Food Analytical Methods

, Volume 6, Issue 4, pp 1016–1023

Performance of Two Real-Time RT-PCR Assays for the Quantification of GI and GII Noroviruses and Hepatitis A Virus in Environmental Water Samples

  • Ann De Keuckelaere
  • Ambroos Stals
  • Leen Baert
  • Mieke Uyttendaele
Article

Abstract

In this study, the performance of two real-time reverse transcription polymerase chain reaction (RT-qPCR) assays for the detection of hepatitis A viruses (HAV) and GI and GII noroviruses (NoV) was tested in the presence of an environmental matrix by analyzing 15 inoculated environmental water samples. For the detection of HAV, an in-house two-step RT-qPCR from literature was compared with a commercial one-step real-time RT-PCR of Ceeram (La Chapelle-sur-Erdre, France). For the detection of GI and GII NoV, an in-house duplex two-step RT-qPCR assay was used and compared with the results obtained using two commercial singleplex one-step RT-qPCR assays of Ceeram (France). The performance of the two RT-qPCR assays was determined by comparing (1) standard curves, (2) the number of detected genomic copies, and (3) the influence of inhibition by RNA dilution. Both assays for the detection of GI and GII NoV performed likewise. For the detection of HAV, the differences in genomic copies detected were to some extent more apparent and in favor of the commercial one-step assay. When the HAV RT-qPCR assays were compared in terms of inhibition, the performance of the commercial one-step RT-qPCR kit was less affected for the detection of HAV in undiluted RNA in comparison to the in-house two-step RT-qPCR assay. On the other hand, inhibition had only a marginal influence on the performance of both assays for detection of HAV in the 1/10 diluted RNA. In conclusion, only minor differences were observed between the in-house RT-qPCR assays and the commercial one-step assays for the detection of HAV and NoV in environmental water samples.

