Method 1615 

Measurement of Enterovirus and Norovirus 

Occurrence in Water by Culture and RT-qPCR

Office of Research and Development 

National Exposure Research Laboratory, Cincinnati, OH 

EPA/600/R-10/181 | December 2010 | www.epa.gov/ord
METHOD 1615. Enterovirus and Norovirus occurrence in water

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Cover: 

Top picture: Prairie Du Sac, WI Pump house, courtesy of Dr. Mark Borchardt 

Bottom left picture: norovirus, courtesy of Fred P. Williams; Bar = 50 nanometers 

Bottom right picture: poliovirus, courtesy of Fred P. Williams; Bar = 50 nanometers 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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METHOD 1615 

MEASUREMENT OF ENTEROVIRUS AND NOROVIRUS 

OCCURRENCE IN WATER BY CULTURE AND RT-qPCR 

Version 1.0 

December 2010 

AUTHORS 

G. S. Fout,1 N. E. Brinkman,1* J. L. Cashdollar,1* S. M. Griffin,1* B. R. McMinn,1* E. R. 

Rhodes,1* E.A. Varughese,1* M. R. Karim,1 A. G. Grimm,1 S. K. Spencer,2 and M. A. Borchardt2

U.S. Environmental Protection Agency, Office of Research and Development, 

National Exposure Research Laboratory, Cincinnati, OH;1 and USDA-Agricultural 

Research Service,2

 Marshfield, WI 

*NEB, JLC, SMG, BRM, ERR, and EAV contributed equally to the molecular method 

development 

DISCLAIMER

This method has been reviewed by the U.S. Environmental Protection Agency (EPA)’s Office of 

Research and Development (ORD) and approved for publication. Mention of trade names or 

commercial products does not constitute endorsement or recommendation for use. 

ACKNOWLEDGEMENTS 

The authors thank Drs. Sandhya Parshionikar, Keya Sen, and Carrie Miller from EPA’s 

Office of Water, and Drs. George Di Giovanni from the Texas AgriLife Research Center, El 

Paso, TX; Paul Hazelton from the University of Manitoba, Winnipeg, Manitoba, Timothy Straub 

from the Pacific Northwest National Laboratory, Richland, WA, and Susan Boutros from 

Analytical Associates, Ithaca, NY for reviewing this manuscript and for providing helpful 

comments. Mohammad R. Karim was supported through an appointment to the Research 

Participation Program at ORD’s National Exposure Research Laboratory, which was 

administered by the Oak Ridge Institute for Science and Education through an Interagency 

Agreement between the U.S. Department of Energy and EPA. The authors thank Dr. Skip 

Virgin of Washington University School of Medicine, St. Louis, MO for Murine norovirus. 

Questions regarding this method or its application should be addressed to: 

G. Shay Fout, Ph.D. 

U.S. Environmental Protection Agency 

Office of Research and Development 

National Exposure Research Laboratory 

Microbiological and Chemical Exposure 

 Assessment Research Division 

Cincinnati, OH 45268-1320 

(513) 569-7387 

(513) 569-7117 (fax) 

fout.shay@epa.gov 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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ABBREVIATIONS USED 

BGM Buffalo Green monkey kidney cells 

BSA Bovine serum albumin 

Cat. No. Catalog number 

cDNA Complementary DNA 

CL Confidence limit 

Ct Cycle threshold 

Cp Crossing point 

Cq Quantitation point 

CPE Cytopathic effect 

CV Coefficient of variation 

D Volume of original water sample assayed 

dH2O Distilled or deionized water 

DNA Deoxyribonucleic acid 

EPA United States Environmental Protection Agency 

EV Enteroviruses belonging to the genus, Enterovirus

FCSV Final concentrated sample volume 

GC Genome copy 

HGV Hepatitis G virus

ICR Information Collection Rule 

LIMS Laboratory information management system 

MEM Minimal essential medium 

MPN Most probable number 

MSDS Material Safety Data Sheet 

Negative FCSV Final concentrated sample volume from a negative QC sample 

NoV GI Genogroup I noroviruses belonging to the genus, Norovirus

NoV GII Genogroup II noroviruses belonging to the genus, Norovirus

NPT National pipe thread 

NTU Nephelometric Turbidity Units 

ORD Office of Research and Development 

OSHA Occupational Safety and Health Administration 

OW Office of Water 

PBS Phosphate buffered saline 

PCR Polymerase chain reaction 

PE Performance evaluation 

QC Quality control 

qPCR Quantitative polymerase chain reaction 

RT-PCR Reverse transcription-polymerase chain reaction 

RT-qPCR Reverse transcription-quantitative polymerase chain reaction 

RNA Ribonucleic acid 

RPM Revolutions per minute 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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RT Reverse transcription 

S Assay sample volume 

SOP Standard operating procedure 

TCVA Total cultural virus assay 

TSV Total sample volume 

U.S. United States 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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TABLE OF CONTENTS 

ABBREVIATIONS USED ................................................................................................ iii 

SCOPE AND APPLICATION ............................................................................................1 

BACKGROUND ...........................................................................................................1 

METHOD CONSTRAINTS ..........................................................................................3 

SUMMARY OF METHOD .................................................................................................3 

DEFINITIONS .....................................................................................................................3 

INTERFERENCES ..............................................................................................................6 

SAFETY ..............................................................................................................................7 

EQUIPMENT AND SUPPLIES ..........................................................................................8 

REAGENTS, MEDIA, AND STANDARDS ....................................................................12 

QUALITY ASSURANCE .................................................................................................16 

SAMPLE COLLECTION, PRESERVATION, AND STORAGE....................................20 

SAMPLE COLLECTION ............................................................................................20 

SHIPMENT OF SAMPLES ........................................................................................23 

LABORATORY HOLDING TIME AND TEMPERATURE ....................................24 

FILTER ELUTION PROCEDURE ...................................................................................24 

ELUTION EQUIPMENT SETUP ...............................................................................24 

ELUTION ....................................................................................................................25 

ORGANIC FLOCCULATION CONCENTRATION PROCEDURE ..............................26 

ORGANIC FLOCCULATION ....................................................................................26 

RECONCENTRATED ELUATE ................................................................................26 

TOTAL CULTURABLE VIRUS ASSAY ........................................................................29 

QUANTAL ASSAY ....................................................................................................29 

VIRUS QUANTITATION ..........................................................................................33 

ENTEROVIRUS AND NOROVIRUS MOLECULAR ASSAY ......................................34 

PRELIMINARY PROCEDURES ...............................................................................35 

TERTIARY CONCENTRATION ...............................................................................35 

NUCLEIC ACID ISOLATION ...................................................................................36 

REVERSE TRANSCRIPTION (RT) ..........................................................................37 

REAL-TIME QUANTITATIVE PCR (qPCR) ...........................................................38 

METHOD PERFORMANCE ............................................................................................42 

CULTURABLE ASSAY .............................................................................................42 

MOLECULAR PROCEDURE ....................................................................................43 

STERILIZATION AND DISINFECTION ........................................................................44 

TABLES AND FIGURES .................................................................................................47 

DATA SHEETS .................................................................................................................56

REFERENCES ..................................................................................................................62
METHOD 1615. Enterovirus and Norovirus occurrence in water

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LIST OF TABLES 

Table 1. Viruses Detected by EPA Method 1615 ..............................................................47

Table 2. Specified and Recommended Sample Volumes ..................................................47

Table 3. MPN Program Default Settings for MPN Calculator ..........................................48

Table 4. Primers and TaqMan® Probes for Virus Detection by RT-qPCR ........................48

Table 5. RT Master Mix 1 and 2 ........................................................................................49

Table 6. PCR Master Mix for Enterovirus Assay ..............................................................50

Table 7. PCR Master Mix for Norovirus GIA Assay ........................................................50

Table 8. PCR Master Mix for Norovirus GIB Assay.........................................................51

Table 9. PCR Master Mix for Norovirus GII Assay ..........................................................51

Table 10. PCR Master Mix for Hepatitis G Assay ............................................................52

Table 11. Mean Recovery and Coefficient of Variation Range .........................................52

LIST OF FIGURES

Figure 1. Uninfected BGM Cells .......................................................................................53

Figure 2. Early CPE from Poliovirus .................................................................................53

Figure 3. Sample Filtration Apparatus ...............................................................................54

Figure 4. Elution of an Electropositive Filter with Beef Extract .......................................54

Figure 5. RT-qPCR Schematic ..........................................................................................55
METHOD 1615. Enterovirus and Norovirus occurrence in water

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1. SCOPE AND APPLICATION

1.1. BACKGROUND 

1.1.1. Introduction 

1.1.1.1. EPA Method 1615 provides culture and molecular procedures 

for detecting human enteroviruses, human noroviruses, and 

mammalian orthoreoviruses (culture procedure only) in water 

(Table 1). The cell culture procedure detects enterovirus and 

orthoreovirus species that are capable of infecting and 

producing cytopathic effects (CPE) in the Buffalo Green 

monkey kidney (BGM) cell line (16, 19). Although this cell 

line is considered a “gold standard” for detection of infectious 

waterborne viruses, noroviruses and a number of enteroviruses 

do not replicate in BGM cells. There is no established cell line 

for detection of infectious human noroviruses, but a prototype 

research method is under development (37, 38). The molecular 

procedure incorporated into EPA Method 1615 detects the 

noroviruses and enteroviruses shown in Table 1, including 

those enteroviruses that do not replicate on BGM cells. 

However, it should be noted that a positive result indicates the 

presence of viral nucleic acid and not the infectivity status of 

the detected virus. 

1.1.1.2. Enteroviruses and noroviruses are enteric viruses that replicate 

within the gastrointestinal tract and are spread through the 

fecal-oral route. They cause a variety of waterborne infections 

through exposure to contaminated drinking and recreational 

waters. Infections may be asymptomatic or result in mild 

gastroenteritis, febrile illness, or respiratory symptoms. They 

can also cause a variety of serious diseases such as aseptic 

meningitis, encephalitis, flaccid paralysis, hand, foot and 

mouth disease, hemorrhagic conjunctivitis, myocarditis, 

neonatal sepsis-like disease, or severe gastroenteritis (3, 20, 

25). Enteroviruses and noroviruses have not only been found 

in drinking and recreational waters, but have caused 

waterborne outbreaks (4, 5, 18, 26, 27, 41). Due to public 

health concern, these viruses are on EPA’s Contaminant 

Candidate List 3 (http://www.epa.gov/safewater/ccl/ccl3.html). 

The Mammalian orthoreovirus species is not associated with 

any known waterborne outbreaks and usually does not cause 

disease in humans (13, 15). If desired, orthoreoviruses can be 

assayed using the molecular method found in Fout et al. (18). 

