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

Kata Farkas,Davey L Jones,Alexis De Rougemont,Shelagh K Malham,James E Mcdonald,Dafydd E Peters

doi:10.1007/s12560-017-9293-5

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.

Highlights

Enteric viruses represent a major risk to human health, being responsible for numerous local and global disease outbreaks
We developed and validated two multiplex quantitative reverse transcription PCR (qRT-PCR) assays for aquatic sediment and shellfish samples targeting viruses that are a common cause of gastroenteritis, two emerging viruses, along with mengovirus (MgV), which is often used as a sample process control for the assessment of RNA extraction efficiency
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

Summary

Introduction

Enteric viruses represent a major risk to human health, being responsible for numerous local and global disease outbreaks. 3 million acute gastroenteritis cases in the UK and 20 million cases in the USA each year (Centers for Disease Control and Prevention 2015; Tam et al 2012) placing an enormous burden on health care systems and the wider economy. Hepatitis A and E viruses (HAV and HEV), causing acute liver disease, have been shown to be associated with large disease outbreaks. HAV, along with NoV, still represents the major cause of foodborne viral outbreaks (Bosch et al 2016), including those associated with shellfish (Bellou et al 2013). HEV was responsible for the largest viral waterborne outbreak in New Delhi in December 1955– January 1956, where more than 29,000 cases were

Methods

Discussion

Conclusion