Journal of Microbiology and Biotechnology
The Korean Society for Microbiology and Biotechnology publishes the Journal of Microbiology and Biotechnology.

2017 ; Vol.27-4: 816~824

AuthorJu Eun Yoo, Cheonghoon Lee, SungJun Park, GwangPyo Ko
Place of dutyDepartment of Environmental Health Sciences, Graduate School of Public Health,
TitleEvaluation of Various Real-Time Reverse Transcription Quantitative PCR Assays for Norovirus Detection
PublicationInfo J. Microbiol. Biotechnol.2017 ; Vol.27-4
AbstractHuman noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR’s robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D’s probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.
Full-Text
Key_wordNorovirus, RT-qPCR, detection limit, sensitivity, internal quencher
References
  1. Ahmed SM, Hall AJ, Robinson AE, Verhoef L, Premkumar P, Parashar UD, et al. 2014. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and metaanalysis. Lancet Infect. Dis. 14: 725-730.
    CrossRef
  2. Patel MM, Widdowson MA, Glass RI, Akazawa K, Vinjé J, Parashar UD. 2008. Systematic literature review of role of noroviruses in sporadic gastroenteritis. Emerg. Infect. Dis. 14:1224-1231.
    Pubmed CrossRef Pubmed Central
  3. Teunis PFM, Moe CL, Liu P, Miller SE, Lindesmith L, Baric RS, et al. 2008. Norwalk virus: how infectious is it? J. Med. Virol. 80: 1468-1476.
    Pubmed CrossRef
  4. Atmar RL, Openkun AR, Gilger MA, Estes MK, Crawford SE, Neill FH, Graham DY. 2008. Norwalk virus shedding after experimental human infection. Emerg. Infect. Dis. 14:1553-1557.
    Pubmed CrossRef Pubmed Central
  5. Cheesbrough JS, Green J, Gallimore CI, Wright PA, Brown DWG. 2000. Widespread environmental contamination with Norwalk-like viruses (NLV) detected in a prolonged hotel outbreak of gastroenteritis. Epidemiol. Infect. 125: 93-98.
    Pubmed CrossRef Pubmed Central
  6. Lopman B, Gastañaduy P, Park GW, Hall AJ, Parashar UD, Vinjé J. 2012. Environmental transmission of norovirus gastroenteritis. Curr. Opin. Virol. 2: 96-102.
    Pubmed CrossRef
  7. Vinjé J. 2015. Advances in laboratory methods for detection and typing of norovirus. J. Clin. Microbiol. 53: 373-381.
    Pubmed CrossRef Pubmed Central
  8. Stals A, Mathijs E, Baert L, Bottledoorn N, Denayer S, Mauroy A, et al. 2012. Molecular detection and genotyping of noroviruses. Food Environ. Virol. 4: 153-167.
    Pubmed CrossRef
  9. Green K. 2013. Caliciviridae: The Noroviruses, pp. 583-609. In Knipe DM, Howley PM, Cohen JI, Griffin DE, Lamb RA, Martin MA, Racaniello VR, Roizman B (eds). Fields Virology, 6th Ed. Vol 1. Lippincott Williams & Wilkins, Philadelphia, PA, USA.
  10. Kageyama T, Kojima S, Shinohara M, Uchida K, Fukushi S, Hoshino FB, et al. 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.
    Pubmed CrossRef Pubmed Central
  11. Jothikumar N, Lowther JA, Henshilwood K, Lees DN, Hill VR, Vinjé, J. 2005. 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: 1870-1875.
    Pubmed CrossRef Pubmed Central
  12. Park YB, Cho YH, Ko GP. 2011. A duplex real-time RT-PCR assay for the simultaneous genogroup-specific detection of noroviruses in both clinical and environmental specimens. Virus Genes 43: 192-200.
    Pubmed CrossRef
  13. Ministry of Food and Drug Safety. 2013. Guideline for Investigation on the Cause of Food Poisoning, Ch. 5. Ministry of Food and Drug Safety, Chungcheonbuk-do, Republic of Korea. [In Korean].
  14. Mattison K, Grudeski E, Auk B, Brassard J, Charest H, Dust K, et al. 2011. Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories. J. Clin. Virol. 50: 109-113