1.1.1.3. Molecular procedures, such as polymerase chain reaction 

(PCR) and reverse transcription-PCR (RT-PCR), provide the 

flexibility to detect all waterborne human enteric viruses for 

which genome sequence data is available (18). The advent of 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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real time quantitative PCR (qPCR) has resulted in additional 

advantages over conventional PCR in that quantitative results 

can be obtained in a very short time (21). These molecular 

methods have been widely used to detect viruses in 

environmental waters (7, 8, 22, 27, 28, 33, 39). Despite the 

advantages, molecular techniques are subject to three main 

limitations. First, PCR methods assay smaller volumes than 

culture methods, resulting in lower detection limits. Second, 

these methods are sensitive to inhibitors that are present in 

some environmental samples. To address this problem, 

controls are used to determine whether negative results are true 

negative or false negative values. Finally, molecular methods 

do not distinguish between infectious and noninfectious 

viruses. Therefore, a positive PCR assay for a particular 

pathogen in drinking water does not directly address issues of 

public health. Research is ongoing on several promising 

approaches to detect infectious viruses (31, 34). However, 

PCR is still a useful public health tool in spite of these 

problems. Because there is a strong relationship between 

indicator measurements by qPCR and health effects in 

recreational waters (40), EPA is considering using qPCR to set 

new criteria for monitoring recreational beaches. At the very 

least, positive PCR virus findings provide a warning of 

possible contamination issues, but recent studies have indicated 

a direct relationship between health effects and positive RTqPCR findings for human viruses (Borchardt et al., manuscript 

in preparation). 

1.1.2. Development of the ICR Total Culturable Virus Assay - In the 1990s EPA 

issued an Information Collection Rule (ICR; Federal Register 61:24353-

24388) that required all drinking water utilities serving a population over 

100,000 to monitor their source water for viruses monthly for a period of 

18 months. The Rule also required that systems finding greater than one 

infectious enteric virus particle per liter of source water to monitor their 

finished water on a monthly basis. One of the purposes of the Rule was to 

obtain national data on virus levels in source waters to determine the 

adequacy of treatment requirements. To support the Rule, a virus 

monitoring protocol was developed by virologists at the EPA and 

modified to reflect consensus agreements from the scientific community 

and public comments to the draft rule (19). This standard ICR Total 

Culturable Virus method, along with quality assurance and laboratory 

approval procedures (http://www.epa.gov/microbes/icrmicro.pdf), was 

incorporated into the ICR by reference. The results of the ICR survey 

indicated that culturable viruses were present in 24% of the source waters 

throughout the nation. Since the end of the ICR, the ICR Total Culturable 

Virus method has continued to be used in the U.S. and in international 

settings for the detection of culturable viruses in surface, ground, and 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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treated waters (14, 19, 24), but the high cost of collecting and analyzing 

virus samples has limited the method’s widespread use. 

1.1.3. Development of Method 1615 – In the past few years, an alternative 

sampling protocol that significantly reduces the cost of sampling has been 

found to be equivalent in performance to the ICR method (24). Method 

1615 is a modification of the ICR protocol. It incorporates the alternative 

sampling procedure and reduces the number of required cell culture 

replicates required by the ICR protocol. It also includes a molecular 

procedure that is a modification of a method used to survey groundwaters 

for enteric viruses in Wisconsin (9, 27). 

1.2. METHOD CONSTRAINTS 

1.2.1. This method is for use by analysts skilled in virus concentration, elution, 

cell culture, and molecular techniques. 

1.2.2. Analysts must not deviate from any of the procedures described in this 

method if the data is being generated to fulfill EPA regulatory 

requirements. For example, alternative procedures for elution, secondary 

and tertiary concentration, and analyses by culture and RT-qPCR must not 

be used without prior approval by EPA. 

2. SUMMARY OF METHOD

Viruses that may be present in environmental or finished drinking waters are concentrated 

by passage through electropositive filters. Viruses are eluted from the filters with a beef 

extract reagent and concentrated using organic flocculation. A portion of the concentrated 

eluate is then inoculated onto replicate flasks of BGM cells to measure infectious viruses. 

Cultures are examined for the development of cytopathic effects for two weeks and then repassaged onto fresh cultures for confirmation. Virus concentration in each sample is 

calculated in terms of the most probable number (MPN) of infectious units per liter using 

EPA’s MPN calculator. For molecular assays, the concentrated eluate is concentrated 

again by centrifugal ultrafiltration. The RNA is extracted from the concentrate and tested 

for enterovirus and norovirus RNA using real time quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR). Virus concentrations for the molecular assay are 

calculated in terms of genomic copies of viral RNA per liter based upon a standard curve.

3. DEFINITIONS

3.1. ANALYSIS BATCH 

3.1.1. All virus samples processed by an analyst within one week's span shall be 

considered a "batch". A week is defined as a 7-day period. 

3.1.2. Each sample result must be associated with a batch number. 

3.1.3. A positive and negative Quality Control (QC) sample set must be 

processed with each batch. 

3.1.3.1. Failure to obtain both a positive value in the positive QC 

sample and a negative value in the negative QC sample results 

in failure of the entire batch. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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3.1.3.2. Obtaining a positive value in the positive QC sample and a 

negative value in the negative QC sample results in acceptance 

of the data from the entire batch. 

3.2. BUFFALO GREEN MONKEY KIDNEY (BGM) CELLS 

This is a stable cell line of monkey kidney cells that were originally developed at 

the University of Buffalo for clinical isolation of enteroviruses and later adapted for 

use in detecting infectious viruses in environmental samples (16). BGM cells form 

a monolayer of cells when propagated in tissue culture vessels. Figure 1 is a 

micrograph of uninfected BGM cells growing as a monolayer. 

3.3. CONTAMINANT CANDIDATE LIST (CCL) 

A list of chemicals and microbial agents under consideration for regulatory action 

by EPA. The current list may be obtained from 

http://water.epa.gov/scitech/drinkingwater/dws/ccl/. 

3.4. CYTOPATHIC EFFECT (CPE) 

The degeneration of cells caused by virus replication. It often involves the 

complete disintegration of cells. It may also be identified through changes in cell 

morphology. However, care must be taken in the use of changes in cell 

morphology as evidence of CPE, because uninfected BGM cells change 

morphology during mitosis. True CPE is always progressive and can be rated on a 

0-4 scale, with the values 0, 1, 2, 3, and 4 indicating that 0% (Figure 1), 25% 

(Figure 2), 50%, 75%, and 100% of the monolayer is showing CPE, respectively. 

Additional examples of CPE can be found in Malherbe and Strickland-Cholmley 

(29). 

3.5. CYTOTOXICITY 

Cytotoxicity is the development of CPE from toxic components in the matrix. 

Cytotoxicity can be distinguished from viral CPE by its early development after 

sample inoculation or by the failure to observe CPE in the second passage required 

by this method. Unlike CPE, which begins as small clusters of killed cells (see 

Figure 2) after two or more days of incubation, cytotoxicity usually develops 

uniformly in all inoculated cell culture replicates within 24 hours of inoculation. 

3.6. DETECTION LIMIT 

The number of virus particles or genome copy numbers that can be detected in a 

given volume by a method with 95% confidence. 

3.7. ENTERIC VIRUSES 

Viruses that primarily infect and replicate in the gastrointestinal tract are known as 

enteric viruses. These include enteroviruses, noroviruses, rotaviruses, hepatitis A 

virus, adenoviruses, and reoviruses, among others. Enteric viruses are capable of 

causing a wide range of illnesses such as gastroenteritis, paralysis, aseptic 

meningitis, respiratory illness, fevers, myocarditis, and neonatal enteroviral sepsis. 

Enteric viruses can be present in human and animal feces, which can contaminate 

recreational and drinking water sources. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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3.8. ENTEROVIRUS 

Enteroviruses are a genus in the Picornaviridae family. These viruses are among 

the most common viruses infecting humans worldwide. Enteroviruses are small 

(approximately 30 nm), nonenveloped, single-stranded, positive sense RNA viruses 

with an icosahedral capsid. Traditionally, human enterovirus serotypes have been 

classified into echoviruses, coxsackieviruses group A and B, and polioviruses. 

Current taxonomy based on molecular typing divides human enteroviruses into four 

species, Human enterovirus A, B, C, and D. 

3.9. GROWTH AND MAINTENANCE MEDIUM 

Growth medium consisting of a 50/50 (v/v) mixture of Minimum Essential Medium 

(MEM) and Leibovitz's L-15 medium with antibiotics and 10% bovine serum is 

recommended. Maintenance medium consists of a 50/50 mixture of Eagle's 

minimum essential medium (MEM) and Leibovitz's L-15 medium with antibiotics 

and 2% bovine serum. 

3.10. INOCULATION 

Inoculation is the placement of concentrated water samples onto a monolayer of 

cells in a culture vessel for growing or replication of viruses in the cells. 

3.11. MATERIAL SAFETY DATA SHEETS (MSDS) 

These sheets contain written information provided by vendors concerning a 

chemical’s toxicity, health hazards, physical properties, fire, and reactivity data, 

including storage, spill, and handling precautions. 

3.12. MONOLAYER 

A single confluent layer of cells covering the bottom of a tissue culture dish or flask 

(Figure 1). 

3.13. NOROVIRUS 

Noroviruses constitute a genus in the Caliciviridae family. The genus is divided 

into five genogroups (GI-GV) and 29 genetic clusters (42). Noroviruses are 

recognized as a leading cause of nonbacterial gastroenteritis in humans. 

Noroviruses are small (approximately 27 nm) and the genome possess a positive 

sense, single stranded RNA in a nonenveloped icosahedral capsid. Due to the 

absence of a standardized and validated infectivity assay for human noroviruses, the 

presence of noroviruses in environmental waters must be measured using molecular 

methods. 

3.14. PERFORMANCE EVALUATION/PERFORMANCE TEST SAMPLE (PE) 

A sample containing Sabin poliovirus type 3 at a concentration unknown to the 

analysts. PE samples are typically sent to all laboratories performing the method 

from a single outside source when performance evaluation is required by EPA. The 

purpose of the PE sample is to demonstrate that all analysts performing the method 

can meet minimum performance standards. 

3.15. QUALITY CONTROL SAMPLE (QC) 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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This is a sample containing Sabin poliovirus type 3 at a known concentration. QC 

samples will typically be sent to all laboratories performing the method from a 

single outside source when performance evaluation is required by EPA. The 

purposes of the QC sample are to give laboratories a standard sample for training 

new analysts and to give EPA and laboratory QA officials a tool to evaluate method 

performance for all laboratory analysts. 

3.16. QUANTITATIVE CYCLE [(Cq), also called Cycle Threshold (Ct) or Crossing 

Point (Cp)] 

The cycle at which the fluorescence of a quantitative PCR assay crosses the 

threshold that defines a positive reaction or at which the second derivative 

maximum is reached (10, 11). 

3.17. QUANTITATIVE POLYMERASE CHAIN REACTION (qPCR) and 

REVERSE TRANSCRIPTION-qPCR (RT-qPCR) 

This is a procedure for quantitatively detecting the levels of specific DNA in a 

sample, or following reverse transcription [RT, e.g., the synthesis of complementary 

DNA (cDNA) from RNA], the levels of specific RNA (e.g., viral) in a sample. 

3.18. REAGENT WATER 

This is deionized or distilled reagent grade water (dH2O) with a resistivity greater 

than 1 Siemens per meter (S/m), i.e., 1 megohms-cm at 25oC; if available, reagent 

grade water with a resistivity greater than 0.1 S/m (10 megohms-cm) is preferred 

(2). 

