Development and Evaluation of EPA Method 1615 for Detection of Enterovirus and Norovirus in Water

Abstract

bThe U.S. EPA developed a sample concentration and preparation assay in conjunction with the total culturable virus assay for concentrating and measuring culturable viruses in source and drinking waters as part of the Information Collection Rule (ICR) promulgated in 1996. In an effort to improve upon this method, the U.S. EPA recently developed Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Method 1615 uses a culturable virus assay with reduced equipment and labor costs compared to the costs associated with the ICR virus method and introduces a new molecular assay for the detection of enteroviruses and noroviruses by reverse transcription-quantitative PCR. In this study, we describe the optimization of several new components of the molecular assay and examine virus recovery from ground, reagent-grade, and surface water samples seeded with poliovirus type 3 and murine norovirus. For the culturable virus and molecular assays, mean poliovirus recovery using the complete method was 58% and 20% in groundwater samples, 122% and 39% using low-titer spikes in reagent-grade water, 42% and 48% using high-titer spikes in reagent-grade water, and 11% and 10% in surface water with high turbidity, respectively. Murine norovirus recovery by the molecular assay was 30% in groundwater samples, less than 8% in both low-and high-titer spikes in reagent-grade water, and 6% in surface water with high turbidity. This study demonstrates the effectiveness of Method 1615 for use with groundwater samples and highlights the need for further research into its effectiveness with surface water.

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

“…None of these resulted in a significant improvement in sample volume (data not shown). , 3) USEPA Lawrence and Lowell, MA study (unpublished data), 4) USEPA Mississippi River study (unpublished data), 5) USEPA drinking water treatment plant study (unpublished data), 6) USEPA Method 1615 evaluation study 25 , and 7) the first three months of the UCMR3 monitoring (unpublished data).…”

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

“…Murine norovirus was used as a surrogate in the method evaluation due to a lack of human norovirus stocks with a virus concentration sufficient for LFSM samples. For groundwater samples the mean poliovirus recovery was 20%, with a standard error of 2%, 14 while mean murine norovirus recovery was 30%, with a standard error of 3% (Figure 3). The regular field groundwater sample for each LFSM had no detectable enterovirus or norovirus.…”

“…The hepatitis G reagent used in this method satisfies this requirement by providing a constant level of viral RNA in all reactions and an RT-qPCR assay for estimating inhibition. When the best of the inhibitor removal approaches fail to remove inhibition, sample concentrates can be diluted as long as virus concentrations are higher than inhibitor concentrations 14,15 . The standard curve procedure described herein has both advantages and a major limitation.…”

“…The purpose of this paper is to describe the molecular assay component of EPA Method 1615 13,14 . This assay uses RT-qPCR to provide a quantitative estimate of enterovirus and norovirus genomic copies (GC) per liter based upon the original volume of the environmental or drinking water passed through an electropositive filter.…”

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

“…The purpose of this paper is to describe Method 1615's procedures for elution of five-inch electropositive cartridge filters, secondary concentration, and measurement of total culturable viruses. An evaluation of the overall method is described in Cashdollar et al 16 .…”