Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples

Abstract

Human enteric viruses are responsible for waterborne and shellfish-associated disease outbreaks worldwide. Quantitative reverse transcription PCR (qRT-PCR) is often used to assess the health risks associated with shellfish and environmental water, but viral titres in sediments are less commonly investigated. In this study, we developed and validated two multiplex qRT-PCR assays for aquatic sediment and shellfish samples targeting viruses that are a common cause of gastroenteritis (norovirus GI, GII and hepatitis A virus), two emerging viruses (sapovirus and hepatitis E virus), along with mengovirus (MgV), which is often used as a sample process control for the assessment of RNA extraction efficiency. Singleplex and multiplex assays demonstrated comparable PCR efficiencies and gave reliable results over a wide concentration range. The multiplex assays showed remarkable sensitivity with a limit of detection of 1 RNA copy/µL nucleic acid extract for all target viruses and limits of quantification of 3-18 RNA copies/µL for the targeted human pathogenic viruses and 20-40 RNA copies/µL for MgV. The results demonstrated the veracity of multiplex qRT-PCR for the estimation of viral titres in sediment and shellfish, allowing the rapid assessment of viral infection risks associated with environments exposed to wastewater contamination.

Keywords: Enteric viruses; Multiplex real-time reverse transcription PCR; Nucleic acid quantification; Sediment; Shellfish.

Fig. 1

Standard curves of the qRT-PCR assays for the target viruses in singleplex (black filled circle) and multiplex (open circle) qRT-PCR assays. Norovirus GII (NoV GII), hepatitis A virus (HAV and mengovirus (MgV) assays were run using the annealing temperature (Ta) of 60 °C, whereas NoV GI, sapovirus (SaV) and Hepatitis E virus (HEV) assays were run using Ta of 56 °C. The grey circle represents the results for the norovirus GI standards when a Ta of 60 °C was used. Symbols and error bars represent the mean and standard deviation of the triplicated experiments