3.19. STANDARD OPERATING PROCEDURE 

A Standard Operating Procedure (SOP) is a set of written instructions that document a 

routine or repetitive activity followed by an organization. The development and use of 

SOPs are an integral part of a successful quality system as it provides individuals with 

the information to perform a job properly, and facilitates consistency in the quality and 

integrity of data. EPA guidance on developing SOPs can be obtained from 

www.epa.gov/quality/qs-docs/g6-final.pdf.

4. INTERFERENCES

4.1. REAGENTS 

To minimize cross contamination, Analytical Reagent or ACS grade chemicals 

(unless specified otherwise) and reagent water should be used to prepare all media 

and reagents. It is recommended that water, media, and other reagent solutions be 

purchased from commercial sources and that tissue culture grade water be used for 

preparation of tissue culture media. 

4.2. MATRIX INTERFERENCE 

Matrix interferences may lead to false negative results and are caused by colloidal, 

suspended, or dissolved substances that are present in the water sample. Matrix 

interference can vary across different water sources and even across time in the 

same source. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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4.2.1. Matrix interference due to colloidal or suspended solids may reduce the 

water volume that can be passed through the positively charged filters 

used in this method. Prefilters (item 6.1.5) or more than one 

electropositive filter must be used to overcome this type of interference. 

4.2.2. Matrix interference may be identified by its effects on the culture or 

molecular assays. This may be expressed as the development of 

cytotoxicity in culture assays and or by inhibition of molecular assays. 

4.3. OTHER INTERFERENCE 

4.3.1. Failure to dechlorinate treated tap water samples during sampling or 

prolonged exposure to ambient temperatures during sample transportation 

or in the laboratory can lead to virus loss. 

4.3.2. Inadequate disinfection of the sampling apparatus and contamination of 

reagents and supplies can lead to sample contamination. Inadequate 

disinfection of the sampling apparatus is identified using negative Quality 

Control samples (Section 8.4.3). 

4.3.3. Inadequate physical separation and controlled workflow may lead to PCR 

interference due to false positive results from contamination. EPA’s 

guidance for processing and handling these samples must be followed to 

minimize this interference (36). 

5. SAFETY

5.1. SAFETY PLAN 

The biohazard associated with, and the risk of infection from, human enteric viruses 

is high in this method because potentially infectious viruses are handled. This 

method does not purport to address all the safety issues associated with its use. 

Each laboratory is responsible for establishing a safety plan that addresses 

appropriate safety and health practices prior to using this method. In particular, 

laboratory staff must know and observe the safety procedures required in a 

microbiology laboratory that handles pathogenic organisms while preparing, using, 

and disposing of sample concentrates, reagents and materials, and while operating 

sterilization equipment. Minimum requirements have been published by the U.S. 

Department of Health and Human Services (1). 

5.2. SHIPMENT OF SAMPLES 

The samples collected using this method may be shipped as non-infectious 

materials, unless they are known to contain virus or other infectious materials. If 

they are known to contain infectious materials, laboratories are responsible for 

packaging and shipping them according to all Department of Transportation, 

Centers for Disease Control and Prevention, and State regulations. 

5.3. CHEMICAL SAFETY 

Each laboratory is responsible for the safe handling of the chemicals used in this 

method. The OSHA laboratory standards can be found on line at 

http://www.osha.gov/SLTC/laboratories/standards.html. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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6. EQUIPMENT AND SUPPLIES

References to specific brands or catalog numbers are included as examples only and do not 

imply endorsement of the product. These references do not preclude the use of other 

vendors or supplies. However, equivalent method performance should be demonstrated for 

substitutions of the electropositive filter, beef extract, and qPCR reagents. All equipment 

should be cleaned according to the manufacturers’ recommendations. Disposable supplies 

should be used wherever possible to reduce the possibility of cross contamination. Note: 

Storage of samples and reagents at temperatures below 0°C must be done using manual 

defrost freezers. 

6.1. SAMPLE FILTRATION APPARATUS 

Figure 3 shows the Sample Filtration Apparatus which has been modified from that 

given in Fout et al. (19) for use with the NanoCeram® electropositive cartridge 

filter. The modification also increases the efficacy for disinfecting the apparatus. 

The sample filtration apparatus shown in Figure 1 is recommended, but that shown 

in Figure VIII-1 of Fout et al. (19) also may be used for virus monitoring. The 

apparatus may have to be modified using appropriate size housings and adapters for 

use with other equivalent filters, such as the ten-inch 1MDS Virosorb cartridge 

filter. The current configuration does not use a pressure regulator or pressure 

gauge, as these components are difficult to disinfect and subject to corrosion; 

however, laboratories are responsible for ensuring that water pressure at sampling 

sites does not exceed the pressure ratings of the cartridge housings used (125 psi for 

item 6.1.2.2). 

6.1.1. Intake Module 

6.1.1.1. Backflow regulator (Watts Regulator Series 8 C Hose 

Connection Vacuum Breaker; this component is optional) 

6.1.1.2. Swivel female insert with garden hose threads (United States 

Plastic Cat. No. 63003) 

6.1.1.3. 1⁄2” tubing (Cole-Parmer Cat. No. 06601-03) and hose clamps 

(Cole-Parmer Cat. No. 06403-10) 

6.1.1.4. 1⁄2” hose barb quick disconnect body (Cole Parmer Cat. No. 

31307-11) 

6.1.1.5. Thread tape (Cole Parmer Cat. No. 08270-34) 

6.1.2. Cartridge Housing Module for NanoCeram filters 

6.1.2.1. 1⁄2” NPT (M) quick disconnect insert (Cole Parmer Cat. No. 

31307-31; connected to the inlet port of the cartridge housing) 

6.1.2.2. Cartridge housing (Argonide Cat. No. H2.5-5) 

6.1.2.3. 1⁄2” NPT (M) quick disconnect body (Cole Parmer Cat. No. 

31307-06; connected to the outlet port of the cartridge housing) 

6.1.2.4. 5” NanoCeram cartridge filter (Argonide Cat. No. VS2.5-5). 

6.1.3. Discharge Module 
METHOD 1615. Enterovirus and Norovirus occurrence in water

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6.1.3.1. 1⁄2” NPT (M) quick disconnect insert (Cole Parmer Cat. No. 

31307-31) 

6.1.3.2. 1⁄2” NPT (F) straight connector (Cole Parmer Cat. No. 06349-

03) 

6.1.3.3. Flow meter (Flow Technology Cat. No. FT6-8NENWULEG-3) 

– Note: the flow meter calibration at the flow rates used for 

sampling should be confirmed before use and after every 

month of use. The calibration can be performed by measuring 

the time to fill a 4 L or larger graduated cylinder (Cole Parmer 

Cat. No. 06135-90; e.g., 24± 1 second for the 10 liter per 

minute rate). 

6.1.3.4. Rate/Totalizer (Flow Technology Cat. No. BR30-5-A-4) 

6.1.3.5. 1⁄2” NPT (F) straight connector (Cole Parmer Cat. No. 06349-

03) 

6.1.3.6. 3⁄4” NPT (M) × 1⁄2” NPT (M) reduction nipple (Cole Parmer 

Cat. No. 06349-87) 

6.1.3.7. 3⁄4” NPT (F) bronze globe valve (Cole Parmer Cat. No. 98675-

09) 

6.1.3.8. 3⁄4” NPT (M) × GHT(M) fitting (United States Plastic Cat. No. 

63016) 

6.1.3.9. A garden hose of sufficient length to reach a drain. 

6.1.4. Injector module – this module, prepared using the components below, 

should only be used when it is necessary to add thiosulfate or other 

additives to water during sampling. It can be modified into a double 

injector module for the addition of more than one additive by installing a 

small 1⁄4” NPT (F) TEE (Cole Parmer Cat. No. 06349-51) on top of the 

larger TEE (item 6.1.4.2) via the male reducer (item 6.1.4.4) and placing 

items 6.1.4.5 and 6.1.4.6 on each end of the smaller TEE using a 1⁄4” NPT 

(M) Nipple (Cole Parmer Cat No. 30623-25). 

6.1.4.1. 3⁄8” NPT (M) quick disconnect insert (Cole Parmer Cat. No. 

31307-30, attached to the left port of the TEE) 

6.1.4.2. 3⁄8” NPT (F) TEE (Cole Parmer Cat. No. 06482-84) 

6.1.4.3. 3⁄8” NPT (M) quick disconnect body (Cole Parmer Cat. No. 

31307-05, attached to the right port of the TEE) 

6.1.4.4. 3⁄8” NPT (M) × 1⁄4” NPT (M) male reducer (Cole Parmer Cat. 

No. 30623-42; this and following components are connected to 

the top port of the TEE) 

6.1.4.5. 1⁄4” NPT (F) metallic check valve (CV; Cole Parmer Cat. No. 

98676-00) 
METHOD 1615. Enterovirus and Norovirus occurrence in water

10 

6.1.4.6. 1⁄4” NPT (M) × 1⁄4” tubing ID male pipe adaptor elbow (Cole 

Parmer Cat. No. 30622-97) 

6.1.4.7. 15-gallon chemical tank (Grainger Cat. No. 4UP09) with 

tubing. This item is for injecting 2% thiosulfate (item 7.3) into 

water containing a disinfectant. The container size can be 

adjusted to meet the anticipated need. 

6.1.4.8. Chemical metering pump (Grainger Cat. No. 4UP03; before 

first use, adjust the chemical metering pump to deliver 2.4 or 6 

mL/minute (i.e., 0.6 mL × L of disinfected water passing 

through the sample filtration apparatus each minute) for flow 

rates of 4 or 10 L/minute, respectively. Using a graduated 

cylinder to measure the flow rate, and record or mark the pump 

setting for each rate. 

6.1.5. Prefilter module – for use with waters exceeding 75 nephelometric 

turbidity units (NTU) or for other conditions that prevent the minimum 

sampling volumes from being obtained. Note: The NanoCeram filter is 

more susceptible to clogging than the 1MDS filter. A prefilter module 

may be required for some matrices even when the turbidity is considerably 

below 75 NTU. 

6.1.5.1. Prepare the prefilter module as described step 6.1.2, except use 

the filter described in step 6.1.5.2 

6.1.5.2. 10 μm Polypropylene Prefilter Cartridge (Parker Hannifin Cat. 

No. M19R5-A) 

6.1.6. Assemble the modules as shown in Figure 3 using thread tape (item 

6.1.1.5) on all connections. Sterilize the regulator, prefilter housing, and 

cartridge housings with chlorine as described in Section 15. Using aseptic 

technique, add a sterile NanoCeram or 1MDS cartridge to the cartridge 

housing and, if needed, a presterilized polypropylene cartridge to the 

prefilter housing. Cover the ends with sterile aluminum foil. 

6.2. OTHER SAMPLING AND SHIPPING MATERIALS 

6.2.1. Peristaltic or chemical resistant pump capable of pumping water at 4-10 

liters/min and appropriate connectors (for use where garden hose-type 

pressurized taps for the source or finished water to be monitored are 

unavailable and for QC samples). Follow the manufacturer's 

recommendations for pump priming. 

