Development of a Tetraplex qPCR for the Molecular Identification and Quantification of Human Enteric Viruses, NoV and HAV, in Fish Samples

Abstract

Human enteric viruses such as norovirus (NoV) and hepatitis A virus (HAV) are some of the most important causes of foodborne infections worldwide. Usually, infection via fish consumption is not a concern regarding these viruses, since fish are mainly consumed cooked. However, in the last years, raw fish consumption has become increasingly common, especially involving the use of seabass and gilthead seabream in dishes like sushi, sashimi, poke, and carpaccio. Therefore, the risk for viral infection via the consumption of raw fish has also increased. In this study, a virologic screening was performed in 323 fish specimens captured along the Portuguese coast using a tetraplex qPCR optimised for two templates (plasmid and in vitro transcribed RNA) to detect and quantify NoV GI, NoV GII and HAV genomes. A difference of approximately 1-log was found between the use of plasmid or in vitro transcribed RNA for molecular-based quantifications, showing an underestimation of genome copy-number equivalents using plasmid standard-based curves. Additionally, the presence of NoV genomic RNA in a pool of seabass brains was identified, which was shown to cluster with a major group of human norovirus sequences from genogroup I (GI.1) by phylogenetic analysis. None of the analysed fish revealed the presence of NoV GII or HAV. This result corroborates the hypothesis that enteric viruses circulate in seawater or that fish were contaminated during their transportation/handling, representing a potential risk to humans through raw or undercooked fish consumption.

A Comparison of Two Methods for Detection of Norovirus RNA in Environmental Swab Samples

“…Quantification of the Lenticule ® reference material against the IVT ssRNA standard yielded an increase in the genome copy number of between 1.5 and 2 log. This can be explained by the relative poor efficiency of the RT step, as the sensitivity-limiting step in the reaction [20]. The implications of this are that the sensitivity of RT-PCR methods commonly reported against DNA standards may be overestimated.…”