Skip to main content

Advertisement

SpringerLink
Log in

Detection of Potential Infectious Enteric Viruses in Fresh Produce by (RT)-qPCR Preceded by Nuclease Treatment

  • Original Paper
  • Published:
Food and Environmental Virology Aims and scope Submit manuscript

Abstract

Foodborne illnesses associated with contaminated fresh produce are a common public health problem and there is an upward trend of outbreaks caused by enteric viruses, especially human noroviruses (HNoVs) and hepatitis A virus (HAV). This study aimed to assess the use of DNase and RNase coupled to qPCR and RT-qPCR, respectively, to detect intact particles of human adenoviruses (HAdVs), HNoV GI and GII and HAV in fresh produce. Different concentrations of DNase and RNase were tested to optimize the degradation of free DNA and RNA from inactivated HAdV and murine norovirus (MNV), respectively. Results indicated that 10 µg/ml of RNase was able to degrade more than 4 log10 (99.99%) of free RNA, and 1 U of DNase degraded the range of 0.84–2.5 log10 of free DNA depending on the fresh produce analysed. The treatment with nucleases coupled to (RT)-qPCR was applied to detect potential infectious virus in organic lettuce, green onions and strawberries collected in different seasons. As a result, no intact particles of HNoV GI and GII were detected in the 36 samples analysed, HAdV was found in one sample and HAV was present in 33.3% of the samples, without any reasonable distribution pattern among seasons. In conclusion, RT-qPCR preceded by RNase treatment of eluted samples from fresh produce is a good alternative to detect undamaged RNA viruses and therefore, potential infectious viruses. Moreover, this study provides data about the prevalence of enteric viruses in organic fresh produce from Brazil.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Albinana-Gimenez, N., Miagostovich, M. P., Calgua, B., Huguet, J. M., Matia, L., & Girones, R. (2009). Analysis of adenoviruses and polyomaviruses quantified by qPCR as indicators of water quality in source and drinking-water treatment plants. Water Research, 43(7), 2011–2019. doi:10.1016/j.watres.2009.01.025.

    Article  CAS  PubMed  Google Scholar 

  • Anonymous. (2013). ISO/TS 15216. Microbiology of food and animal stuffs—Horizontal method for detection of hepatis A virus and norovirus in food using real-time RT-PCR. International Organization for Standardization.

  • Baert, L., Wobus, C. E., Coillie, E. Van, Thackray, L. B., Debevere, J., & Uyttendaele, M. (2008). Detection of murine norovirus 1 by using plaque assay, transfection assay, and real-time reverse transcription-PCR before and after heat exposure. Applied and Environmental Microbiology, 74(2), 543–546. doi:10.1128/AEM.01039-07.

    Article  CAS  PubMed  Google Scholar 

  • Bosch, A., Sanchez, G., Abbaszadegan, M., Carducci, A., Guix, S., Le Guyader, F. S., et al. (2011). Analytical methods for virus detection in water and food. Food Analytical Methods, 4(1), 4–12.

    Article  Google Scholar 

  • Callejón, R. M., Rodríguez-Naranjo, M. I., Ubeda, C., Hornedo-Ortega, R., Garcia-Parrilla, M. C., & Troncoso, A. M. (2015). Reported foodborne outbreaks due to fresh produce in the United States and European Union: Trends and causes. Foodborne Pathogens and Disease, 12, 32–38. doi:10.1089/fpd.2014.1821.

    Article  PubMed  Google Scholar 

  • Cannon, J. L., Papafragkou, E., Park, G. W., & Osborne, J. (2006). Surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus. Journal of Food Protection, 69(11), 2761–2765.

    Article  PubMed  Google Scholar 

  • Collier, M. G., Khudyakov, Y. E., Selvage, D., Adams-cameron, M., Epson, E., Cronquist, A., et al. (2014). Outbreak of hepatitis A in the USA associated with frozen pomegranate arils imported from Turkey: An epidemiological case study. The Lancet Infectious Diseases, 14(10), 976–981. doi:10.1016/S1473-3099(14)70883-7.

    Article  PubMed  Google Scholar 

  • Colombet, J., Robin, A., Lavie, L., Bettarel, Y., Cauchie, H. M., & Sime-Ngando, T. (2007). Virioplankton “pegylation”: Use of PEG (polyethylene glycol) to concentrate and purify viruses in pelagic ecosystems. Journal of Microbiological Methods, 71(3), 212–219.

