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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 (UGent) , Ambroos Stals (UGent) , Leen Baert (UGent) and Mieke Uyttendaele (UGent)
(2013) FOOD ANALYTICAL METHODS. 6(4). p.1016-1023
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VEG-I-TRADE (Impact of climate change and globalisation on safety of fresh produce – governing a supply chain of uncompromised food sovereignty)
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, water, real-time RT-PCR, REVERSE TRANSCRIPTION-PCR, SHELLFISH SAMPLES, FOODBORNE VIRUSES, OUTBREAKS, STANDARDIZATION, INHIBITION, PATHOGENS, GERMANY

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Citation

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MLA
De Keuckelaere, Ann, Ambroos Stals, Leen Baert, et al. “Performance of Two Real-time RT-PCR Assays for the Quantification of GI and GII Noroviruses and Hepatitis A Virus in Environmental Water Samples.” FOOD ANALYTICAL METHODS 6.4 (2013): 1016–1023. Print.
APA
De Keuckelaere, A., Stals, A., Baert, L., & Uyttendaele, M. (2013). Performance of two real-time RT-PCR assays for the quantification of GI and GII noroviruses and hepatitis A virus in environmental water samples. FOOD ANALYTICAL METHODS, 6(4), 1016–1023.
Chicago author-date
De Keuckelaere, Ann, Ambroos Stals, Leen Baert, and Mieke Uyttendaele. 2013. “Performance of Two Real-time RT-PCR Assays for the Quantification of GI and GII Noroviruses and Hepatitis A Virus in Environmental Water Samples.” Food Analytical Methods 6 (4): 1016–1023.
Chicago author-date (all authors)
De Keuckelaere, Ann, Ambroos Stals, Leen Baert, and Mieke Uyttendaele. 2013. “Performance of Two Real-time RT-PCR Assays for the Quantification of GI and GII Noroviruses and Hepatitis A Virus in Environmental Water Samples.” Food Analytical Methods 6 (4): 1016–1023.
Vancouver
1.
De Keuckelaere A, Stals A, Baert L, Uyttendaele M. Performance of two real-time RT-PCR assays for the quantification of GI and GII noroviruses and hepatitis A virus in environmental water samples. FOOD ANALYTICAL METHODS. 2013;6(4):1016–23.
IEEE
[1]
A. De Keuckelaere, A. Stals, L. Baert, and M. Uyttendaele, “Performance of two real-time RT-PCR assays for the quantification of GI and GII noroviruses and hepatitis A virus in environmental water samples,” FOOD ANALYTICAL METHODS, vol. 6, no. 4, pp. 1016–1023, 2013.
@article{3527065,
  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.},
  author       = {De Keuckelaere, Ann and Stals, Ambroos and Baert, Leen and Uyttendaele, Mieke},
  issn         = {1936-9751},
  journal      = {FOOD ANALYTICAL METHODS},
  keywords     = {norovirus,hepatitis A virus,water,real-time RT-PCR,REVERSE TRANSCRIPTION-PCR,SHELLFISH SAMPLES,FOODBORNE VIRUSES,OUTBREAKS,STANDARDIZATION,INHIBITION,PATHOGENS,GERMANY},
  language     = {eng},
  number       = {4},
  pages        = {1016--1023},
  title        = {Performance of two real-time RT-PCR assays for the quantification of GI and GII noroviruses and hepatitis A virus in environmental water samples},
  url          = {http://dx.doi.org/10.1007/s12161-013-9647-z},
  volume       = {6},
  year         = {2013},
}

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