself, rather than the matrix,

filter type or sample volume, is more important in pre-

dicting the performance of a method for detection of

viruses in water [20�]. Additionally, the mechanism of

virus adsorption to food or virus behavior in water is

poorly understood since no systematic investigations have

been performed. The latter would help to clarify the

difference in recoveries observed between methods

and virus types.

The recovery can be also impacted by the presence of

inhibitory substances, such as polysaccharides, proteins

and fatty acids compounds [25–28]. To mitigate RT-

qPCR inhibition a 10-fold dilution of ribonucleic acid

(RNA) is commonly applied. For the detection of HAV

and MNV-1 in lettuce, Coudray et al. [18] obtained higher

recoveries using a 10-fold dilution compared to an un-

diluted RNA. However, viral RNA copy number is close

to the assay detection limit, the diluted RNA will give a

negative result, demonstrating the importance of analyz-

ing diluted and undiluted RNA [18], as recommended in

ISO/TS 15216 [22��,23��]. These inhibitors should be

removed and controlled before molecular detection to

avoid false negative results.

An alternative means of comparing methods might be to

consider the detection limit achieved by each method. It

is difficult to make robust comparisons based on detection

limits as these are defined differently in different studies

e.g. RT-qPCR units (RT-qPCRU), Plaque Forming

Current Opinion in Virology 2014, 4:66–70

68 Environmental virology

Units (PFU), RNA copies or Tissue Culture Infectious

Dose 50% (TCID50). For several viruses it is known that

there is a lack of correlation between infective viruses

(expressed as PFU or TCID50) and viral genomes

(expressed as RT-PCRU or RNA copies) [29–31].

The establishment of a reference method as currently in

preparation by CEN/TC 275/TAG 4 is very important, as

newly developed viral methods can be compared against

the proposed standard. It facilitates the performance

evaluation of the method itself and benchmarks the

method relative to other existing methods. As an

example, Coudray et al. [18] evaluated a novel extraction

method for lettuce against the proposed virus standard.

Additionally, a full validation of any proposed method is

needed as required for alternative microbial methods

described in ISO 16140:2003 [32] to prove the robustness

and reproducibility of the newly developed method. A

similar approach was performed by Schultz et al. [33�]where virus concentration methods in bottled water were

evaluated. An intra-laboratory study was carried out by

comparing the newly developed method against the

proposed reference method. Subsequently, an inter-

laboratory study was conducted where a noteworthy

variation in the detection limit between laboratories

was observed. The authors suggested that this may be

due to the lack of adequate standardized virus stocks and

suggested the use of standardized control viruses and

RNA for validation studies.

The harmonization of methods will enable the reliable

comparison of prevalence data from different studies.

This would facilitate the use of the data for risk assess-

ment and, if possible, eventually to define an acceptable

threshold of virus presence [34]. In such circumstances,

the accuracy of quantitative data would have to be

Figure 1

Sampling

Opportunities:

• Testing environmental samples may prove more relevant than testing produce directly

• Sampling for an «index» virus could be used to build trust in certain supply chains

Complexities:

• No specific mention of sampling for human enteric viruses in current standards

• Only limited information on the prevalence of viruses in produce

Complexities and opportunities of virus detection prior to molecular amplific

Current Opinion in Virology 2014, 4:66–70

assessed. Therefore, the question arises as to whether

or not the quantitative result is corrected for the extrac-

tion efficiency of the process virus. Because of the

inherent difficulties associated with virus analytics, there

have not been a substantial number of risk assessments

performed for a number of matrices. Exceptionally, com-

prehensive risk assessments were conducted for shellfish

[35,36,37]. Such studies are needed for the food industry

to address the viral risk associated with food and water

and to set appropriate standards for raw materials and the

production environment.

One neglected concern in most of the virus extraction

methods is internalization, particularly for fresh produce

where virus particles may be internalized within plant

tissue. Indeed, some studies have demonstrated the

capacity of viral particles to be trapped inside vegetables

[38–41]. Hirneisen et al. [42] have observed internaliz-

ation of MNV-1 and HAV in plants grown in contami-

nated hydroponic systems but found this phenomenon

rare when plants were grown in soil. As highlighted by

Bosch et al. [7�], this needs further investigation to be

confirmed and could in the future modify the way viruses

are detected/recovered from fruits and vegetables.