    Article  CAS  PubMed  Google Scholar 

  • Cook, N., & D’Agostino, M. (2013). Prevalence and control of hepatitis A virus in fresh produce. In H. Budka, H. B. Frans, J. M. Smulders, & B. Nørrung (Eds.), Foodborne viruses and prions and their significance for public health (Vol. 6, pp. 169–180). Wageningen: Wageningen Academic Publishers. doi:10.3920/978-90-8686-780-6.

    Chapter  Google Scholar 

  • De Paula, V. S., Diniz-mendes, L., Villar, L. M., Luz, S. L. B., Silva, L. A., Jesus, M. S., et al. (2007). Hepatitis A virus in environmental water samples from the Amazon Basin. Water Research, 41, 1169–1176. doi:10.1016/j.watres.2006.11.029.

    Article  PubMed  Google Scholar 

  • Deboosere, N., Pinon, A., Caudrelier, Y., Delobel, A., Merle, G., Perelle, S., et al. (2012). Adhesion of human pathogenic enteric viruses and surrogate viruses to inert and vegetal food surfaces. Food Microbiology, 32(1), 48–56. doi:10.1016/j.fm.2012.04.007.

    Article  PubMed  Google Scholar 

  • Diez-Valcarce, M., Kovac, K., Raspor, P., Rodríguez-Lázaro, D., & Hernández, M. (2011). Virus genome quantification does not predict norovirus infectivity after application of food inactivation processing technologies. Food and Environmental Virology, 3, 141–146. doi:10.1007/s12560-011-9070-9.

    Article  CAS  Google Scholar 

  • Donnan, E. J., Fielding, J. E., Gregory, J. E., Lalor, K., Rowe, S., Goldsmith, P., et al. (2012). A multistate outbreak of hepatitis A associated with semidried tomatoes in Australia, 2009. Clinical Infectious Diseases, 54, 775–781. doi:10.1093/cid/cir949.

    Article  PubMed  Google Scholar 

  • EFSA & ECDC. (2014). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA Journal, 12, 3547. doi:10.2903/j.efsa.2014.3547

  • EFSA & ECDC. (2015). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014. EFSA Journal, 13, 4329

  • EFSA & ECDC. (2016). The Euroean Union summary report on trends and sources of zoonsoes, zoonotic agents and food-borne outbreaks in 2015. EFSA Journal, 14, 4634. doi:10.2903/j.efsa.2015.4329

  • Elizaquível, P., Aznar, R., & Sánchez, G. (2013). Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field. Journal of Applied Microbiology, 116, 1–13. doi:10.1111/jam.12365.

    Article  PubMed  Google Scholar 

  • Escudero-Abarca, B. I., Rawsthorne, H., Goulter, R. M., Suh, S. H., & Jaykus, L. A. (2014). Molecular methods used to estimate thermal inactivation of a prototype human norovirus: More heat resistant than previously believed? Food Microbiology, 41, 91–95. doi:10.1016/j.fm.2014.01.009.

    Article  CAS  PubMed  Google Scholar 

  • Ettayebi, K., Crawford, S. E., Murakami, K., Broughman, J. R., Karandikar, U., Tenge, V. R., et al. (2016). Replication of human noroviruses in stem cell— derived human enteroids. Science, 353, 1387–1393.

    Article  PubMed  PubMed Central  Google Scholar 

  • Fongaro, G., do Nascimento, M. A., Rigotto, C., Ritterbusch, G., da Silva, A., Esteves, P. A., & M Barardi, C. R. (2013). Evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays. Virology Journal, 10, 166–175

  • Hall, A., Wikswo, M., Pringle, K., Gouldd, L., & Parashar, U. (2014). Vital Signs: foodborne norovirus outbreaks—United States, 2009–2012. MMWR Morb Mort Wkly Rep, Recomm Rep, 63, 491–495

  • Hernroth, B. E., Girones, R., & Allard, A. K. (2002). Environmental factors influencing human viral pathogens and their potential indicator organisms in the Blue Mussel, Mytilus edulis: The first scandinavian report. Applied and Environmental Microbiology, 68(9), 4523–4533. doi:10.1128/AEM.68.9.4523.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jothikumar, N., Cromeans, T. L., Sobsey, M. D., & Robertson, B. H. (2005). Development and evaluation of a broadly reactive TaqMan assay for rapid detection of Hepatitis A virus. Applied and Environmental Microbiology, 71(6), 3359–3363. doi:10.1128/AEM.71.6.3359.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kageyama, T., Kojima, S., Shinohara, M., Uchida, K., Fukushi, S., Hoshino, F. B., et al. (2003). Broadly reactive and highly sensitive assay for Norwalk-Like viruses based on Real-Time quantitative reverse transcription-PCR. Journal of Clinical Microbiology, 41(4), 1548–1557. doi:10.1128/JCM.41.4.1548.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim, K., Katayama, H., Kitajima, M., Tohya, Y., & Ohgaki, S. (2011). Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure. Water Science and Technology, 63, 502–507. doi:10.2166/wst.2011.249.