6.2.2. One L polypropylene wide-mouth bottles (Nalgene Cat. No. 2104-0032) 

6.2.3. Collapsible 10 L LDPE cubitainer (Cole Parmer Cat. No. 06100-30) 

6.2.4. Portable pH and temperature probe (Omega Cat. No. PHH-830) 

6.2.5. Portable turbidity meter (Omega Cat. No. TRB-2020) 

6.2.6. Portable Chlorine (Free & Total), Pocket Colorimeter II Test Kit with 

reagents (Hach Cat. No. 5870062). 
METHOD 1615. Enterovirus and Norovirus occurrence in water

11 

6.2.7. Commercial ice packs (Cole-Parmer Cat. No. 06345-20) 

6.2.8. iButtons (Maxim Cat. No. DS1921G) 

6.2.9. Insulated shipping box with carrying strap (17" × 17" × 16"; Cole-Parmer 

Cat. No. 03742-00 and 03742-30) or insulated storage and transport chest 

(Fisher Scientific Cat. No. 39) 

6.2.10. Miscellaneous - aluminum foil, surgical gloves, waterproof marker

6.3. EQUIPMENT AND SUPPLIES FOR THE ORGANIC FLOCCULATION 

PROCEDURE 

6.3.1. Dispensing pressure vessels, 5 and 20 L capacity (Millipore Corp. Cat. 

No. XX6700P05 and XX6700P20) 

6.3.2. Pressure source - laboratory positive pressure air line (equipped with oil 

filter), compressed nitrogen, peristaltic pump (e.g., Cole Parmer Cat. No. 

07523-60), or self-priming pump (e.g., Cole Parmer Cat. No. 07036-10) 

6.3.3. pH meter with combination-type electrode and an accuracy of at least 0.1 

pH units 

6.3.4. Magnetic stirrer and stir bars 

6.3.5. Refrigerator, set at 4oC ± 3oC 

6.3.6. Refrigerated centrifuge capable of attaining 4,000 ×g 

6.3.6.1. Screw-capped centrifuge bottles with 100 to 1000 mL capacity. 

Each bottle must be rated for the relative centrifugal force 

used. 

6.3.6.2. Appropriate centrifuge buckets for centrifuge bottles 

6.3.7. Sterilizing filter – 0.22 μm Acrodisc filter with prefilter (VWR Cat. No. 

28143-295) for use at step 11.2.3.8. 

6.3.8. 47 mm disc filter holder (Millipore Corp. Product No. SX0004700) 

6.3.9. Sterilizing filter stack – Place a 0.22 μm pore-size membrane filter 

(Millipore Corp. Product No. GSWP04700) on the bottom of a 47 mm 

disc filter holder. 

6.3.9.1. Place an AP15 prefilter (Millipore Corp. AP1504700) on top of 

the 0.22 μm filter and an AP20 prefilter (Millipore Corp. 

AP2004700) on top of the AP15 prefilter. 

6.3.9.2. Assemble the filter holder unit and sterilize as defined in 

section 15.2.2. 

6.3.9.3. Note: Disassemble the filter stack after each use to check the 

integrity of the 0.22 μm filter. Refilter any media filtered with 

a damaged stack using another sterile sterilizing filter stack. 

6.3.9.4. Note: The sterilizing filter stack is optional, but should be used 

for samples that are difficult to filter using item 6.3.7. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

12 

6.3.10. Sterilizing filter – 0.22 μm (Corning Cat. No. 431219) for use at step 

12.1.4.2. 

6.4. EQUIPMENT AND SUPPLIES FOR THE TOTAL CULTURABLE VIRUS 

ASSAY 

6.4.1. Mechanical rocking platform (Daigger Product No. EF4907G) 

6.4.2. Incubator capable of maintaining the temperature of cell cultures at 

36.5±1°C 

6.4.3. Flasks or bottles for BGM culture maintenance and quantal assays (e.g., 

2100 cm2

 roller bottles, Fisher Cat. No. 73520-420; 850 cm2 roller bottles, 

Corning Cat. No. 430849; 25 cm2 flasks, Sigma Cat. No. C6231) 

6.5. EQUIPMENT FOR THE TERTIARY CONCENTRATION PROCEDURE 

AND THE MOLECULAR ASSAYS 

6.5.1. UV-Vis Spectrophotometer (Thermo Fisher Scientific Cat. No. NanoDrop 

2000) 

6.5.2. Vivaspin 20 – 30,000 MWCO (Sartorius-Stedim Cat. No. VS2022; other 

centrifugal concentrators with 30,000 MWCO may be substituted for this 

item, if equivalent recoveries are demonstrated) 

6.5.3. 50 mL polypropylene centrifuge tube and appropriate buckets for 

centrifuge (item 6.3.6) 

6.5.4. Microcentrifuge capable of 20,000 x g (Fisher Scientific Cat. No. 05-406-

11) 

6.5.5. 1.5 mL microcentrifuge tubes (Fisher Scientific Cat. No. 02-682-550) 

6.5.6. Vortex mixer (Fisher Scientific Cat. No. 02-216-100) 

6.5.7. Thermal cycler (Applied Biosystems Cat. No. 4314879) 

6.5.8. Optical reaction plate (Applied Biosystems Cat. No. 4314320) 

6.5.9. Real-Time PCR system (Applied Biosystems Cat. No. 4351405) 

7. REAGENTS, MEDIA, AND STANDARDS

The amount of reagents, media, and standards prepared for each step of the method may be 

adjusted proportionally to the number of samples to be analyzed. For any given section of 

this method only media, reagents, and standards that are not described in previous sections 

are listed. 

7.1. REAGENTS FOR THE SAMPLING PROCEDURE 

7.1.1. Hydrochloric acid (HCl) – Prepare 0.12, 1.2, and 6 M solutions by mixing 

50, 100, or 50 mL of concentrated HCl (Note: HCl at 37% concentration 

is about 12 M) with 4950, 900, or 50 mL of dH2O, respectively. Prepare 

solutions to be used for adjusting the pH of water samples at least 24 h 

before use. HCl solutions can be stored for several months at room 

temperature. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

13 

7.1.2. QC stock – This is a stock of Sabin poliovirus type 3 containing 200 

MPN/mL. This stock may be prepared by the analytical laboratory or, if 

available, obtained from a contractor designated by the US EPA. 

7.1.3. Matrix spike – This is a stock of Sabin poliovirus type 3 containing 1000 

MPN/mL. This stock may be prepared by the analytical laboratory or, if 

available, obtained from a contractor designated by the US EPA. 

7.1.4. Sodium hypochlorite (NaClO) – Prepare a 0.525% NaClO solution by 

diluting household bleach 1:10 in water. 0.525% NaClO solutions can be 

stored for one week at room temperature. 

7.1.5. 1 M sodium thiosulfate (Na2S2O3) pentahydrate – Prepare a 1 M solution 

by dissolving 248.2 g of Na2S2O3 in 1 liter of dH2O. Sodium thiosulfate 

solutions may be stored for six months at room temperature. 

7.1.6. 2 % sodium thiosulfate (Na2S2O3) pentahydrate – Prepare 2% thiosulfate 

by dissolving 1 kg of sodium thiosulfate pentahydrate into 49 L of sterile 

water. Sodium thiosulfate solutions may be stored for six months at room 

temperature. 

7.2. REAGENTS FOR THE ELUTION AND ORGANIC FLOCCULATION 

PROCEDURES 

7.2.1. Sodium hydroxide (NaOH) – Prepare 0.1, 1, and 5 M solutions by 

dissolving 0.4, 4, or 20 g of NaOH in a final volume of 100 mL of dH2O, 

respectively. NaOH solutions may be stored for several months at room 

temperature. 

7.2.2. 0.5% iodine – Prepare a 0.5% iodine solution by dissolving 5 g of iodine 

in 1 L of 70% ethanol. Iodine solutions can be stored for one year at room 

temperature. 

7.2.3. 1.5% beef extract, pH 9.0 

7.2.3.1. Prepare buffered 1.5% beef extract by dissolving 30 g of beef 

extract, desiccated (BD Bacto Cat. No. 211520) powder and 

7.5 g of glycine (final glycine concentration = 0.05 M) in 1.9 L 

of dH2O. 

7.2.3.2. Adjust the pH to 9.0 with 1 or 5 M NaOH and bring the final 

volume to 2 L with dH2O. 

7.2.3.3. Autoclave the beef extract solution at 121°C for 15 minutes 

and use at room temperature. Beef extract solutions may be 

stored overnight at room temperature or 4°C, for one week at 

4°C, or for longer periods at -20°C. 

7.2.3.4. Warning: Desiccated beef extract lots show considerable 

variation in virus recovery. Therefore, each new lot of beef 

extract must be screened before use in the Organic Flocculation 

Concentration Procedure to determine whether virus recoveries 

are adequate. Perform the screening by spiking one liter of 
METHOD 1615. Enterovirus and Norovirus occurrence in water

14 

beef extract solution with 1 mL of a QC sample. Process the 

spiked sample according to the Organic Flocculation and Total 

Culturable Virus Assay procedures given below. The mean 

recovery of poliovirus for three trials should be greater than 

50%. 

7.2.4. 1.5% beef extract, pH 7.0 – 7.5 - Prepare 1.5% beef extract by dissolving 

7.5 g of beef extract, desiccated powder and 1.88 g of glycine in 0.5 L of 

dH2O. Autoclave the beef extract solution at 121°C for 15 minutes and 

use at room temperature. This beef extract solution may be stored for up 

to six months at room temperature, but must be discarded if there is 

evidence of microbial growth or any other change in appearance. 

7.2.5. 0.15 M sodium phosphate, pH 9.0 – Prepare 0.15M sodium phosphate by 

dissolving 40.2 g of sodium phosphate, dibasic (Na2HPO4 • 7H2O) in a 

final volume of 1000 mL dH2O. Adjust the pH to 9.0 with HCl, if 

necessary. Autoclave at 121°C for 15 minutes. Sodium phosphate 

solutions (items 7.9 and 7.10) may be stored at room temperature for up to 

12 months. 

7.2.6. 0.15 M sodium phosphate, pH 7.0-7.5 – Prepare by dissolving 40.2 g of 

sodium phosphate, dibasic (Na2HPO4 • 7H2O) in a final volume of 1000 

mL dH2O. Adjust the pH to 7.0 to 7.5 with HCl. Autoclave at 121°C for 

15 minutes. 

7.2.7. Antifoam (Sigma Cat. No. A8311) 

7.3. REAGENTS FOR THE TOTAL CULTURABLE VIRUS ASSAY 

7.3.1. Cell culture test vessels containing confluent monolayers of BGM cells – 

BGM cells between passages 117 and 250 must be used. Cells should be 

passaged and maintained using the standard procedures available in the 

most recent version of the USEPA Manual of Methods for Virology (6), 

available at http://www.epa.gov/microbes/about.html. BGM cells from 

various sources and other standard tissue culture techniques and media 

may be used as long as analysts meet the acceptance criteria listed in 

section 14. Cell cultures used for virus assay are generally found to be at 

their most sensitive level between the third and sixth days after their most 

recent passage; those older than seven days must not be used. 

