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

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

U.S. Environmental Protection Agency

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water.

Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. Detection and genotypic characterization of noroviruses is commonly performed by reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing. However, sequencing of products amplified from environmental water samples is often hindered by the co-amplification of non-specific cDNA. To overcome this issue, a generic microarray was evaluated to genotype noroviruses by probe hybridization. RT-PCR amplicons are used in a single base extension (SBE) reaction where genotype-specific probes are labeled and then hybridized to an Affymetrix GeneChip GenFlex Tag Array for detection. Using a standardized, multiplex SBE reaction, genotyping of representative strains was accomplished through the identification of genotype-specific patterns of positive hybridization results. Furthermore, the SBE-GenFlex array method was successful in the genotype identification of noroviruses seeded into tap and Ohio River water samples. This study shows the utility of using a microarray to genotype noroviruses in complex environmental matrices.

Development and evaluation of a generic tag array to detect and genotype noroviruses in water.

The 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.

Development and evaluation of EPA method 1615 for detection of enterovirus and norovirus in water.

Enteroviruses, noroviruses and adenoviruses are among the most common viruses infecting humans worldwide. These viruses are shed in the feces of infected individuals and can accumulate in wastewater, making wastewater a source of a potentially diverse group of enteric viruses. In this study, two procedures were evaluated to concentrate noroviruses, adenoviruses and enteroviruses from primary effluent of wastewater. In the first procedure, indigenous enteroviruses, noroviruses and adenoviruses were concentrated using celite (diatomaceous earth) followed by centrifugation through a 30K MWCO filter and nucleic acid extraction. The second procedure used celite concentration followed by nucleic acid extraction only. Virus quantities were measured using qPCR. A second set of primary effluent samples were seeded with Coxsackievirus A7, Coxsackievirus B1, poliovirus 1 or enterovirus 70 before concentration and processed through both procedures for recovery evaluation of enterovirus species representatives. The pairing of the single step extraction procedure with the celite concentration process resulted in 47-98% recovery of examined viruses, while the celite concentration process plus additional centrifugal concentration before nucleic acid extraction showed reduced recovery (14-47%). The celite concentration process followed by a large volume nucleic acid extraction technique proved to be an effective procedure for recovering these important human pathogens from wastewater.

Evaluation of methods using celite to concentrate norovirus, adenovirus and enterovirus from wastewater.

The objective of this study was to compare three nucleic acid extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approaches for norovirus (NoV) detection in drinking water with respect to performance, costs, and analysis time. The approaches evaluated were: (A) an approach that utilizes the QIAamp DNA Blood Mini Kit and multiplex primers and probes for detection; (B) a procedure which includes the NucliSENS Magnetic Extraction Kit and other components of a proposed European Union standard method for NoV detection in foods; and (C) a commercialized assay which uses NucliSENS extraction and Cepheid SmartCycler® technologies. Each approach was evaluated by most probable number (MPN) analysis for detection of GI.1 and GII.4 NoVs from human stool. Furthermore, recoveries of spiked primary effluent in tap water concentrates were compared for each approach. Few significant differences were observed between approaches with regard to performance. However, Approach C was the most time consuming and expensive to perform. This research presents a case study of how molecular-based approaches for detection of NoVs can be compared and how various factors may play a role in which approach laboratories choose to employ.

Nichole E. Brinkman

National Exposure Research Laboratory

U.S. Environmental Protection Agency

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

Nichole E. Brinkman

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U.S. Environmental Protection Agency

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

National Exposure Research Laboratory