Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

Abstract

Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.Noroviruses belong to the family Caliciviridae and contain a positive-sense, single-stranded RNA genome of approximately 7.6 kb (14). Based on sequence information of the genes encoding the viral RNA-dependent RNA polymerase (RdRp) and the capsid protein, the norovirus genus is divided into five genogroups (genogroup I [GGI] to GGV) that can be divided further into genetic clusters, each represented by a prototype virus. The diversity of norovirus variants increases continually due to the generation of new variants, with two groups of strains predominating in the past 5 years (22,29,33,34,39). Noroviruses that cluster within GGI and GGII are mostly human pathogens. They are the most common agents involved in outbreaks of gastroenteritis. Outbreaks originating from contaminated drinking water as well as from recreational water have been described previously (4,11,18,21,37).Due to the lack of a cell culture system, noroviruses are currently detected mainly by molecular techniques. With the increase in knowledge of norovirus sequences, many molecular detection assays that detect a wide variety of norovirus strains have been developed, e.g., reverse line blot hybridization (47), reverse transcriptase PCR (RT-PCR) (1,13,46), and nucleic acid sequence-based amplification (NASBA) (17,36). In the last decade, several different regions of the norovirus ...

Evaluation of the Persistence of Infectious Human Noroviruses on Food Surfaces by Using Real-Time Nucleic Acid Sequence-Based Amplification

“…Several recent studies have explored the ORF1-ORF2 junction as a target because it is the most conserved part of the NoV genome (23,25,36). In an effort to improve NoV GII primer specificity, Jothikumar et al (21) designed the broadly reactive forward NoV GII primer JJV2F, which targets the junction domain and contains eight bases at the 3Ј end that are 100% identical among the majority of NoV GII sequences.…”

Analytical Methods for Virus Detection in Water and Food

“…The alternative molecular technique nucleic acid sequence-based amplification (NASBA) was shown to be less prone to environmental PCR inhibitors present in large volumes of surface water samples (Rutjes et al 2006b). …”

Adsorption-Elution Techniques and Molecular Detection of Enteric Viruses from Water

“…Therefore rt PCR has increased speed due to reduced cycle number, is more sensitive due to higher sensitivity of fluorescent dyes used for the detection of the amplicon and more specific by use of DNA/ RNA strand specific probes like Taqman or molecular beacons [33,35,36]. Real time quantitative (rt qRT-PCR/PCR) provides numerical data on the presence of enteric viral genomes which is important in estimating the public health risks of low levels of enteric viruses in environmental samples [11].…”

“…Gel filtration resins like Sephadex or Sepharose are generally effective in removing inhibitors but there is loss of nucleic acids in the process [33,39]. To avoid loss of nucleic acids that can arise from chromatographic techniques and binding resins, bovine serum albumin (BSA) has been added to PCR mixtures to reduce inhibition by binding to inhibitors [8,11,39]. Methods including the use of internal or external controls are also available for identifying and monitoring inhibition [18,19,36,39].…”

“…Detection and quantification of viral pathogen levels in water is rarely performed because of the technical challenges involved in viral assays [9]. One of the major difficulties encountered in the detection of viruses in water sources is the occurrence of low virus particle numbers in water [10,11]. This makes analysis of viruses in water samples a complex and more expensive process than detecting bacteria.…”