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Food and Environmental Virology

, Volume 9, Issue 4, pp 395–405 | Cite as

Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing

  • Emmy Borgmästars
  • Mehrdad Mousavi Jazi
  • Sofia Persson
  • Linda Jansson
  • Peter Rådström
  • Magnus Simonsson
  • Johannes Hedman
  • Ronnie Eriksson
Original Paper

Abstract

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C q shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.

Keywords

Norovirus Hepatitis A virus Surface water Ultrafiltration RT-qPCR RT-qPCR inhibition 

Notes

Acknowledgements

Thanks are due to colleagues at the National Food Agency; Rikard Dryselius for collecting water from Görväln, Karin Jacobsson for valuable comments, Melle Säve-Söderbergh for help with statistical analyses, and Sofia Lindström for standard curve and EC RNA preparations. Finally, we thank Almunge and Görväln water treatment plants for surface water samples.

Funding

This study was funded by the Swedish Civil Contingencies Agency, project “Stärkt beredskapskapacitet via rationell laboratoriediagnostik samt förenklad provberedning, pre-PCR processing”, Grants SOFÄ-2013-02, SOFÄ-2013-03, SOFÄ-2015-04, the Swedish Research Council (Grant 621-2013-5999) and Sweden’s innovation agency, UDI SEAL Grant.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

