Article

Food Analytical Methods

, Volume 6, Issue 4, pp 1016-1023

First online:

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 KeuckelaereAffiliated withDepartment of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University Email author 
  • , Ambroos StalsAffiliated withDepartment of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University
  • , Leen BaertAffiliated withDepartment of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University
  • , Mieke UyttendaeleAffiliated withDepartment of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University

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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