Virologica Sinica

, Volume 32, Issue 2, pp 139–146 | Cite as

Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II

  • Kitwadee Rupprom
  • Porntip Chavalitshewinkoon-Petmitr
  • Pornphan Diraphat
  • Leera KittigulEmail author
Research Article


Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (103 DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.


norovirus genogroup real-time RT-PCR quantification 



This work was supported by research grant from the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. program (Grant No. PHD/0085/2554), and the Thai Government Budget through Mahidol University, fiscal year 2015‒2017. The authors thank The Language Center, Faculty of Graduate Studies, Mahidol University for editorial assistance.

Author Contributions

KDR performed the experiments and analyzed the data. PCP and PPD participated in the experiments. LRK designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.

Compliance with Ethics Guidelines

All the authors declare that they have no competing interests. The study was approved by the Ethical Review Committee for Human Research, Faculty of Public Health, Mahidol University. The protocol complied with a “Research with Exemption” category.


  1. Aw TG, Gin KY, Ean Oon LL, Chen EX, Woo CH. 2009. Prevalence and genotypes of human noroviruses in tropical urban surface waters and clinical samples in Singapore. Appl Environ Microbiol, 75: 4984–4992.CrossRefGoogle Scholar
  2. Baert L, Uyttendaele M, Debevere J. 2007. Evaluation of two viral extraction methods for the detection of human noroviruses in shellfish with conventional and real-time reverse transcriptase PCR. Lett Appl Microbiol, 44: 106–111.CrossRefGoogle Scholar
  3. Bok K, Abente EJ, Realpe-Quintero M, Mitra T, Sosnovtsev SV, Kapikian AZ, Green KY. 2009. Evolutionary dynamics of GII.4 noroviruses over a 34-year period. J Virol, 83: 11890–11901.CrossRefGoogle Scholar
  4. Bull RA, Tanaka MM, White PA. 2007. Norovirus recombination. J Gen Virol, 88: 3347–3359.CrossRefGoogle Scholar
  5. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 55: 611–622.CrossRefGoogle Scholar
  6. 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: 90–94.CrossRefGoogle Scholar
  7. Costafreda MI, Bosch A, Pintó 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: 3846–3855.CrossRefGoogle Scholar
  8. Dunbar NL, Bruggink LD, Marshall JA. 2014. Evaluation of the RIDAGENE real-time PCR assay for the detection of GI and GII norovirus. Diagn Microbiol Infect Dis, 79: 317–321.CrossRefGoogle Scholar
  9. Espy MJ, Uhl, JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JD, Wengenack NL, Rosenblatt J E, Cockerill F R 3rd, Smith T F. 2006. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev, 19: 165–256.CrossRefGoogle Scholar
  10. Farkas T, Singh A, Le Guyader FS, La Rosa G, Saif L, McNeal M. 2015. Multiplex real-time RT-PCR for the simultaneous detection and quantification of GI, GII and GIV noroviruses. J Virol Methods, 223: 109–114.CrossRefGoogle Scholar
  11. Fuentes C, Guix S, Pérez-Rodriguez FJ, Fuster N, Carol M, Pintó RM, Bosch A. 2014. Standardized multiplex one-step qRT-PCR for hepatitis A virus, norovirus GI and GII quantification in bivalve mollusks and water. Food Microbiol, 40: 55–63.CrossRefGoogle Scholar
  12. Green KY. 2013. Caliciviridae: the noroviruses. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE. (Eds.), Fields Virology, 6th ed. Philadelphia: Lippincott Williams & Wilkins, pp. 582–608.Google Scholar
  13. Hall AJ, Wikswo ME, Manikonda K, Roberts VA, Yoder JS, Gould LH. 2013. Acute gastroenteritis surveillance through the national outbreak reporting system, United States. Emerg Infect Dis, 19: 1305–1309.CrossRefGoogle Scholar
  14. Hyun J, Kim, HS, Kim HS, Lee KM. 2014. Evaluation of a new real-time reverse transcription polymerase chain reaction assay for detection of norovirus in fecal specimens. Diagn Microbiol Infect Dis, 78: 40–44.CrossRefGoogle Scholar
  15. 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: 1548–1557.CrossRefGoogle Scholar
  16. Kittigul L, Pombubpa K, Taweekate Y, Diraphat P, Sujirarat D, Khamrin P, Ushijima H. 2010. Norovirus GII-4 2006b variant circulating in patients with acute gastroenteritis in Thailand during a 2006–2007 study. J Med Virol, 82: 854–860.CrossRefGoogle Scholar
  17. Kittigul L, Panjangampatthana A, Pombubpa K, Taweekate Y, Pungchitton S, Diraphat P, Siripanichgon K. 2012. Detection and genetic characterization of norovirus in environmental water samples in Thailand. Southeast Asian J Trop Med Public Health, 43: 323–332.PubMedGoogle Scholar
  18. Kittigul L, Thamjaroen A, Chiawchan S, Chavalitshewinkoon-Petmitr P, Pombubpa K, Diraphat P. 2016. Prevalence and molecular genotyping of noroviruses in market oysters, mussels, and cockles in Bangkok, Thailand. Food Environ Virol, 8: 133–140.CrossRefGoogle Scholar