References

  1. Abbaszadegan, M., Huber, M. S., Gerba, C. P., & Pepper, I. L. (1993). Detection of enteroviruses in groundwater with the polymerase chain reaction. Applied and Environmental Microbiology, 59(5), 1318–1324.PubMedPubMedCentralGoogle Scholar
  2. Abu Al-Soud, W., & Rådström, P. (1998). Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Applied and Environmental Microbiology, 64(10), 3748–3753.PubMedPubMedCentralGoogle Scholar
  3. Alouini, S., & Sobsey, M. D. (1995). Evaluation of an extraction-precipitation method for recovering Hepatitis-A virus and poliovirus from Hardshell Clams (Mercenaria-Mercenaria). Water Science and Technology, 31(5–6), 465–469.Google Scholar
  4. Baar, C., d’Abbadie, M., Vaisman, A., Arana, M. E., Hofreiter, M., Woodgate, R., et al. (2011). Molecular breeding of polymerases for resistance to environmental inhibitors. Nucleic Acids Research, 39(8), e51.CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bitler, E. J., Matthews, J. E., Dickey, B. W., Eisenberg, J. N., & Leon, J. S. (2013). Norovirus outbreaks: A systematic review of commonly implicated transmission routes and vehicles. Epidemiology and Infection, 141(8), 1563–1571.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chigor, V. N., & Okoh, A. I. (2012). Quantitative RT-PCR detection of Hepatitis A Virus, rotaviruses and enteroviruses in the buffalo river and source water dams in the Eastern Cape province of South Africa. International Journal of Environmental Research and Public Health, 9(11), 4017–4032.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Core Team, R. (2016). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing.Google Scholar
  8. Costafreda, M. I., Bosch, A., & Pinto, 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. Applied and Environmental Microbiology, 72(6), 3846–3855.CrossRefPubMedPubMedCentralGoogle Scholar
  9. da Silva, A. K., Le Saux, J. C., Parnaudeau, S., Pommepuy, M., Elimelech, M., & Le Guyader, F. S. (2007). Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: Different behaviors of genogroups I and II. Applied and Environmental Microbiology, 73(24), 7891–7897.CrossRefPubMedPubMedCentralGoogle Scholar
  10. D’Ugo, E., Marcheggiani, S., Fioramonti, I., Giuseppetti, R., Spurio, R., Helmi, K., et al. (2016). Detection of human enteric viruses in freshwater from European countries. Food and Environmental Virology, 8(3), 206–214.CrossRefPubMedGoogle Scholar
  11. Francy, D. S., Stelzer, E. A., Brady, A. M., Huitger, C., Bushon, R. N., Ip, H. S., et al. (2013). Comparison of filters for concentrating microbial indicators and pathogens in lake water samples. Applied and Environment Microbiology, 79(4), 1342–1352.CrossRefGoogle Scholar
  12. Guzman-Herrador, B., Carlander, A., Ethelberg, S., Freiesleben de Blasio, B., Kuusi, M., Lund, V., et al. (2015). Waterborne outbreaks in the Nordic countries, 1998 to 2012. Euro Surveillance, 20, 24.Google Scholar
  13. Hedman, J., Knutsson, R., Ansell, R., Rådström, P., & Rasmusson, B. (2013). Pre-PCR processing in bioterrorism preparedness: Improved diagnostic capabilities for laboratory response networks. Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science, 11(Suppl 1), S87–101.CrossRefGoogle Scholar
  14. Hedman, J., Nordgaard, A., Dufva, C., Rasmusson, B., Ansell, R., & Rådström, P. (2010). Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis. Analytical Biochemistry, 405(2), 192–200.CrossRefPubMedGoogle Scholar
  15. Hedman, J., Nordgaard, A., Rasmusson, B., Ansell, R., & Rådström, P. (2009). Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles. BioTechniques, 47(5), 951–958.CrossRefPubMedGoogle Scholar
  16. Hill, V. R., Kahler, A. M., Jothikumar, N., Johnson, T. B., Hahn, D., & Cromeans, T. L. (2007). Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples. Applied and Environmental Microbiology, 73(13), 4218–4225.CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hill, V. R., Mull, B., Jothikumar, N., Ferdinand, K., & Vinjé, J. (2010). Detection of GI and GII noroviruses in ground water using ultrafiltration and TaqMan real-time RT-PCR. Food and Environmental Virology, 2(4), 218–224.CrossRefGoogle Scholar
  18. Hoehne, M., & Schreier, E. (2006). Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3′-minor groove binder-DNA probe. BMC Infectious Diseases, 6, 69.CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kermekchiev, M. B., Kirilova, L. I., Vail, E. E., & Barnes, W. M. (2009). Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples. Nucleic Acids Research, 37(5), e40.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kotwal, G., & Cannon, J. L. (2014). Environmental persistence and transfer of enteric viruses. Current Opinion in Virology, 4, 37–43.CrossRefPubMedGoogle Scholar
