Isolation and Detection of Enterovirus RNA from Large-Volume Water Samples by Using the NucliSens miniMAG System and Real-Time Nucleic Acid Sequence-Based Amplification

Abstract

Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.

Analytical Methods for Virus Detection in Water and Food

“…Contamination of surface water with enteric viruses through disposal of human waste is a concern for public health, especially if these surface waters are used for recreational water, irrigation water, and the production of drinking water (Rutjes et al 2005). The surveillance of irrigation water and water for washing the fresh produce is therefore essential to facilitate correct management procedures for the protection of fruit and vegetable growers and the health of farm workers and the consumers.…”

“…Most of these kits are based on guanidinium lysis and then the capture of nucleic acids on a column or bead of silica (commonly called Boom's method (1990)). Although most perform well, differences can be found depending on the virus and/or matrix analyzed (Rutjes et al 2005;Hourfar et al 2005;Knepp et al 2003;Kok et al 2000;Le Guyader et al 2009;Burgener et al 2003). In the last years, automated nucleic acid extraction platforms have been developed by commercial companies, which have been shown to be suitable for the analysis of virus in water samples (Rutjes et al 2005;Perelle et al 2009).…”

“…Quantification can also be performed as most probable number by conventional PCR (Rutjes et al 2005(Rutjes et al , 2006a. Qualitative real-time PCR producing a positive or negative result is most appropriate when testing matrices that are unlikely to be contaminated with virus as it is least expensive and straightforward.…”

“…The method is prone to inhibition, favoring false negative results and demonstrating the need for proper quality control (QC). Several ways have been described to overcome this inhibition, such as the analysis of samples dilutions, smaller sample sizes, adaptation of the PCR by, e.g., the addition of Tween, BSA, or commercial reagents (Rutjes et al 2005;Butot et al 2007;Al-Soud and Radstrom 2001). …”

Current Methods for Extraction and Concentration of Enteric Viruses from Fresh Fruit and Vegetables: Towards International Standards

“…Pores in the membrane, varying from 10 to 100kDa, permit passage of liquids and low molecular mass particles in solution and exclude viruses and macromolecules. Ultrafiltration has been described for the concentration of viruses from several foods including fruits and vegetables (Le Guyader et al 2004b;Finance et al 1981;Kostenbader and Cliver 1981;Rutjes et al 2005). Release of virus particles from food surfaces prior to ultrafiltration is achieved by using either demineralized water adjusted to pH 9.0 or a 0.05 to 0.09M glycine-containing elution buffer (pH 8.5 to 9.5) and is generally followed by a clarification step using centrifugation.…”