  19. Lees D, CEN WG6 TAG4. 2010. International standardisation of a method for detection of human pathogenic viruses in molluscan shellfish. Food Environ Virol, 2: 146–155.CrossRefGoogle Scholar
  20. Loisy F, Atmar RL, Guillon P, Le Cann P, Pommepuy M, Le Guyader FS. 2005. Real-time RT-PCR for norovirus screening in shellfish. J Virol Methods, 123: 1–7.CrossRefGoogle Scholar
  21. Lu J, Fang L, Zheng H, Lao J, Yang F, Sun L, Xiao J, Lin J, Song T, Ni T, Raghwani J, Ke C, Faria NR, Bowden TA, Pybus OG, Li H. 2016. The evolution and transmission of epidemic GII.17 noroviruses. J Infect Dis, 214: 556–64.CrossRefGoogle Scholar
  22. Mathijs E, Stals A, Baert L, Botteldoorn N, Denayer S, Mauroy A, Scipioni A, Daube G, Dierick K, Herman L, Van Coillie E, Uyttendaele M, Thiry E. 2012. A review of known and hypothetical transmission routes for noroviruses. Food Environ Virol, 4: 131–152.CrossRefGoogle Scholar
  23. Mattison K, Grudeski E, Auk B, Brassard J, Charest H, Dust K, Gubbay J, Hatchette TF, Houde A, Jean J, Jones T, Lee BE, Mamiya H, McDonald R, Mykytczuk O, Pang X, Petrich A, Plante D, Ritchie G, Wong J, Booth TF. 2011. Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories. J Clin Virol 50: 109–113.CrossRefGoogle Scholar
  24. Myers TW, Gelfand DH. 1991. Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry, 30: 7661–7666.CrossRefGoogle Scholar
  25. Neesanant P, Sirinarumitr T, Chantakru S, Boonyaprakob U, Chuwongkomon K, Bodhidatta L, Sethabutr O, Abente EJ, Supawat K, Mason CJ. 2013. Optimization of one-step real-time reverse transcription-polymerase chain reaction assays for norovirus detection and molecular epidemiology of noroviruses in Thailand. J Virol Methods, 194: 317–325.CrossRefGoogle Scholar
  26. Patel MM, Widdowson MA, Glass RI, Akazawa K, Vinje J, Parashar UD. 2008. Systematic literature review of role of noroviruses in sporadic gastroenteritis. Emerg Infect Dis, 14: 1224–1231.CrossRefGoogle Scholar
  27. Pang X, Lee BE. 2015. Laboratory diagnosis of noroviruses: present and future. Clin Lab Med, 35: 345–362.CrossRefGoogle Scholar
  28. Poddar SK, Sawyer MH, Connor JD. 1998. Effect of inhibitors in clinical specimens on Taq and Tth DNA polymerase-based PCR amplification of influenza A virus. J Med Microbiol, 47: 1131–1135.CrossRefGoogle Scholar
  29. Schultz AC, Vega E, Dalsgaard A, Christensen LS, Norrung B, Hoorfar J, Vinje J. 2011. Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses. J Clin Virol, 50: 230–234.CrossRefGoogle Scholar
  30. Siebenga JJ, Vennema H, Zheng DP, Vinjé J, Lee BE, Pang XL, Ho EC, Lim W, Choudekar A, Broor S, Halperin T, Rasool NB, Hewitt J, Greening GE, Jin M, Duan ZJ, Lucero Y, O’Ryan M, Hoehne M, Schreier E, Ratcliff RM, White PA, Iritani N, Reuter G, Koopmans M. 2009. Norovirus illness is a global problem: emergence and spread of norovirus GII.4 variants, 2001–2007. J Infect Dis, 200: 802–812.CrossRefGoogle Scholar
  31. Stals A, Uyttendaele M, Van Coillie E. 2013. The need for harmonization in detection of human noroviruses in food. J AOAC Int, 96: 998–1005.CrossRefGoogle Scholar
  32. Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M. 2010. A practical approach to RT-qPCR-publishing data that conform to the MIQE guidelines. Methods, 50: S1–S5.CrossRefGoogle Scholar
  33. Van Stelten A, Kreman TM, Hall N, Desjardin LE. 2011. Optimization of a real-time RT-PCR assay reveals an increase of genogroup I norovirus in the clinical setting. J Virol Methods, 175: 80–84.CrossRefGoogle Scholar
  34. Vinjé J. 2015. Advances in laboratory methods for detection and typing of norovirus. J Clin Microbiol, 53: 373–381.CrossRefGoogle Scholar
  35. Wenzel JJ, Panning M, Kaul KL, Mangold KA, Revell PA, Luna RA, Zepeda H, Perea L, Vazquez-Perez JA, Young S, Rodic-Polic B, Eickmann M, Drosten C, Jilg W, Reischl U. 2010. Analytical performance determination and clinical validation of the novel Roche RealTime Ready Influenza A/H1N1 Detection Set. J Clin Microbiol, 48: 3088–3094.CrossRefGoogle Scholar
  36. Yan Y, Wang HH, Gao L, Ji JM, Ge ZJ, Zhu XQ, He PY, Chen ZW. 2013. A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV. J Virol Methods, 189: 277–282.CrossRefGoogle Scholar

Copyright information

© Wuhan Institute of Virology, CAS and Springer Science+Business Media Singapore 2017

Authors and Affiliations

  • Kitwadee Rupprom
    • 1
  • Porntip Chavalitshewinkoon-Petmitr
    • 2
  • Pornphan Diraphat
    • 1
  • Leera Kittigul
    • 1
    Email author
  1. 1.Department of Microbiology, Faculty of Public HealthMahidol UniversityBangkokThailand
  2. 2.Department of Protozoology, Faculty of Tropical MedicineMahidol UniversityBangkokThailand

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