  21. Le Guyader, F. S., Parnaudeau, S., Schaeffer, J., Bosch, A., Loisy, F., Pommepuy, M., et al. (2009). Detection and quantification of noroviruses in shellfish. Applied and Environmental Microbiology, 75(3), 618–624.CrossRefPubMedGoogle Scholar
  22. Loisy, F., Atmar, R. L., Guillon, P., Le Cann, P., Pommepuy, M., & Le Guyader, F. S. (2005). Real-time RT-PCR for norovirus screening in shellfish. Journal of Virological Methods, 123(1), 1–7.CrossRefPubMedGoogle Scholar
  23. Medema, G. (2012). Microbial risk assessment of pathogens pathogen in water. In R. A. Meyers (Ed.), Encyclopedia of sustainability science and technology (pp. 6605–6633). New York, NY: Springer.CrossRefGoogle Scholar
  24. Mull, B., & Hill, V. R. (2012). Recovery of diverse microbes in high turbidity surface water samples using dead-end ultrafiltration. Journal of Microbiological Methods, 91(3), 429–433.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Nenonen, N. P., Hannoun, C., Larsson, C. U., & Bergström, T. (2012). Marked genomic diversity of norovirus genogroup I strains in a waterborne outbreak. Applied and Environmental Microbiology, 78(6), 1846–1852.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Pinto, R. M., Costafreda, M. I., & Bosch, A. (2009). Risk assessment in shellfish-borne outbreaks of Hepatitis A. Applied and Environmental Microbiology, 75(23), 7350–7355.CrossRefPubMedPubMedCentralGoogle Scholar
  27. Rådström, P., Knutsson, R., Wolffs, P., Lovenklev, M., & Löfström, C. (2004). Pre-PCR processing: Strategies to generate PCR-compatible samples. Molecular Biotechnology, 26(2), 133–146.CrossRefPubMedGoogle Scholar
  28. Robilotti, E., Deresinski, S., & Pinsky, B. A. (2015). Norovirus. Clinical Microbiology Reviews, 28(1), 134–164.CrossRefPubMedPubMedCentralGoogle Scholar
  29. Rutjes, S. A., Italiaander, R., van den Berg, H. H., Lodder, W. J., & de Roda Husman, A. M. (2005). Isolation and detection of enterovirus RNA from large-volume water samples by using the NucliSens miniMAG system and real-time nucleic acid sequence-based amplification. Applied and Environmental Microbiology, 71(7), 3734–3740.CrossRefPubMedPubMedCentralGoogle Scholar
  30. Schneider, U. V., Mikkelsen, N. D., Lindqvist, A., Okkels, L. M., Johnk, N., & Lisby, G. (2012). Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers. PLoS ONE, 7(6), e38451.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Schwab, K. J., De Leon, R., & Sobsey, M. D. (1995). Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses, Hepatitis A virus, and Norwalk virus by reverse transcription-PCR. Applied and Environmental Microbiology, 61(2), 531–537.PubMedPubMedCentralGoogle Scholar
  32. Sidstedt, M., Jansson, L., Nilsson, E., Noppa, L., Forsman, M., Rådström, P., et al. (2015). Humic substances cause fluorescence inhibition in real-time polymerase chain reaction. Analytical Biochemistry, 487, 30–37.CrossRefPubMedGoogle Scholar
  33. Svraka, S., Duizer, E., Vennema, H., de Bruin, E., van der Veer, B., Dorresteijn, B., et al. (2007). Etiological role of viruses in outbreaks of acute gastroenteritis in The Netherlands from 1994 through 2005. Journal of Clinical Microbiology, 45(5), 1389–1394.CrossRefPubMedPubMedCentralGoogle Scholar
  34. Teunis, P. F., Moe, C. L., Liu, P., Miller, S. E., Lindesmith, L., Baric, R. S., et al. (2008). Norwalk virus: How infectious is it? Journal of Medical Virology, 80(8), 1468–1476.CrossRefPubMedGoogle Scholar
  35. Tornevi, A., Simonsson, M., Forsberg, B., Säve-Söderbergh, M., & Toljander, J. (2016). Efficacy of water treatment processes and endemic gastrointestinal illness—A multi-city study in Sweden. Water Research, 102, 263–270.CrossRefPubMedGoogle Scholar
  36. Wilrich, C., & Wilrich, P. T. (2009). Estimation of the POD function and the LOD of a qualitative microbiological measurement method. Journal of AOAC International, 92(6), 1763–1772.PubMedGoogle Scholar
  37. Yezli, S., & Otter, J. A. (2011). Minimum infective dose of the major human respiratory and enteric viruses transmitted through food and the environment. Food and Environmental Virology, 3(1), 1–30.Google Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Emmy Borgmästars
    • 1
  • Mehrdad Mousavi Jazi
    • 1
  • Sofia Persson
    • 1
    • 2
  • Linda Jansson
    • 3
  • Peter Rådström
    • 3
  • Magnus Simonsson
    • 1
  • Johannes Hedman
    • 3
    • 4
  • Ronnie Eriksson
    • 1
  1. 1.Science Division, Biology DepartmentNational Food AgencyUppsalaSweden
  2. 2.Department of Medical ScienceUppsala UniversityUppsalaSweden
  3. 3.Applied MicrobiologyLund UniversityLundSweden
  4. 4.Swedish National Forensic CentreLinköpingSweden

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