Keywords

Norovirus Hepatitis A virus Real-time RT-PCR Water 

References

  1. Abu Al-Soud W, Radstrom P (1998) Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Appl Environ Microbiol 64:3748Google Scholar
  2. Albinana-Gimenez N, Clemente-Casares P, Cagua B, Huguet JM, Courtois S, Girones R (2009) Comparison of methods for concentrating human adenoviruses, polyomavirus JC and noroviruses in source waters and drinking water using quantitative PCR. J Virol Methods 158:104CrossRefGoogle Scholar
  3. APHA (1998) Standard methods for the examination of water and wastewater. American Public Health Association, New York, p 1220Google Scholar
  4. Baert L, Vandekinderen I, Devlieghere F, Van Coillie E, Debevere J, Uyttendaele M (2009) Efficacy of sodium hypochlorite and peroxyacetic acid to reduce murine norovirus 1, B40-8, Listeria monocytogenes, and Escherichia coli O157:H7 on shredded iceberg lettuce and in residual wash water. J Food Prot 72:1047Google Scholar
  5. Butot S, Le Guyader FS, Krol J, Putallaz T, Amoroso R, Sanchez G (2010) Evaluation of various real-time RT-PCR assays for the detection and quantitation of human norovirus. J Virol Methods 167:90CrossRefGoogle Scholar
  6. Cao Y, Griffith JF, Dorevitch S, Weisberg SB (2012) Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters. J Appl Microbiol 113:66CrossRefGoogle Scholar
  7. Costafreda MI, Bosch A, Pinto RM (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 72:3846CrossRefGoogle Scholar
  8. Costa-Mattioli M, Monpoeho S, Nicand E, Aleman MH, Billaudel S, Ferre V (2002) Quantification and duration of viraemia during hepatitis A infection as determined by real-time RT-PCR. J Viral Hepat 9:101CrossRefGoogle Scholar
  9. Croci L, Dubois E, Cook N, de Medici D, Schultz AC, China B, Rutjes SA, Hoorfar J, Van der Poel WHM (2008) Current methods for extraction and concentration of enteric viruses from fresh fruit and vegetables: towards international standards. Food Anal Methods 1:73CrossRefGoogle Scholar
  10. de Bruin E, Duizer E, Vennema H, Koopmans MPG (2006) Diagnosis of norovirus outbreaks by commercial ELISA or RT-PCR. J Virol Methods 137:259CrossRefGoogle Scholar
  11. De Keuckelaere A, Baert L, Duarte A, Stals A, Uyttendaele M (2013) Evaluation of viral concentration methods from irrigation and processing water. J Virol Methods 187:294CrossRefGoogle Scholar
  12. EFSA (2012) The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in the European Union in 2010. EFSA J 10. www.efsa.europa.eu/efsajournal. Accessed 16 April 2012
  13. FAO/WHO (2008) Viruses in food: scientific advice to support risk management activities, meeting report. Microbiological Risk Assessment series no 13 RomeGoogle Scholar
  14. Forbes BA, Hicks KE (1996) Substances interfering with direct detection of Mycobacterium tuberculosis in clinical specimens by PCR: effects of bovine serum albumin. J Clin Microbiol 34:2125Google Scholar
  15. Fout GS, Martinson BC, Moyer MWN, Dahling DR (2003) A multiplex reverse transcription-PCR method for detection of human enteric viruses in groundwater. Appl Environ Microbiol 69:3158CrossRefGoogle Scholar
  16. Gibson KE, Schwab KJ (2011) Tangential-flow ultrafiltration with integrated inhibition detection for recovery of surrogates and human pathogens from large-volume source water and finished drinking water. Appl Environ Microbiol 77:385CrossRefGoogle Scholar
  17. Hamza IA, Jurzik L, Stang A, Sure K, Uberla K, Wilhelm M (2009) Detection of human viruses in rivers of a densely-populated area in Germany using a virus adsorption elution method optimized for PCR analyses. Water Res 43:2657CrossRefGoogle Scholar
  18. Haugland RA, Siefring S, Lavender J, Varma M (2012) Influences of sample interference and interference controls on quantification of enterococci fecal indicator bacteria in surface water samples by the qPCR method. Water Res 46:5989CrossRefGoogle Scholar
  19. Hohne M, Schreier E (2004) Detection and characterization of norovirus outbreaks in Germany: application of a one-tube RT-PCR using a fluorogenic real-time detection system. J Med Virol 72:312CrossRefGoogle Scholar
  20. Huggett J, Novak T, Garson J, Green C, Morris-Jones S, Miller R, Zumla A (2008) Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon. BMC Res Notes 1Google Scholar
  21. Jothikumar N, Cromeans TL, Sobsey MD, Robertson BH (2005a) Development and evaluation of a broadly reactive TaqMan assay for rapid detection of hepatitis A virus. Appl Environ Microbiol 71:3359CrossRefGoogle Scholar