The detection of viruses necessitates the use of molecular

methods due to the inability to cultivate them, and

various detection approaches have been investigated

intensively over the last decade. Nevertheless, the mol-

ecular-based detection methods can only indicate the

presence of viral nucleic acids and thus no information

about their infectivity is provided [43]. Consequently, the

real viral risk linked to a positive RT-qPCR result is

unknown [3]. Moreover, most of the extraction and con-

centration methods seek to recover intact viral particles as

well as RNA [16]. Pretreatments with proteinase K and

RNase A, with RNase alone, with intercalating agents

Sample preparation

Complexities:

• High variability of virus recovery

• Low extraction efficiency• Large spectrum of matrices• Broad diversity of methods

Opportunities:

• Comparison versus ISO/TS15216 and harmonization of available methods are needed

• Methods to differentiate infectious versus non-infectious virus particles are needed

Current Opinion in Virology

ation techniques.

www.sciencedirect.com

Sample preparation prior to molecular amplification Butot, Zuber and Baert 69

(ethidium monoazide and propidium monoazide) or using

integrated RT-qPCR approaches [7�,16,44–46] have been

studied in the attempt to correlate detection of viral RNA

with infectivity. Validation is needed to verify these

approaches in food and water. Shellfish may represent

an exception, as bioaccumulation of RNA in shellfish

appears to be insignificant [47]. That suggests that shell-

fish detection methods mostly detect intact particles, but

this also needs to be verified [16].

ConclusionAn overview of complexities and opportunities of virus

detection prior to molecular amplification techniques is

presented in Figure 1. Sampling for enteric human

viruses in water and food should not necessarily follow

bacterial sampling plans since important differences are

evident, such as the low level of viral contamination, the

inability to enrich viruses and the complexity and high

cost of assays. As there is currently no specific mention of

virus sampling in any of the available standards from

international bodies, there is room for the development

of new approaches to incorporate recommendations for

the sampling of enteric viruses into current standards.

The development of a reference detection method,

which includes matrix specific sample preparation proto-

cols as proposed in ISO/TS 15216 [22��,23��], is a mile-

stone to facilitate the evaluation of the performance and

eventually validation of future virus detection methods.

The ability to link viral infectivity to a positive PCR

result is a remaining issue. Pretreatments allowing the

detection of infectious viruses only would be useful for

future risk assessments.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest�� of outstanding interest

1. D’Agostino M, Cook N, Rodriguez-Lazaro D, Rutjes S: Nucleicacid amplification-based methods for detection of entericviruses: definition of controls and interpretation of results.Food EnvironVirol 2011, 3:55-60.

2. Koopmans M, Duizer E: Foodborne viruses: an emergingproblem. Int J Food Microbiol 2004, 90:23-41.

3. Rodriguez-Lazaro D, Cook N, Ruggeri FM, Sellwood J, Nasser A,Nascimento MS, D’Agostino M, Santos R, Saiz JC, Rzezutka A,Bosch A, Girones R, Carducci A, Muscillo M, Kovac K, Diez-Valcarce M, Vantarakis A, von Bonsdorff CH, de RodaHusman AM, Hernandez M, van der Poel WH: Virus hazards fromfood, water and other contaminated environments. FEMSMicrobiol Rev 2012, 36:786-814.

4. Lowther JA, Gustar NE, Powell AL, Hartnell RE, Lees DN: Two-year systematic study to assess norovirus contamination inoysters from commercial harvesting areas in the UnitedKingdom. Appl Environ Microbiol 2012, 78:5812-5817.

5. EFSA: Scientific opinion on an update on the presentknowledge on the occurrence and control of foodborneviruses. EFSA J 2011, 9:2190-2286.

6.��

EFSA: Scientific opinion on norovirus (NoV) in oysters:methods, limits and control options. EFSA J 2012,10:2500-2539.

www.sciencedirect.com

A scientific opinion assessing the relationship between the number ofinfectious virus particles and the number of virus genome copies detectedby quantitative PCR in oysters, showing that in this commodity, microbialcriteria for norovirus are useful for validation and verification of HACCP-based processes and procedures.

7.�

Bosch A, Sanchez G, Abbaszadegan M, Carducci A, Guix S, LeGuyader FS, Netshikweta R, Pinto RM, van der Poel WHM,Rutjes S, Sano D, Taylor MB, van Zyl WB, Rodroguez-Lazaro D,Kovac K, Sellwood J: Analytical methods for virus detection inwater and food. Food Anal Methods 2011, 4:4-12.

The authors carried out a survey of the available methods to detectviruses in food and environmental matrices and discussed potential waysof addressing the issues related to these methods.

8. ICMSF: Microorganisms in foods, vol. 7: Microbiological testing infood safety management. Kluwer Academic/Plenum Publishers;2002.

9.�

European Union: Commission Implementing Regulation (EU)1235/2012 of 19 December 2012 amending Annex I toRegulation 669/2009 implementing Regulation 882/2004 of theEuropean Parliament and of the Council as regards theincreased of official controls on imports of certain food andfeed of non-animal origin. Official J Eur Union 20/12/12,L350/44 2012.

This represents the first regulation to specifically incorporate the sam-pling and testing for norovirus and hepatitis A virus in foods.