7.3.2. Cell culture media 

7.3.2.1. Hank’s balanced salt solution (Invitrogen Cat. No. 14170-112) 

7.3.2.2. Fetal bovine serum, certified, heat-inactivated (Invitrogen Cat. 

No. 10082-139) 

7.3.2.3. Trypsin with EDTA (Invitrogen Cat. No. 25300-062) 

7.3.2.4. Fungizone (Invitrogen Cat. No. 15290-018), PenicillinStreptomycin (Invitrogen Cat. No. 15140-122) 
METHOD 1615. Enterovirus and Norovirus occurrence in water

15 

7.3.2.5. Minimum essential medium (MEM) with Hanks' salts and Lglutamine (Sigma Aldrich Cat. No. M1018) 

7.3.2.6. Leibovitz's L-15 medium with L-glutamine (Sigma Aldrich 

Cat. No. L4386). 

7.3.2.7. 7.5% Sodium bicarbonate (Sigma Aldrich Cat. No. S8761) 

7.3.2.8. Trypan blue (Sigma-Aldrich Cat. No. T8154) 

7.3.3. Positive Assay Control – Prepare by diluting the QC stock in sodium 

phosphate, pH 7.0 - 7.5, to give a concentration of 20 PFU per Inoculation 

Volume (see step 11.2.4.5 for a definition of Inoculum Volume). 

7.4. REAGENTS FOR THE TERTIARY CONCENTRATION PROCEDURE 

AND THE MOLECULAR ASSAYS 

7.4.1. Dulbecco’s Phosphate Buffered Saline, without CaCl2 and MgCl2 (U.S 

Biological Cat. No. D9820) 

7.4.2. BSA, crystalline (United States Biochemical Cat. No. 10856) 

7.4.3. 0.2 μm sterilizing filter (Sigma Cat. No. F-9768) 

7.4.4. 5% BSA – Prepare 5% BSA by dissolving 5 g of in 100 mL of dH2O. 

Sterilize by passing the solution through a sterilizing filter (item 7.4.3). 

Store at 4°C. 

7.4.5. PBS, 0.2% BSA – Prepare by adding 4 mL of 5% BSA to 96 mL of PBS. 

Sterilize by passing the solution through a 0.2 μm sterilizing. Store at 

4°C. 

7.4.6. QIAamp DNA Blood Mini Kit (Qiagen Cat. No. 51104 or 51106) 

7.4.7. Buffer AVL (Qiagen Cat. No. 19073; carrier RNA is supplied with this 

reagent) 

7.4.8. Buffer AVE (Qiagen Cat. No. 1026956) 

7.4.9. Collection tubes, 2 mL (Qiagen Cat. No. 19201 

7.4.10. Absolute ethanol (Fisher Scientific Cat. No. BP2818-100) 

7.4.11. Random Primers, 0.5 μg/μL (Promega Cat. No. C1181) 

7.4.12. 10X PCR Buffer II with 25 mM MgCl2 (Applied Biosystems Cat. No. 

N808130) 

7.4.13. Deoxyribonucleotides, 10 mM (NTPs; Promega Cat. No. C1141) 

7.4.14. Dithiothreitol, 100 mM (DTT; Promega Cat. No. P1171) 

7.4.15. RNasin® Plus RNase Inhibitor, 40 units/μL (Promega Cat. No. N2615) 

7.4.16. SuperScript II Reverse Transcriptase, 200 units/μL (Invitrogen Cat. No. 

18064) 

7.4.17. LightCycler 480 Probes Master kit (Roche Diagnostics Cat. No. 

04707494001) 
METHOD 1615. Enterovirus and Norovirus occurrence in water

16 

7.4.18. ROX reference dye, 25 mM (Invitrogen Cat. No. 12223) 

7.4.19. Hepatitis G virus Armored RNA® (Asuragen Cat. No. 42024) 

7.4.20. Enterovirus, norovirus GI, norovirus GII Armored RNA (Asuragen 

custom order giving >1010 genomic copies at a defined concentration) 

8. QUALITY ASSURANCE

This section describes the minimum quality assurance (QA) requirements. Laboratories are 

encouraged to institute additional QC practices that go beyond these minimum criteria to 

meet their needs. 

8.1. QUALITY ASSURANCE PLAN 

All laboratories analyzing samples with this method must adhere to defined quality 

assurance procedures that ensure analytical data which are scientifically valid and 

which demonstrate acceptable precision and specificity. Each laboratory must have a 

written Quality Assurance Plan that addresses the following: 

8.1.1. Laboratory organization and responsibility – This section must include the 

following: 1) a list that identifies the laboratory QA manager(s) and key 

individuals who are responsible for ensuring the production of valid 

measurements and the routine assessment of QC data, 2) specify who is 

responsible for internal audits and reviews of the implementation of the 

QA plan and its requirements, and 3) include a chart showing the 

laboratory organization and line authority. 

8.1.2. Personnel – This section must list each analyst’s academic background 

and experience, describe how each analyst is trained to perform the 

method, and describe how training is documented. 

8.1.3. Facilities – This section must describe the arrangement and size of 

laboratories, workflow patterns to minimize cross contamination, air 

system; laboratory reagent water system, and waste disposal system [see 

reference (36)]. 

8.1.4. Field sampling procedures – This section must describe the laboratory 

chain-of-custody procedures, including the sample identification and 

information recording system; and describe how field samples are 

collected and transported, including transportation time and temperature. 

8.1.5. Laboratory sample handling procedures – This section must describe 

sample-holding times and temperature during analyses and the procedures 

for maintaining the integrity of the samples, i.e., logging and tracking of 

samples from receipt through analyses and disposal. 

8.1.6. Equipment – This section must describe the specifications, calibration 

procedures, preventive maintenance, and how quality control records are 

maintained for each item used during the performance of the method. All 

calibrations must be traceable to national standards, when they are 

available. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

17 

8.1.7. Supplies – This section must describe the specifications, storage 

conditions, and how catalog and lot numbers are documented for 

chemicals, reagents, and media. 

8.1.8. Laboratory practices – This section must describe the preparation of 

reagent-grade water, glassware washing and preparation procedures, and 

sterilization procedures. It should also describe the workflow 

requirements among laboratories to prevent cross contamination, 

especially for molecular procedures. The work flow and other 

recommended requirements are described in detail in Sen et al. (36). 

8.1.9. Analytical procedures – This section must reference this method and 

identify available laboratory SOPs. 

8.1.10. Quality control checks – This section must describe all laboratory 

procedures that are implemented to ensure the quality of each analyst’s 

data. 

8.1.11. Data reduction, verification, and reporting – This section must describe 

any procedures for converting raw to final data, identify procedures for 

ensuring the accuracy of data transcription and calculations, and describe 

the laboratory’s procedures for reporting all data to EPA. 

8.1.12. Corrective actions – This section must describe how the laboratory will 

respond to PE and QC failures and from failures of its own internal QC 

procedures, identify the person(s) responsible for taking corrective action, 

and describe how the effectiveness of the actions will be documented. 

8.1.13. Record keeping – This section must describe how records are maintained 

(e.g., hard copy, electronic or LIMS, etc.), how long records are kept, and 

where records are stored. 

8.2. LABORATORY PERSONNEL 

8.2.1. Principal Analyst/Supervisor – Laboratories must have a principal analyst 

who may also serve as a supervisor if an additional analyst(s) is to be 

involved. The principal analyst/supervisor oversees or performs the entire 

analyses and carries out QC performance checks to evaluate the quality of 

work performed by analysts and technicians. This person must be an 

experienced microbiologist with at least a B.A./B.S. degree in 

microbiology or a closely related field. The person must also have a 

minimum of three years continuous bench experience in cell culture 

propagation, processing and analysis of virus samples, and in performing 

PCR, and at least six months of experience in performing RT-qPCR. This 

analyst must have analyzed a PE sample set and results must fall within 

acceptance limits. The principal analyst must also demonstrate acceptable 

performance during any on-site performance audits. 

8.2.2. Analyst – The analyst performs at the bench level under the supervision of 

a principal analyst and can be involved in all aspects of analysis, including 

preparation of sampling equipment, filter extraction, sample processing, 

cell culture, virus assay, qPCR, and data handling. The analyst must have 
METHOD 1615. Enterovirus and Norovirus occurrence in water

18 

two years of college lecture and laboratory course work in microbiology 

or a closely related field. The analyst must have at least six months bench 

experience in cell culture, animal virus analyses and PCR, including three 

months experience in filter extraction of virus samples and sample 

processing. Six months of additional bench experience in the above areas 

may be substituted for the two years of college. Each analyst must have 

analyzed a PE sample set and results must fall within acceptance limits. 

The analyst must also demonstrate acceptable performance during any an 

on-site audits. Should laboratories choose to use teams of analysts who 

specialize in performing the culture or molecular portions of this method, 

analysts only need to meet the educational requirement of the portion they 

perform. Laboratories using analyst teams must ensure that the PE 

samples are analyzed by the appropriate team member. 

8.2.3. Technician – The technician extracts filters, processes samples, and 

performs qPCR under the supervision of an analyst, but does not perform 

cell culture work, virus detection, or enumeration. The technician must 

have at least three months experience in filter extraction, processing of 

virus samples to participate in the cultural portion of this method and three 

months of experience with PCR to participate in the molecular portion of 

the method. 

8.2.4. Samplers – The sampler collects water samples and ships them to the 

analytical laboratory. The sampler must be familiar with the sample 

collection process and have at least training by means of a video or written 

instructions demonstrating proper sampling technique. Laboratories are 

responsible for ensuring that samplers have adequate training. 

8.3. LABORATORY PERFORMANCE 

8.3.1. Laboratories using this method must evaluate the ability of analysts to 

perform the method using known quality control (QC) and unknown 

performance evaluation (PE) samples as defined in Section 8.3.1.2 and 

8.3.1.3. EPA may also require laboratories to be approved. 

8.3.1.1. Laboratory approval – Laboratories must have a Quality 

Assurance Plan, adequately trained staff, proper equipment, 

and at least one approved analyst to be approved. 

8.3.1.2. Initial analyst approval/initial demonstration of capability – 

Each analyst must demonstrate the ability to perform the 

method using QC and PE samples as part of an initial approval 

process. New analysts must initially use QC samples to gain 

method proficiency followed by the analysis of a PE sample 

set. 

8.3.1.3. Ongoing analyst approval/ongoing demonstration of capability 

– Each analyst must analyze one QC sample set for every 

analysis batch (see section 3) and one PE sample set per month 

following initial approval to remain approved. A QC sample 

set is comprised of a negative and a positive QC sample (see 
METHOD 1615. Enterovirus and Norovirus occurrence in water

19 

section 8.4). A PE sample set is comprised of two PE samples 

that may or may not contain virus at various levels (section 

8.5). For initial and ongoing approval, analysts must meet the 

method performance characteristics defined in section 14 or in 

any additional guidance from EPA. Any analyst who does not 

meet the initial or ongoing demonstration of capability must 

not process samples until the cause of the failure has been 

identified and corrected. 

