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Application of long-range and binding reverse transcription-quantitative PCR to indicate the viral integrities of noroviruses

Dan Li (UGent) , Ann De Keuckelaere (UGent) and Mieke Uyttendaele (UGent)
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Abstract
This study intends to establish and apply methods evaluating both viral capsid and genome integrities of human noroviruses (NoVs), which thus far remain nonculturable. Murine norovirus 1 (MNV-1) and human NoV GII.4 in phosphate-buffered saline suspensions were treated with heat, UV light, or ethanol and detected by reverse transcription-quantitative PCR (RT-qPCR), long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR. For MNV-1 heated at 60 degrees C for 2 and 30 min, limited reductions of genomic copies (<0.3-log) were obtained by RT-qPCR and long-range RT-qPCR, while the cell-binding pretreatments obtained higher reductions (>1.89-log reduction after 60 degrees C for 30 min by binding long-range RT-qPCR). The human NoV GII.4 was found to be more heat resistant than MNV-1. For both MNV-1 and human NoV GII.4 after UV treatments of 20 and 200 mJ/cm(2), no significant difference (P > 0.05) was observed between the dose-dependent reductions obtained by the four detection methodologies. Treatment of 70% ethanol for 1 min was shown to be more effective for inactivation of both MNV-1 and human NoV GII. 4 than the heat and UV treatments used in this study. Subsequently, eight raspberry and four shellfish samples previously shown to be naturally contaminated with human NoVs by RT-qPCR (GI and GII; thus, 24 RT-qPCR signals) were subjected to comparison by this method. RT-qPCR, long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR detected 20/24, 14/24, 24/24, and 23/24 positive signals, respectively, indicating the abundant presence of intact NoV particles.
Keywords
FELINE CALICIVIRUS, MURINE NOROVIRUS, ENTERIC VIRUSES, RT-PCR, INACTIVATION, WATER, INFECTIVITY, OYSTERS, CELLS, REPLICATION

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Citation

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MLA
Li, Dan, Ann De Keuckelaere, and Mieke Uyttendaele. “Application of Long-range and Binding Reverse Transcription-quantitative PCR to Indicate the Viral Integrities of Noroviruses.” APPLIED AND ENVIRONMENTAL MICROBIOLOGY 80.20 (2014): 6473–6479. Print.
APA
Li, Dan, De Keuckelaere, A., & Uyttendaele, M. (2014). Application of long-range and binding reverse transcription-quantitative PCR to indicate the viral integrities of noroviruses. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 80(20), 6473–6479.
Chicago author-date
Li, Dan, Ann De Keuckelaere, and Mieke Uyttendaele. 2014. “Application of Long-range and Binding Reverse Transcription-quantitative PCR to Indicate the Viral Integrities of Noroviruses.” Applied and Environmental Microbiology 80 (20): 6473–6479.
Chicago author-date (all authors)
Li, Dan, Ann De Keuckelaere, and Mieke Uyttendaele. 2014. “Application of Long-range and Binding Reverse Transcription-quantitative PCR to Indicate the Viral Integrities of Noroviruses.” Applied and Environmental Microbiology 80 (20): 6473–6479.
Vancouver
1.
Li D, De Keuckelaere A, Uyttendaele M. Application of long-range and binding reverse transcription-quantitative PCR to indicate the viral integrities of noroviruses. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2014;80(20):6473–9.
IEEE
[1]
D. Li, A. De Keuckelaere, and M. Uyttendaele, “Application of long-range and binding reverse transcription-quantitative PCR to indicate the viral integrities of noroviruses,” APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 80, no. 20, pp. 6473–6479, 2014.
@article{5832866,
  abstract     = {This study intends to establish and apply methods evaluating both viral capsid and genome integrities of human noroviruses (NoVs), which thus far remain nonculturable. Murine norovirus 1 (MNV-1) and human NoV GII.4 in phosphate-buffered saline suspensions were treated with heat, UV light, or ethanol and detected by reverse transcription-quantitative PCR (RT-qPCR), long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR. For MNV-1 heated at 60 degrees C for 2 and 30 min, limited reductions of genomic copies (<0.3-log) were obtained by RT-qPCR and long-range RT-qPCR, while the cell-binding pretreatments obtained higher reductions (>1.89-log reduction after 60 degrees C for 30 min by binding long-range RT-qPCR). The human NoV GII.4 was found to be more heat resistant than MNV-1. For both MNV-1 and human NoV GII.4 after UV treatments of 20 and 200 mJ/cm(2), no significant difference (P > 0.05) was observed between the dose-dependent reductions obtained by the four detection methodologies. Treatment of 70% ethanol for 1 min was shown to be more effective for inactivation of both MNV-1 and human NoV GII. 4 than the heat and UV treatments used in this study. Subsequently, eight raspberry and four shellfish samples previously shown to be naturally contaminated with human NoVs by RT-qPCR (GI and GII; thus, 24 RT-qPCR signals) were subjected to comparison by this method. RT-qPCR, long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR detected 20/24, 14/24, 24/24, and 23/24 positive signals, respectively, indicating the abundant presence of intact NoV particles.},
  author       = {Li, Dan and De Keuckelaere, Ann and Uyttendaele, Mieke},
  issn         = {0099-2240},
  journal      = {APPLIED AND ENVIRONMENTAL MICROBIOLOGY},
  keywords     = {FELINE CALICIVIRUS,MURINE NOROVIRUS,ENTERIC VIRUSES,RT-PCR,INACTIVATION,WATER,INFECTIVITY,OYSTERS,CELLS,REPLICATION},
  language     = {eng},
  number       = {20},
  pages        = {6473--6479},
  title        = {Application of long-range and binding reverse transcription-quantitative PCR to indicate the viral integrities of noroviruses},
  url          = {http://dx.doi.org/10.1128/AEM.02092-14},
  volume       = {80},
  year         = {2014},
}

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