10. CEFAS WU. 2012:. Available at http://www.eurlcefas.org/softprotocol.asp (accessed 24.09.13).

11.��

Rzezutka A, Carducci A: Sampling strategies for virus detectionin foods, food-processing environments, water and air. InViruses in Food and Water: Risks, Surveillance and Control. Editedby Cook N. Woodhead Publishing; 2013:79-96.

A comprehensive chapter on the problematic of sampling strategies forvirus detection in different supply chains, proposing interesting alterna-tives to food sampling, such as surface and water sampling.

12. Wyer MD, Wyn-Jones AP, Kay D, Au-Yeung HKC, Girones R,Lopez-Pila J, de Roda Husman AM, Rutjes S, Schneider O:Relationships between human adenoviruses and faecalindicator organisms in European recreational waters. WaterRes 2012, 46:4130-4141.

13. D’Agostino M, Cook N, Di Bartolo I, Ruggeri FM, Berto A, Martelli F,Banks M, Vasickova P, Kralik P, Pavlik I, Kokkinos P, Vantarakis A,Soderberg K, Maunula L, Verhaelen K, Rutjes S, de RodaHusman AM, Hakze R, Van der Poel W, Kaupke A, Kozyra I,Rzezutka A, Prodanov J, Lazic S, Petrovic T, Carratala A,Girones R, Diez-Valcarce M, Hernandez M, Rodriguez-Lazaro D:Multicenter collaborative trial evaluation of a method fordetection of human adenoviruses in berry fruit. Food AnalMethods 2012, 5:1-7.

14. Bofill-Mas S, Rusinol M, Fernandez-Cassi X, Carratala A,Hundesa A, Girones R: Quantification of human and animalviruses to differentiate the origin of the fecal contaminationpresent in environmental samples. Bio Med Res Int 2013.

15. Haramoto E, Kitajima M, Kishida N, Konno Y, Katayama H,Asami M, Akiba M: Occurrence of pepper mild mottle virus indrinking water sources in Japan. Appl Environ Microbiol 2013.

16. Knight A, Li D, Uyttendaele M, Jaykus LA: A critical review ofmethods for detecting human noroviruses and predicting theirinfectivity. Crit Rev Microbiol 2013, 39:295-309.

17. Stals A, Baert L, Van CE, Uyttendaele M: Extraction of food-borne viruses from food samples: a review. Int J Food Microbiol2012, 153:1-9.

18. Coudray C, Merle G, Martin-Latil S, Guillier L, Perelle S:Comparison of two extraction methods for the detection ofhepatitis A virus in lettuces using the murine norovirus as aprocess control. J Virol Methods 2013, 193:96-102.

19. Blaise-Boisseau S, Hennechart-Collette C, Guillier L, Perelle S:Duplex real-time qRT-PCR for the detection of hepatitis Avirus in water and raspberries using the MS2 bacteriophage asa process control. J Virol Methods 2010, 166:48-53.

20.�

Cashdollar JL, Wymer L: Methods for primary concentration ofviruses from water samples: a review and meta-analysis ofrecent studies. J Appl Microbiol 2013, 115:1-11.

Current Opinion in Virology 2014, 4:66–70

70 Environmental virology

The authors compared the recoveries of process controls giving usefulinformation on the performance of a virus detection method.

21. Hennechart-Collette C, Martin-Latil S, Guillier L, Perelle S:Multiplex real-time RT-qPCR for the detection of Norovirus inbottled and tap water using murine norovirus as a processcontrol. J Appl Microbiol 2013.

22.��

ISO/TS15216-1: Microbiology of food and animal feed – Horizontalmethod for determination of hepatitis A virus and norovirus in foodusing real-time RT-PCR Part 1: Method for quantification. 2013.

23.��

ISO/TS15216-2: Microbiology of food and animal feed – Horizontalmethod for determination of hepatitis A virus and norovirus in foodusing real-time RT-PCR – Part 2: Method for qualitative detection.2013.

ISO/TS15216-1 and ISO/TS15216-2 are the proposed reference methodsfor detection hepatitis A virus and norovirus in foodstuffs. This metho-dology should be used as a reference to evaluate alternative virusdetection protocols.

24. Di Pasquale S, Paniconi M, Auricchio B, Orefice L, Schultz AC, DeMedici D: Comparison of different concentration methodsfor the detection of hepatitis A virus and calicivirus frombottled natural mineral waters. J Virol Methods 2010,165:57-63.

25. Rijpens NP, Herman LMF: Molecular methods for identificationand detection of bacterial food pathogens. J AOAC Int 2002,85:984-995.

26. Schwab KJ, McDevitt JJ: Development of a PCR-enzymeimmunoassay oligoprobe detection method for toxoplasmagondii oocysts, incorporating PCR controls. Appl EnvironMicrobiol 2003, 69:5819-5825.