  22. Jothikumar N, Lowther JA, Henshilwood K, Lees DN, Hill VR, Vinje J (2005b) Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples. Appl Environ Microbiol 71:1870CrossRefGoogle Scholar
  23. Kageyama T, Kojima S, Shinohara M, Uchida K, Fukushi S, Hoshino FB, Takeda N, Katayama K (2003) Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J Clin Microbiol 41:1548CrossRefGoogle Scholar
  24. Katayama H, Shimasaki A, Ohgaki S (2002) Development of a virus concentration method and its application to detection of enterovirus and Norwalk virus from coastal seawater. Appl Environ Microbiol 68:1033CrossRefGoogle Scholar
  25. Koopmans M, Duizer E (2004) Foodborne viruses: an emerging problem. Int J Food Microbiol 90:23CrossRefGoogle Scholar
  26. Le Guyader FS, Bon F, DeMedici D, Parnaudeau S, Bertone A, Crudeli S, Doyle A, Zidane M, Suffredini E, Kohli E, Maddalo F, Monini M, Gallay A, Pommepuy M, Pothier P, Ruggeri FM (2006) Detection of multiple noroviruses associated with an international gastroenteritis outbreak linked to oyster consumption. J Clin Microbiol 44:3878CrossRefGoogle Scholar
  27. Le Guyader FS, Parnaudeau S, Schaeffer J, Bosch A, Loisy F, Pommepuy M, Atmar RL (2009) Detection and quantification of noroviruses in shellfish. Appl Environ Microbiol 75:618CrossRefGoogle Scholar
  28. Lees D (2010) International standardisation of a method for detection of human pathogenic viruses in molluscan shellfish. Food Environ Virol 2:146CrossRefGoogle Scholar
  29. Nasser AM, Metcalf TG (1987) Production of cytopathology in Frhk-4 cells by Bs-C-1-passaged hepatitis A-virus. Appl Environ Microbiol 53:2967Google Scholar
  30. Opel KL, Chung D, Mccord BR (2009) A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 55:25CrossRefGoogle Scholar
  31. Pontiroli A, Travis ER, Sweeney FP, Porter D, Gaze WH, Mason S, Hibberd V, Holden J, Courtenay O, Wellington EMH (2011) Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition. PLoS One 6Google Scholar
  32. Sarvikivi E, Roivainen M, Maunula L, Niskanen T, Korhonen T, Lappalainen M, Kuusi M (2012) Multiple norovirus outbreaks linked to imported frozen raspberries. Epidemiol Infect 140:260CrossRefGoogle Scholar
  33. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM (2011) Foodborne illness acquired in the United States—major pathogens. Emerg Infect Dis 17:7Google Scholar
  34. Schrader C, Schielke A, Ellerbroek L, Johne R (2012) PCR inhibitors—occurrence, properties and removal. J Appl Microbiol 113:1014CrossRefGoogle Scholar
  35. Schriewer A, Wehlmann A, Wuertz S (2011) Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8. J Microbiol Methods 85:16CrossRefGoogle Scholar
  36. Seymour IJ, Appleton H (2001) Foodborne viruses and fresh produce. J Appl Microbiol 91:759CrossRefGoogle Scholar
  37. Stals A, Baert L, Botteldoorn N, Werbrouck H, Herman L, Uyttendaele M, Van Coillie E (2009) Multiplex real-time RT-PCR for simultaneous detection of GI/GII noroviruses and murine norovirus 1. J Virol Methods 161:247CrossRefGoogle Scholar
  38. Stals A, Baert L, Van Coillie E, Uyttendaele M (2012) Extraction of food-borne viruses from food samples: a review. Int J Food Microbiol 153:1CrossRefGoogle Scholar
  39. Vinje J, Hamidjaja RA, Sobsey MD (2004) Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses. J Virol Methods 116:109CrossRefGoogle Scholar
  40. Wei T, Lu GJ, Clover G (2008) Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR, demonstrated using an assay for detection of three strawberry viruses. J Virol Methods 151:132CrossRefGoogle Scholar
  41. Wheeler C, Vogt TM, Armstrong GL, Vaughan G, Weltman A, Nainan OV, Dato V, Xia GL, Waller K, Amon J, Lee TM, Highbaugh-Battle A, Hembree C, Evenson S, Ruta MA, Williams IT, Fiore AE, Bell BP (2005) An outbreak of hepatitis A associated with green onions. N Engl J Med 353:890CrossRefGoogle Scholar
  42. Wyn-Jones AP, Carducci A, Cook N, D’Agostino M, Divizia M, Fleischer J, Gantzer C, Gawler A, Girones R, Holler C, Husman AMD, Kay D, Kozyra I, Lopez-Pila J, Muscillo M, Nascimento MS, Papageorgiou G, Rutjes S, Sellwood J, Szewzyk R, Wyer M (2011) Surveillance of adenoviruses and noroviruses in European recreational waters. Water Res 45:1025CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Ann De Keuckelaere
    • 1
  • Ambroos Stals
    • 1
  • Leen Baert
    • 1
  • Mieke Uyttendaele
    • 1
  1. 1.Department of Food Safety and Food Quality, Faculty of Bioscience EngineeringGhent UniversityGhentBelgium

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