8.4. QC SAMPLE SET 

A QC sample set consists of a negative and a positive QC sample. QC stocks (item 

7.1.2) with a titer of 200 MPN/mL should be prepared by the analytical laboratories 

and stored in aliquots containing about 1.1 mL at -70°C. QC sample results must 

meet the method performance characteristics defined in section 14. 

8.4.1. Note: It is difficult to obtain an accurate pH on pure water. To 

compensate, a buffering agent, such as HEPES (Sigma Aldrich Cat. No. 

H4034), may be added to the water at a concentration up to 0.01M. 

8.4.2. Note: QC samples should use the filter type (e.g., 1MDS or NanoCeram) 

that will be used for collecting samples. If both filter types are being used, 

the QC samples should be performed using the filter type that is most 

frequently analyzed by the analyst. 

8.4.3. Negative QC Sample/Equipment blanks 

8.4.3.1. Place 10 L of reagent grade water in a dispensing pressure 

vessel or polypropylene container (e.g., Cole Parmer Product 

No. EW-06317-53). Adjust the pH to 6.5-7.5 with 0.12 M HCl 

or 0.1 M NaOH, as necessary. 

8.4.3.2. Place a magnetic stir bar into the vessel or container and stir for 

10 minutes at a speed sufficient to create a vortex. 

8.4.3.3. Pass the water through a sterile standard filter apparatus 

(Section 6.1) containing a sterile electropositive filter using a 

flow rate of approximately 10 L/minute. Note: to meet 

ongoing QC requirements standard filter apparatuses that have 

been sterilized after use in the field must be used. 

8.4.3.4. Process and analyze the filter using the Filter Elution (section 

10), Organic Flocculation (section 11), Total Culturable Virus 

Assay (section 12) and Enterovirus and Norovirus Molecular 

(section 13) procedures. 

8.4.4. Positive QC Sample 

8.4.4.1. Place 10 L of reagent grade water in a dispensing pressure 

vessel or polypropylene container (e.g., Cole Parmer Product 

No. EW-06317-53). Adjust the pH to 6.5-7.5 with 0.12 M HCl 

or 0.1 M NaOH, as necessary. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

20 

8.4.4.2. Add 1.0 mL of a QC sample (see item 7.1.2) to the water. 

8.4.4.3. Place a magnet into the vessel or container and stir for 10 

minutes at a speed sufficient to create a vortex. 

8.4.4.4. Pass the water through a sterile standard apparatus containing a 

sterile electropositive filter using a flow rate of approximately 

10 L/minute. 

8.4.4.5. Process and analyze the filter using the Elution (step 10), 

Organic Flocculation (step 11), Total Culturable Virus Assay 

(step 12) and Enterovirus and Norovirus Molecular (step 13) 

procedures. 

8.4.5. QC sample stocks (item 7.1.2) are also to be used for the Positive Assay 

Control (see item 7.3.3 and step 12.1.2.3.2). 

8.5. PE SAMPLES 

8.5.1. PE samples will be sent to analysts in a randomized fashion and may 

contain no, low, medium, or high levels of Sabin poliovirus type 3 on the 

filter type (e.g., 1MDS or NanoCeram) used for sampling. 

8.5.2. Process and analyze the PE filter using the Elution (section 10), Organic 

Flocculation (section 11), Total Culturable Virus Assay (section 12) and 

Enterovirus and Norovirus Molecular (section 13) procedures and 

according to any additional requirements supplied with the samples. 

8.5.3. PE sample results should meet the method performance characteristics 

defined in section 14. 

8.6. MATRIX SPIKE 

Matrix spike should be run for every sample location initially and then after every 

20th sample from the same location. Matrix spikes duplicates are performed by 

collecting two samples at the sampling location as described in section 9, except 

that the sampling volume of the second sample is reduced by 10 L. The last 10L is 

collected in a 10 L cubitainer (item 6.2.3), shipped back to the laboratory, seeded 

with 1 mL of the matrix spike (item 7.1.3), passed through the duplicate filter, and 

analyzed by the method procedures (steps 10 through 12.2.11). The results of the 

analysis of the matrix spike must meet the performance measures in section 14. 

8.7. RECORD MAINTENANCE 

Laboratories shall maintain all records related to data quality. This shall include a 

record of the analyst name, date, and results of all QA controls performed, records 

of equipment calibration and maintenance, and reagent and material catalog and lot 

numbers used for all analytical procedures. 

9. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

9.1. SAMPLE COLLECTION 

9.1.1. Filter sampling apparatus sterilization 
METHOD 1615. Enterovirus and Norovirus occurrence in water

21 

9.1.1.1. Before each use, analytical laboratories must wash, and then 

sterilize the intake and cartridge housing modules, any 

necessary injector modules, and pumps as described in section 

15.2.4. 

9.1.1.2. Cover the apparatus module ends and the injector port(s) with 

sterile aluminum foil. 

9.1.1.3. Place the injector module and tubing into a sterile bag or 

wrapping in such a way that they may be removed without 

contaminating them. 

9.1.1.4. Ship the filter sampling apparatus components to the 

individuals who will be collecting water samples. 

9.1.2. Preparation for sample collection 

9.1.3. Note: Individuals collecting water samples for virus analysis must wear 

surgical gloves and avoid conditions that can contaminate a sample with 

virus. Gloves should be changed after touching human skin or handling 

components that may be contaminated (e.g., water taps, other 

environmental surfaces). Care must be taken to ensure that cartridge 

filters are properly seated in the housings. Housings with properly seated 

filters must not leak. Filters should be checked for proper seating upon 

opening the housing at the analytical laboratory by examining the gaskets 

for depressions that do not extend beyond the edge of the filter.] 

9.1.3.1. Purge the water tap to be sampled before connecting the filter 

apparatus. Continue purging for 2-3 minutes or until any 

debris that has settled in the line has cleared. 

9.1.3.2. Remove the foil from the backflow regulator. Loosen the 

swivel female insert slightly to allow it to turn freely and 

connect the backflow regulator to the tap. Retighten the swivel 

female insert. Disconnect the cartridge housing module at the 

quick connect, if connected, and cover the open end with sterile 

foil. 

9.1.3.3. Remove the foil from the ends of the discharge module and 

connect it to the regulator module. Place the end of the 

regulator module or the tubing connected to the outlet of the 

regulator module into a 1 L plastic bottle. 

9.1.3.4. Slowly turn on the tap and adjust the globe valve until the flow 

meter/totalizer reads 10 L/min. If the tap is incapable of 

reaching this flow rate, adjust the valve to achieve the 

maximum flow rate. Slower flow rates will result in longer 

sampling times. 

9.1.3.5. Flush the apparatus assembly with at least 75 L of the water to 

be sampled. While the system is being flushed, measure the 
METHOD 1615. Enterovirus and Norovirus occurrence in water

22 

chlorine residual, pH, temperature, and the turbidity of the 

water collecting in and overflowing from the 1 L plastic bottle. 

9.1.3.6. Record the pH, temperature, and turbidity values onto a sample 

data sheet. 

9.1.3.7. Turn off the water at the tap. 

9.1.4. Injector module adjustment (Note: If a NanoCeram filter is being used 

and if the water pH is 9.0 or less and if it does not contain a disinfectant, 

skip to section 9.1.5. If disinfected waters above pH 8.0 are being used 

with a 1MDS cartridge filter, substitute a double injector module for the 

single injector module in the following steps. With 1MDS filters under 

these conditions, use a second metering pump connected to the second 

connection on the double injector module to add 0.12 M HCl at a rate, 

which brings the pH of the water exiting the discharge module to 6.5 to 

7.5.) 

9.1.4.1. If the sample contains a disinfectant, turn off the water at the 

tap and disconnect the discharge module from the regulator 

module. 

9.1.4.2. Remove the foil from the ends of an injector module and 

connect the module to the quick connect of the regulator 

module. 

9.1.4.3. Turn on the metering pump. Set the pump to deliver 2.4 or 

6±0.2 mL/minute for flow rates of 4 or 10 L/minute, 

respectively (see Table 2). Measure the flow exiting the 

injector module for several minutes to ensure that the flow rate 

is correct. Measure the chlorine residual and if present, readjust the flow rate until no residual is present. Re-mark the 

setting, if necessary. Turn off the metering pump. 

9.1.5. Virus collection 

9.1.5.1. If connected, remove the discharge module. Remove the foil 

from the cartridge housing module and connect it to the end of 

the regulator module, or if used, the injector module. Connect 

the discharge module to the outlet of the cartridge housing 

module. 

9.1.5.2. If the water sample has turbidity greater than 75 NTU, remove 

the foil from each end of the prefilter module and connect the 

prefilter module between the regulator module or, if used, the 

injector module and the cartridge housing module. 

9.1.5.3. Record the unique sample number, location, date, time of day 

and initial totalizer reading onto a sample data sheet (section 

17.1). 

9.1.5.4. If an injector module is being used, turn on the metering pump. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

23 

9.1.5.5. With the filter housing placed in an upright position, slowly 

open the water tap until it is completely open (Note: If the 

cartridge housing has a vent button, press it while opening the 

tap to expel air from the housing. When the air is totally 

expelled from the housing, release the button, and open the 

sample tap completely. If the housing does not have a vent 

button, allow the housing to fill with water before completely 

opening the tap). 

9.1.5.5.1. After the tap is opened completely, check the flow 

rate and readjust to the recommended rate from 

Table 2, if necessary. 

9.1.5.5.2. Check and readjust the metering pump rate, if 

necessary. 

9.1.5.6. Using the totalizer readings, pass a volume of water through 

the apparatus that equals the volume specified in Table 2. 

9.1.5.7. Turn off the flow of water at the sample tap at the end of the 

sampling period and record the date, time of day, and totalizer 

reading onto a sample data sheet (see section 17.1). Although 

the totalizer reading may be affected by the addition of 

thiosulfate, the effect is insignificant and may be ignored. 

9.1.5.8. Loosen the swivel female insert on the regulator module and 

disconnect the backflow regulator from the tap. Disconnect the 

cartridge housing module and the prefilter housing module, if 

used from the other modules. 

9.1.5.9. Turn the filter housing(s) upside down and allow excess water 

to flow out. Turn the housing(s) upright and cover the quick 

connects on each end of the modules with sterile aluminum 

foil. 

9.2. SHIPMENT OF SAMPLES 

9.2.1. Pack the cartridge housing module(s) into an insulated shipping box. 

9.2.2. Add 6-8 small ice packs (prefrozen at -20°C) or double bagged ice cubes 

around the cartridge housings to keep the sample cool in transit (the 

number of ice packs or bags may have to be adjusted based upon 

experience to ensure that the samples remain cold, but not frozen). 

9.2.2.1. Add an iButton (or other temperature recording devise) to a 

location in the shipping box where it will not come in direct 

contact with the ice packs or bags. 

9.2.2.2. The temperature during shipment must be in the range of 1-

10°C. 

9.2.3. Drain and add the regulator and injector modules used. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

24 

9.2.4. Place the sample data sheet (protected with a closable plastic bag) in with 

the sample. 

9.2.5. Drain and then cover the ends of the discharge module with foil. The 

discharge module may remain at the sampling site, if samples will be 

taken on a routine basis. If not, pack the module into the shipping box. 