27. Escobar-Herrera J, Cancio C, Guzman GI, Villegas-Sepulveda N,Estrada-Garcia T, Garcia-Lozano H, Gomez-Santiago F,Gutierrez-Escolano AL: Construction of an internal RT-PCRstandard control for the detection of human caliciviruses instool. J Virol Methods 2006, 137:334-338.

28. Demeke T, Jenkins GR: Influence of DNA extraction methods,PCR inhibitors and quantification methods on real-time PCRassay of biotechnology-derived traits. Anal Bioanal Chem 2010,396:1977-1990.

29. Baert L, Uyttendaele M, Debevere J: Evaluation of viralextraction methods on a broad range of ready-to-eat foodswith conventional and real-time RT-PCR for norovirus GIIdetection. Int J Food Microbiol 2008, 123:101-108.

30. Gassilloud B, Schwartzbrod L, Gantzer C: Presence of viralgenomes in mineral water: a sufficient condition to assumeinfectious risk? Appl Environ Microbiol 2003, 69:3965-3969.

31. Hewitt J, Greening GE: Effect of heat treatment on hepatitis Avirus and norovirus in New Zealand Greenshell mussels(Perna canaliculus) by quantitative real-time reversetranscription PCR and cell culture. J Food Prot 2006,69:2217-2223.

32. ISO 16140:2003: Microbiology of food and animal feeding stuffs –protocol for the validation of alternative methods. 2013.

33.�

Schultz AC, Perelle S, Di Pasquale S, Kovac K, De MD, Fach P,Sommer HM, Hoorfar J: Collaborative validation of a rapid

Current Opinion in Virology 2014, 4:66–70

method for efficient virus concentration in bottled water. Int JFood Microbiol 2011, 145:S158-S166.

This study compares virus extraction methods with the proposed refer-ence method. Additionally an inter-laboratory study was performed.

34. Schaeffer J, Le Saux JC, Lora M, Atmar RL, Le Guyader FS:Norovirus contamination on French marketed oysters. Int JFood Microbiol 2013, 166:244-248.

35. Dore B, Keaveney S, Flannery J, Rajko-Nenow P: Management ofhealth risks associated with oysters harvested from anorovirus contaminated area, Ireland, February–March 2010.Eurosurveillance 2010, 15:1-4.

36. Lowther JA, Avant JM, Gizynski K, Rangdale RE, Lees DN:Comparison between quantitative real-time reversetranscription PCR results for norovirus in oysters and self-reported gastroenteric illness in restaurant customers. J FoodProt 2010, 73:305-311.

37. Pinto RM, Costafreda MI, Bosch A: Risk assessment in shellfish-borne outbreaks of hepatitis A. Appl Environ Microbiol 2009,75:7350-7355.

38. Oron G, Goemans M, Manor Y, Feyen J: Poliovirus distribution inthe soil–plant system under reuse of secondary wastewater.Water Res 1995, 29:1069-1078.

39. Chancellor DD, Tyagi S, Bazaco MC, Bacvinskas S,Chancellor MB, Dato VM, de MF: Green onions: potentialmechanism for hepatitis A contamination. J Food Prot 2006,69:1468-1472.

40. Carter MJ: Enterically infecting viruses: pathogenicity,transmission and significance for food and waterborneinfection. J Appl Microbiol 2005, 98:1354-1380.

41. Urbanucci A, Myrmel M, Berg I, von Bonsdorff CH, Maunula L:Potential internalisation of caliciviruses in lettuce. Int J FoodMicrobiol 2009, 135:175-178.

42. Hirneisen KA, Kniel KE: Inactivation of internalized and surfacecontaminated enteric viruses in green onions. Int J FoodMicrobiol 2013, 166:201-206.

43. Iker BC, Bright KR, Pepper IL, Gerba CP, Kitajima M: Evaluation ofcommercial kits for the extraction and purification of viralnucleic acids from environmental and fecal samples. J VirolMethods 2013, 191:24-30.

44. Hamza IA, Jurzik L, Uberla K, Wilhelm M: Methods to detectinfectious human enteric viruses in environmental watersamples. Int J Hyg Environ Health 2011, 214:424-436.

45. Sanchez G, Elizaquivel P, Aznar R: Discrimination of infectioushepatitis A viruses by propidium monoazide real-time RT-PCR. Food Environ Virol 2012, 4:21-25.

46. Parshionikar S, Laseke I, Fout GS: Use of propidium monoazidein reverse transcriptase PCR to distinguish betweeninfectious and noninfectious enteric viruses in water samples.Appl Environ Microbiol 2010, 76:4318-4326.

47. Dancer D, Rangdale RE, Lowther JA, Lees DN: Human norovirusRNA persists in seawater under simulated winter conditionsbut does not bioaccumulate efficiently in Pacific oysters(Crassostrea gigas). J Food Prot 2010, 73:2123-2127.

www.sciencedirect.com