    Pubmed CrossRef
  15. Lee SG, Lee SH, Park SW, Suh CI, Jheong WH, Oh S, Paik SY. 2011. Standardized positive controls for detection of norovirus by reverse transcription PCR. Virol. J. 8: 260-267.
    Pubmed CrossRef Pubmed Central
  16. Martínez-Martínez M, Diez-Valcarce M, Hernández M, Rodríguez-Lázaro D. 2011. Design and application of nucleic acid standards for quantitative detection of enteric viruses by real-time PCR. Food Environ. Virol. 3: 92-98.
    Pubmed CrossRef Pubmed Central
  17. Victoria M, Miagostovich MP, Ferreira MSR, Vieira CB, Fioretti JM, Leite JPG, et al. 2009. Bayesian coalescent inference reveals high evolutionary rates and expansion of norovirus populations. Infect. Genet. Evol. 9: 927-932.
    Pubmed CrossRef
  18. Xia H, Gravelsina S, Öhrmalm C, Ottoson J, Blomberg J. 2016. Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II. Biotechniques 60: 28-34.
    Pubmed CrossRef
  19. Kojima S, Kageyama T, Fukushi S, Hoshino FB, Shinohara M, Uchida K, et al. 2002. Genogroup-specific PCR primers for detection of Norwalk-like viruses. J. Virol. Methods 100:107-114.
    CrossRef
  20. Kim SH, Cheon DS, Kim JH, Lee DH, Jheong WH, Heo YJ, et al. 2005. Outbreaks of gastroenteritis that occurred during school excursions in Korea were associated with several waterborne strains of norovirus. J. Clin. Microbiol. 43: 48364839.
    Pubmed CrossRef Pubmed Central
  21. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55: 611-622.
    Pubmed CrossRef
  22. Lopman B, Simmons K, Gambhir M, Vinjé J, Parashar U. 2014. Epidemiological implications of asymptomatic reinfection:a mathematical modeling study of norovirus. Am. J. Epidemiol. 179: 507-512.
    Pubmed CrossRef
  23. Kabue JP, Meader E, Hunter PR, Potgieter N. 2016. Norovirus prevalence and estimated viral load in symptomatic and asymptomatic children from rural communities of Vhembe district, South Africa. J. Clin. Virol. 84: 12-18.
    Pubmed CrossRef Pubmed Central
  24. Stals A, Van Collie E, Uyttendaele M. 2013. Virus genes everywhere: public health implications of PCR-based testing of foods. Curr. Opin. Virol. 3: 69-73.
    Pubmed CrossRef
  25. Butot S, Zuber S, Baert L. 2014. Sample preparation prior to molecular amplification: complexities and opportunities. Curr. Opin. Virol. 4: 66-70.
    Pubmed CrossRef
  26. Zheng D-P, Ando T, Fankhauser RL, Beard RS, Glass RI, Monroe SS. 2006. Norovirus classification and proposed strain nomenclature. Virology 346: 312-323.
    Pubmed CrossRef
  27. Park GW, Collins N, Barclay L, Hu L, Prasad BVV, Lopman BA, Vinjé J. 2016. Strain-specific virolysis patterns of human noroviruses in response to alcohols. PLoS One 11: e0157787
    Pubmed CrossRef Pubmed Central
  28. Jin M, Tan M, Xia M, Wei C, Huang P, Wang L, et al. 2015. Strain-specific interaction of a GII.10 norovirus with HBGAs. Virology 476: 386-394.
    Pubmed CrossRef
  29. Vega E, Barclay L, Gregoricus N, Williams K, Lee D, Vinjé J. 2011. Novel surveillance network for norovirus gastroenteritis outbreaks, United States. Emerg. Infect. Dis. 17: 1389-1395.
    CrossRef
  30. Gonzalez MD, Langley LC, Buchan BW, Faron ML, Maier M, Templeton K, et al. 2016. Multicenter evaluation of the Xpert norovirus assay for detection of norovirus genogroups I and II in fecal specimens. J. Clin. Microbiol. 54: 142-147.
    Pubmed CrossRef Pubmed Central
  31. Bustin SA, Nolan T. 2004. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Tech. 15: 155-166.
    Pubmed Pubmed Central



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