    Article  CAS  PubMed  Google Scholar 

  • Kim, S. Y., & Ko, G. (2012). Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus. Letters in Applied Microbiology, 55, 182–188. doi:10.1111/j.1472-765X.2012.03276.x.

    Article  CAS  PubMed  Google Scholar 

  • Knight, A., Li, D., Uyttendaele, M., & Jaykus, L. (2012). A critical review of methods for detecting human noroviruses and predicting their infectivity. Critical Reviews in Microbiology, 39, 295–309. doi:10.3109/1040841X.2012.709820.

    Article  PubMed  Google Scholar 

  • Lamhoujeb, S., Fliss, I., Ngazoa, S. E., & Jean, J. (2008). Evaluation of the persistence of infectious human noroviruses on food surfaces by using real-time nucleic acid sequence-based. Applied and Environmental Microbiology, 74(11), 3349–3355. doi:10.1128/AEM.02878-07.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lassen, S. G., Soborg, B., Midgley, S. E., Steens, A., & Vold, L. (2013). Ongoing multi-strain food-borne hepatitis A outbreak with frozen berries as suspected vehicle: Four Nordic countries affected, October 2012 to April 2013. Euro Surveillance, 18, 20467.

    Google Scholar 

  • Levy, H. C., Bostina, M., Filman, D. J., & Hogle, J. M. (2010). Catching a virus in the act of RNA release: a novel poliovirus uncoating intermediate characterized by cryo-electron microscopy. Journal of Virology, 84(9), 4426–4441. doi:10.1128/JVI.02393-09.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li, D., Keuckelaere, A. De, & Uyttendaele, M. (2015). Fate of foodborne viruses in the ‘Farm to Fork’ chain of fresh produce. Comprehensive Reviews in Food Science and Food Safety, 14, 755–770. doi:10.1111/1541-4337.12163.

    Article  Google Scholar 

  • Lynch, M. F., Tauxe, R. V., & Hedberg, C. W. (2009). The growing burden of foodborne outbreaks due to contaminated fresh produce: Risks and opportunities. Epidemiology and Infection, 137(3), 307–315. doi:10.1017/S0950268808001969.

    Article  CAS  PubMed  Google Scholar 

  • Marti, E., & Barardi, C. R. M. (2016). Detection of human adenoviruses in organic fresh produce using molecular and cell culture-based methods. International Journal of Food Microbiology, 230, 40–44. doi:10.1016/j.ijfoodmicro.2016.04.018.

    Article  CAS  PubMed  Google Scholar 

  • Maunula, L., Kaupke, A., Vasickova, P., Söderberg, K., Kozyra, I., Lazic, S., et al. (2013). Tracing enteric viruses in the European berry fruit supply chain. International Journal of Food Microbiology, 167, 177–185. doi:10.1016/j.ijfoodmicro.2013.09.003.

    Article  PubMed  Google Scholar 

  • Mormann, S., Dabisch, M., & Becker, B. (2010). Effects of technological processes on the tenacity and inactivation of Norovirus genogroup II in experimentally contaminated foods. Applied and Environmental Microbiology, 76(2), 536–545. doi:10.1128/AEM.01797-09.

    Article  CAS  PubMed  Google Scholar 

  • Nowak, P., Topping, J. R., Bellamy, K., Fotheringham, V., Gray, J. J., Golding, J. P., et al. (2011). Virolysis of Feline calicivirus and human GII.4 Norovirus following chlorine exposure under standardized light soil disinfection conditions. Journal of Food Protection, 74(12), 2113–2118. doi:10.4315/0362-028X.JFP-11-087.

    Article  CAS  PubMed  Google Scholar 

  • Nuanualsuwan, S., & Cliver, D. O. (2003). Capsid functions of inactivated human picornaviruses and Feline calicivirus. Applied and Environmental Microbiology, 69(1), 350–357. doi:10.1128/AEM.69.1.350.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Parshionikar, S., Laseke, I., & Fout, G. S. (2010). Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples. Applied and Environmental Microbiology, 76(13), 4318–4326. doi:10.1128/AEM.02800-09.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pasloske, B. L. (2001). Ribonuclease inhibitors. In C. H. Schien (Ed.), Nuclease methods and protocols (pp. 105–111). Totowa, NJ: Humana Press.