9.2.6. Close the insulated portion of the shipping box and tape to prevent any 

leakage of water. Close and label. 

9.2.7. If the shipping box cannot be directly transported to the laboratory for 

virus analysis by close of business on the day collected or by the next 

morning, ship it to the laboratory by overnight courier. 

9.3. LABORATORY HOLDING TIME AND TEMPERATURE 

9.3.1. Record the date of arrival and the arrival condition on the sample data 

sheet packed with the sample. Print out the transit temperature reading 

from the iButton. 

9.3.1.1. Attach the readout of the iButton or other temperaturerecording device for recording the temperature during shipment 

to the sample data sheet. 

9.3.1.2. Warning: The cartridge filters must arrive from the utility or 

other sampling site in a refrigerated, but not frozen, condition. 

The temperature during shipment must be in the range of 1-

10°C. 

9.3.1.3. Brief transient temperatures outside the acceptable range 

associated with the initial packing and closing of the shipping 

box and its opening at the analytical laboratory may be 

ignored. 

9.3.2. Filters must be refrigerated immediately upon arrival. Ideally, viruses 

should be eluted from filters within 24 h of the start of the sample 

collection, but all filters must be eluted within 72 h of the start of the 

sample collection. 

10. FILTER ELUTION PROCEDURE

10.1. ELUTION EQUIPMENT SETUP 

10.1.1. Attach sections of braided tubing to the inlet and outlet ports of the 

cartridge housing containing the cartridge filter (see Figure 4). Note: If a 

prefilter or more than one electropositive filter was used to collect a 

sample, each filter must be eluted and analyzed separately using the 

procedures below. 

10.1.2. Place the sterile end of the tubing connected to the outlet of the cartridge 

housing into a sterile 2 L glass or polypropylene beaker. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

25 

10.1.3. Connect the free end of the tubing from the inlet port of the cartridge 

housing to the outlet port of a sterile pressure vessel and connect the inlet 

port of the pressure vessel to a positive air pressure source. 

10.2. ELUTION 

10.2.1. First elution 

10.2.1.1. Elute NanoCeram or 1MDS filters with 500 mL or 1,000 mL of 

buffered 1.5% beef extract, pH 9.0 (item 7.2, prewarmed to 

room temperature), respectively, by opening the cartridge 

housing and adding a sufficient amount of beef extract to cover 

the filter. Close the housing and pour the remaining beef 

extract into the pressure container. An acceptable alternative to 

the use of a pressure vessel is to use a peristaltic pump and 

sterile tubing to push the remaining beef extract through the 

filter. 

10.2.1.2. Replace the top of the pressure vessel and close its vent/relief 

valve. 

10.2.1.3. Open the vent/relief valve (if present) on the cartridge housing 

and slowly apply sufficient pressure to fill the housing with 

beef extract. Close the vent/relief valve (if present) as soon as 

the buffered beef extract solution begins to flow from it. 

Carefully observe housings without vents to ensure that all 

trapped air has been purged. 

10.2.1.4. Turn off the pressure and allow the solution to contact the filter 

for 1 minute. Wipe up spilled liquid with disinfectant-soaked 

sponge. 

10.2.1.5. Increase the pressure to force the buffered beef extract solution 

through the filter(s). The solution should pass through the 

filter slowly to maximize the elution contact period. When air 

enters the line from the pressure vessel, elevate and invert the 

filter housing to permit complete evacuation of the solution 

from the filters. Note: Slow passage of the solution also 

minimizes foaming, which may inactivate some viruses; the 

addition of a few drops of sterile antifoam to minimize foaming 

is optional. 

10.2.1.6. Turn off the pressure at the source and open the vent/relief 

valve on the pressure vessel. 

10.2.2. Second elution 

10.2.2.1. For the NanoCeram filter, repeat sections 10.2.1.1 to 10.2.1.6 

using an additional 500 mL of buffered 1.5% beef extract, 

except increase the contact time in section 10.2.1.4 to 15 

minutes. For a 1MDS filter, place the buffered beef extract 
METHOD 1615. Enterovirus and Norovirus occurrence in water

26 

from the 2 L beaker back into the cartridge housing and 

pressure vessel and repeat sections 10.2.1.2 to 10.2.1.6). 

10.2.2.2. Turn off the pressure at the source and open the vent/relief 

valve on the pressure vessel. Combine the two 500 mL 

portions from the elution of NanoCeram filters. Record the 

total volume on the Virus Data Sheet (17.2). Thoroughly mix 

the eluate and proceed to the Organic Flocculation 

Concentration Procedure immediately. 

11. ORGANIC FLOCCULATION CONCENTRATION PROCEDURE

11.1. ORGANIC FLOCCULATION 

11.1.1. Place a sterile stir bar into the beaker containing the buffered beef extract 

eluate from the cartridge filter. Place the beaker onto a magnetic stirrer, 

and stir at a speed sufficient to develop a vortex. Minimize foaming 

(which may inactivate viruses) throughout the procedure by not stirring or 

mixing faster than necessary to develop a vortex. 

11.1.2. Sterilize the electrode of a combination-type pH electrode as described in 

section 15.2.4. Calibrate the pH meter at pH 4 and 7. 

11.1.3. Insert the sterile pH electrode into the beef extract eluate. Add 1.2 M HCl 

to the eluate dropwise while moving the tip of the pipette in a circular 

motion away from the vortex to facilitate mixing. Continue adding 1.2 M 

HCl until the pH reaches 3.5 ± 0.1. 

11.1.4. While continuing to monitor the pH, slowly stir the eluate for 30 minutes 

at room temperature. A precipitate will form during the 30 minutes 

stirring period. If pH falls below 3.4, add 1 M NaOH to bring it back to 

3.5 ± 0.1. Exposure to a pH below 3.4 may result in virus inactivation. 

11.1.5. Remove the electrode from the beaker, and pour the contents of the beaker 

into a centrifuge bottle. To prevent the transfer of the stir bar into a 

centrifuge bottle, hold another stir bar or magnet against the bottom of the 

beaker while decanting the contents. The beef extract suspension will 

usually have to be divided into several centrifuge bottles. 

11.1.6. Cap the bottle and centrifuge the precipitated beef extract suspension at 

2,500 x g for 15 minutes at 4°C. 

11.1.7. Carefully pour off or aspirate the supernatant so as to not disturb the 

pellet, including any loose floc on top of the pellet, and then discard the 

supernatant. 

11.2. RECONCENTRATED ELUATE 

11.2.1. Place a stir bar into the centrifuge bottle that contains the precipitate. 

11.2.2. Add 30 mL of 0.15 M sodium phosphate, pH 9.0. Note: A smaller 

volume (down to 15 mL) may be used, if the analytical laboratory’s PE 

sample sets meet the performance requirements of Section 14. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

27 

11.2.3. Place the bottle onto a magnetic stirrer, and stir slowly until the precipitate 

has dissolved completely. Continue stirring for at least 10 minutes before 

proceeding to step 11.2.3.4. When the centrifugation is performed in more 

than one bottle, dissolve the precipitates in a total of 30 mL and combine 

into one bottle before proceeding to the next step. Significant virus loss 

can occur if the precipitates are not dissolved completely. Precipitates that 

prove to be difficult to dissolve can be treated using any of the following 

techniques: 

11.2.3.1. Break up the precipitate with a sterile spatula before or during 

the stirring procedure. 

11.2.3.2. Use a pipette repeatedly to draw the solution up and down 

during the stirring. 

11.2.3.3. Shake the precipitate at 160 rpm for 20 minutes on an orbital 

shaker in place of stirring. 

11.2.3.4. If the above procedures take longer than 20 minutes to dissolve 

the precipitate or if experience with the water matrix shows 

that precipitates are always difficult to manage, either slowly 

adjust the pH to 7.0 - 7.5 with 1.2 M HCl or resuspend the 

precipitate initially in 0.15 M sodium phosphate, pH 7.0 - 7.5. 

Use one of the above techniques to dissolve the precipitate and 

then slowly re-adjust the pH to 9.0 with 1 M NaOH. Mix for 

10 minutes at room temperature before proceeding. 

11.2.3.5. Check the pH and readjust to 9.0 with 1 M NaOH, as 

necessary. Remove the stir bar. 

11.2.3.6. Centrifuge the dissolved precipitate at 4,000 x g for 10 minutes 

at 4°C. The centrifugation speed may be increased to 10,000 x 

g for 10 minutes at 4°C to facilitate the filtration step below. 

Record the centrifugation speed on the virus data sheet (section 

17.2). 

11.2.3.7. Remove and collect the supernatant and discard the pellet. 

Adjust the pH of the supernatant to 7.0-7.5 slowly with 1.2 M 

HCl. 

11.2.3.8. Pretreat a sterilizing filter (item 6.3.7) or, for samples that are 

difficult to filter, a sterilizing filter stack (item 6.3.9) with 10-

15 mL of 1.5% beef extract (item 7.2.4). Load the supernatant 

into a 50 mL syringe and force it through the filter. If the 

sterilizing filter or filter stack begins to clog badly, empty the 

loaded syringe into the bottle containing the unfiltered 

supernatant, fill the syringe with air, and inject air into filter to 

force any residual sample from it. Continue the filtration 

procedure with another filter. 

11.2.4. Calculation of assay volumes and preparation of subsamples 
METHOD 1615. Enterovirus and Norovirus occurrence in water

28 

11.2.4.1. Record the reconcentrated eluate volume (designated the Final 

Concentrated Sample Volume for cultural assays; FCSV) on 

the virus data sheet (section 17.2). 

11.2.4.2. Calculate the Assay Sample Volume (S) for source and 

finished water samples using equation 1. D (the Volume of 

Original Water Sample Assayed) is the amount of 

reconcentrated eluate that must be assayed by Total Culturable 

Virus Assay (section 12) or processed for the Enterovirus and 

Norovirus Molecular Assay (section 12.2.11). This amount is 

100 L for source water or 500 L for finished or ground waters. 

TSV is the Total Sample Volume from the Virus Data Sheet. 

The Assay Sample Volume is the volume of the 

reconcentrated eluate that represents 100 L of source water or 

500 L of finished or ground waters. Record the S and D onto 

the Virus Data Sheet (section 17.2). 

FCSV

TSV

D S   Equation 1 

11.2.4.3. For example, if 1,800 L of a groundwater sample is passed 

through the NanoCeram filter and subsequently concentrated to 

30 mL, then TSV equals 1,800 L, D equals 500 L, FCSV 

equals 30 mL, and S = (500 L/1,800 L) x 30 mL = 8.33 mL. 

11.2.4.4. Prepare three subsamples of the reconcentrated eluate. Prepare 

subsamples 1 and 2 with a volume equal to 1.2 times the Assay 

Sample Volume. Prepare subsample 3 with the remaining 

volume. Label subsamples 1-3 with appropriate sampling 

information for identification. 

11.2.4.4.1. Hold subsample 1 at 4°C for use with the Total 

Cultural Virus Assay if it can be assayed within 

24 hours, otherwise freeze at -70°C.