    Chapter  Google Scholar 

  • Randazzo, W., López-gálvez, F., Allende, A., Aznar, R., & Sánchez, G. (2016). Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water. International Journal of Food Microbiology, 229, 1–6. doi:10.1016/j.ijfoodmicro.2016.04.010.

    Article  CAS  PubMed  Google Scholar 

  • Rigotto, C., Victoria, M., Moresco, V., Kolesnikovas, C. K., Corrêa, A. A., Souza, D. S. M., et al. (2010). Assessment of adenovirus, hepatitis A virus and rotavirus presence in environmental samples in Florianopolis, South Brazil. Journal of Applied Microbiology, 109(6), 1979–1987. doi:10.1111/j.1365-2672.2010.04827.x.

    Article  CAS  PubMed  Google Scholar 

  • Rodríguez-Lázaro, D., Cook, N., Ruggeri, F. M., Sellwood, J., Nasser, A., Nascimento, M. S. J., et al. (2012). Virus hazards from food, water and other contaminated environments. FEMS Microbiology Reviews, 36(4), 786–814. doi:10.1111/j.1574-6976.2011.00306.x.

    Article  PubMed  Google Scholar 

  • Rzeżutka, A., & Cook, N. (2004). Survival of human enteric viruses in the environment and food. FEMS Microbiology Reviews, 28(4), 441–453. doi:10.1016/j.femsre.2004.02.001.

    Article  PubMed  Google Scholar 

  • Sánchez, G., Elizaquível, P., & Aznar, R. (2012). Discrimination of infectious Hepatitis A viruses by propidium monoazide Real-Time RT-PCR. Food and Environmental Virology, 4, 21–25. doi:10.1007/s12560-011-9074-5.

    Article  PubMed  Google Scholar 

  • Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M., Roy, S. L., et al. (2011). Foodborne illness acquired in the United States—Major pathogens. Emerging Infectious Diseases, 17(1), 7–15. doi:10.3201/eid1701.P11101.

    Article  PubMed  PubMed Central  Google Scholar 

  • Topping, J. R., Schnerr, H., Haines, J., Scott, M., Carter, M. J., Willcocks, M. M., et al. (2009). Temperature inactivation of Feline calicivirus vaccine strain FCV F-9 in comparison with human noroviruses using an RNA exposure assay and reverse transcribed quantitative real-time polymerase chain reaction-A novel method for predicting virus infectivity. Journal of Virological Methods, 156(1–2), 89–95. doi:10.1016/j.jviromet.2008.10.024.

    Article  CAS  PubMed  Google Scholar 

  • Viancelli, A., Garcia, L. A. T., Kunz, A., Steinmetz, R., Esteves, P. A., & Barardi, C. R. M. (2012). Detection of circoviruses and porcine adenoviruses in water samples collected from swine manure treatment systems. Research in Veterinary Science, 93(1), 538–543. doi:10.1016/j.rvsc.2011.07.022.

    Article  CAS  PubMed  Google Scholar 

  • WHO. (2015). WHO estimates of the global burden of foodborne diseases: Foodborne diseases burden epidemiology reference group 2007–2015 (pp. 2007–2015). Geneva: WHO.

    Google Scholar 

Download references

Acknowledgements

E. Marti received a “Bolsa Jovens Talentos (BJT)” scholarship (303491/2014-0) from the Science without Borders Programme from the National Council for Scientific and Technological Development (CNPq; Brazil Government). The same CNPq programme (Project 400183/2014-5) also supported this work.

Author information

Authors and Affiliations

  1. Laboratório de Virologia Aplicada, Departamento de Microbiologia, Imunologia e Parasitologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, CEP: 88040-970, Brazil

    Elisabet Marti, Monique Ferrary-Américo & Célia Regina Monte Barardi

Authors
  1. Elisabet Marti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Monique Ferrary-Américo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Célia Regina Monte Barardi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elisabet Marti.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Marti, E., Ferrary-Américo, M. & Barardi, C.R.M. Detection of Potential Infectious Enteric Viruses in Fresh Produce by (RT)-qPCR Preceded by Nuclease Treatment. Food Environ Virol 9, 444–452 (2017). https://doi.org/10.1007/s12560-017-9300-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12560-017-9300-x

Keywords

Search

Navigation