11.2.4.4.2. Hold subsample 2 at 4°C and analyze using the 

Molecular Assay within 24 hours. Note: Freeze 

thawing leads to norovirus losses. 

11.2.4.4.3. Freeze subsample 3 at -70°C for backup and 

archival purposes. 

11.2.4.5. Determine the Inoculum Volume for the Total Culturable 

Virus Assay (section 12) by dividing the Assay Sample 

Volume by 10. Record the Inoculum Volume onto the Virus 

Data Sheet (section 17.2). 

11.2.4.5.1. For ease of inoculation, a sufficient quantity of 

0.15 M Na2HPO4, pH 7.0 - 7.5, may be added to 

the Inoculum Volume to give a more usable 

working Final Inoculation Volume (e.g., 1.0 

mL). If this option is used, then record the Final 
METHOD 1615. Enterovirus and Norovirus occurrence in water

29 

Inoculation Volume onto the Virus Data Sheet 

(section 17.2) and substitute the term Final 

Inoculation Volume for each use of the 

Inoculum Volume, except where indicated. 

11.2.4.5.2. For example, if an Inoculum Volume of 0.72 mL 

is to be placed onto each of 10 vessels, then for 

10.5 vessels (with the extra 0.5 added to account 

for sample loss on the surface of the vessel used 

for the preparation), 7.56 mL of subsample will be 

needed (10.5 x 0.72 mL). The addition of 2.94 

mL of 0.15 M Na2HPO4, pH 7.0-7.5 (10.5 x (1-

0.72 mL) to the 7.56 mL of subsample results in a 

mixture that contains the required Inoculum 

Volume per 1.0 mL. 

11.2.4.6. Calculate the Assay Sample Volume for QC (section 8.4) 

samples by multiplying FCSV by 0.3. Calculate the Inoculum 

Volume by dividing the Assay Sample Volume by 10. Divide 

the Final Concentrated Sample Volume from QC samples 

into three subsamples and handle as described in step 11.2.4.4.

12. TOTAL CULTURABLE VIRUS ASSAY

12.1. QUANTAL ASSAY 

12.1.1. Preparation of cell culture test vessels 

12.1.1.1. Using 10 cell culture test vessels for every water sample to be 

tested, code each vessel with the water sample number, 

subsample number, analyst initials, and date using an indelible 

marker. 

12.1.1.2. Return the cell culture test vessels to a 36.5 ± 1°C incubator 

and hold at that temperature until the cell monolayer is to be 

inoculated. 

12.1.1.3. Decant and discard the medium from cell culture test vessels. 

12.1.1.4. Wash the test vessels with a balanced salt solution (e.g., item 

7.3.2.1) or maintenance medium without serum using a wash 

volume of at least 0.06 mL/cm2

 of surface area. Rock the wash 

medium over the surface of each monolayer several times and 

then decant and discard the wash medium. Do not disturb the 

cell monolayer during the wash procedure. 

12.1.2. Inoculation of test samples 

12.1.2.1. Rapidly thaw subsample 1, if frozen, and inoculate an amount 

equal to the Inoculum Volume or the Final Inoculation 

Volume (see step 11.2.4.5) onto each of 10 cell culture test 

vessels. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

30 

12.1.2.1.1. Note: Samples should be thawed in a 37°C water 

bath or under warm running water at about 37°C 

with shaking. Samples should be removed from 

the warm water as soon as the last ice crystal melt. 

12.1.2.1.2. Note: The number of cell culture replicates was 

cut from the 20 required by the ICR standard 

method (19) to 10 to reduce labor costs. This 

reduction of replicates results in wider 95% 

confidence limits (c. 20-40%) and reduces the 

maximum virus titer that can be assayed without 

dilutions by about 25%. Laboratories assaying 

samples that are known to have more than about 

20 MPN/100 L may assay subsample 1 using 

more than 10 replicates or by using dilutions as 

described in section 12.1.2.2. 

12.1.2.1.3. Note: The analysis of a second subsample is not 

required for this method. Subsample 2 was used 

in the ICR method (19) to account for 

cytotoxicity. Samples with cytotoxicity should be 

assayed using dilutions as described in section 

12.1.2.2 

12.1.2.1.4. Warning! Use at least a different pipetting tip or 

device for each set of samples to be inoculated. 

12.1.2.2. For QC samples and for water samples known or suspected of 

having virus concentrations greater than 20 MPN/100 L, 

prepare five- and twenty five-fold dilutions of subsample 1 (or 

subsample 3, see section 12.1.2.1.3). 

12.1.2.2.1. Prepare a 1:5 dilution by adding a volume of 

subsample 1 equal to 0.1334 times the Assay 

Sample Volume (amount "a") to a volume of 0.15 

M sodium phosphate (pH 7.0-7.5) equal to 0.5336 

times the Assay Sample Volume (amount "b"). 

Mix thoroughly. 

12.1.2.2.2. Prepare a 1:25 dilution by adding amount "a" of 

the 1:5 diluted sample to amount "b" of 0.15 M 

sodium phosphate (pH 7.0-7.5). 

12.1.2.2.3. Inoculate 10 cell cultures each with undiluted 

subsample 1, subsample 1 diluted 1:5, and 

subsample 1 diluted 1:25, respectively, with an 

amount equal to the Inoculum Volume. 

12.1.2.2.4. Freeze the remaining portions of the 1:25 dilution 

at -70°C until the sample results are known. 

Thaw and perform additional 5-fold dilutions 
METHOD 1615. Enterovirus and Norovirus occurrence in water

31 

using the dilution format above if all replicates of 

the undiluted to 1:25-fold dilutions develop CPE. 

12.1.2.3. Cell culture controls 

12.1.2.3.1. Negative Assay Control – Inoculate three or 

more BGM cultures with a volume of sodium 

phosphate, pH 7.0 - 7.5, equal to the Inoculum 

Volume as a negative control. Run a Negative 

Assay Control with every group of subsamples 

inoculated onto cell cultures. If any Negative 

Assay Control develops CPE, all subsequent 

assays should be halted until the cause of the 

positive result is determined. 

12.1.2.3.2. Positive Assay Control – Inoculate three or more 

BGM cultures with the Positive Assay Control

(see item 7.3.3). Run a positive control with 

every group of subsamples inoculated onto cell 

cultures. This control will provide a measure for 

continued sensitivity of the cell cultures to virus 

infection. If any Positive Assay Control fails to 

develop CPE, all subsequent assays should be 

halted until the cause of the negative result is 

determined. 

12.1.2.4. Record the date of inoculation on the Virus Data Sheet

(section 17.2). 

12.1.2.5. Rock the inoculated cell culture test vessels gently to achieve 

uniform distribution of inoculum over the surface of the cell 

monolayers. Place the cell culture test vessels on a mechanical 

rocking platform set at 1-5 oscillations per minute at room 

temperature. 

12.1.2.6. Continue incubating the inoculated cell cultures for 80 - 120 

minutes at room temperature to permit viruses to adsorb onto 

and infect cells. Note: If a rocking platform is not available, 

the vessels may be placed upon a level laboratory surface, but 

the vessels should be rocked every 15-20 minutes during the 

adsorption period to prevent cell death in the middle of the 

vessels from dehydration. 

12.1.2.7. Add liquid maintenance medium (see Item 3.7) and incubate at 

36.5 ± 1°C. 

12.1.2.7.1. Warm the maintenance medium to 36.5 ± 1°C 

before placing it onto cell monolayers. 

12.1.2.7.2. Add the medium to the side of the cell culture 

vessel opposite the cell monolayer. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

32 

12.1.2.7.3. Warning! Never touch the pipetting device to the 

inside rim of the cell culture test vessels during 

medium addition. This step represents the most 

likely place where cross contamination of cultures 

can occur. Cross contamination will result in 

invalid MPN values and can cause false positive 

results. Laboratories must ensure that analysts 

take great precaution in performing this step. 

12.1.2.7.4. If CPE has not started to develop, the cultures 

may be re-fed with fresh maintenance medium 

after 4-7 days. 

12.1.3. CPE development 

12.1.3.1. Examine each culture microscopically for the appearance of 

CPE daily for the first three days and then every couple of days 

for a total of 14 days. 

12.1.3.2. Freeze cultures at -70°C when more than 75% of the 

monolayer has developed CPE. Freeze all remaining cultures, 

including controls, after 14 days. 

12.1.4. Perform a second passage for confirmation. Confirmation passages may 

be performed in small vessels or multiwell trays, however, it may be 

necessary to distribute the inoculum into several vessels or wells to insure 

that the inoculum volume is less than or equal to 0.04 mL/cm2

 of surface 

area. 

12.1.4.1. Thaw all the cultures, including the negative and positive assay 

controls, to confirm the results of the previous passage. 

Refreeze at least 2 mL of the medium from each vessel at -

70°C for optional analysis by molecular methods (see step 

13.3.2). 

12.1.4.2. Filter at least 10% of the medium from each vessel that was 

positive for CPE through separate 0.22 μm sterilizing filters 

(item 6.3.8). If the medium is difficult to filter, it can be 

centrifuged at 1,500 to 18,000 x g prior to filtration. 

12.1.4.3. Prepare fresh cell culture test vessels as described in step 

12.1.1. Inoculate the fresh cultures with the thawed medium 

from all negative samples (step 12.1.4.1) and the filtered 

medium from step 12.1.4.2 using an inoculation volume that 

represents 10% of the medium from the first passage. 

12.1.4.4. Repeat sections 12.1.2.4 to 12.1.3.1. 

12.1.5. Score cultures that developed CPE in both the first and second passages as 

confirmed positives. 

12.1.5.1. Perform a third passage as described in Step 12.1.3.2 through 

12.1.4.4 on any cell culture vessels that were negative during 
METHOD 1615. Enterovirus and Norovirus occurrence in water

33 

the first passage and positive in the second passage and with 

the negative assay controls. Other vessels that were either 

negative or positive in both the first and second passages do 

not need to be carried through the third passage. 

12.1.5.2. Score cultures that develop CPE in both the second and third 

passages as confirmed positives. 

12.2. VIRUS QUANTITATION 

12.2.1. Record the total number of confirmed and not confirmed positive and 

negative cultures for each subsample onto a Total Culturable Virus Data 

Sheet (section 17.3). 

12.2.2. Transfer the number of cultures inoculated and the number of confirmed 

positive cultures from the Total Culturable Virus Data Sheet for each 

sample to the Quantitation of Total Culturable Virus Data Sheet

(section 17.4). 

12.2.3. Calculate the MPN/mL value (MmL) and the upper (CLumL) and lower 

(CLlmL) 95% confidence limits/mL using the number of confirmed 

positive cultures from step 12.2.2 and the MPN computer program 

supplied by U.S. EPA (www.epa.gov/microbes). If dilutions are required, 

calculate the MPN/mL value and 95% confidence limits using the 

confirmed values from all dilutions, including dilutions with all positive or 

negative samples. 

12.2.3.1. Change the MPN program default settings as shown in Table 3. 

12.2.3.2. Note that the Inoculum Volume rather than the Inoculation 

Volume (see step 11.2.4.5) must be used. 

12.2.3.3. The MPN program will run on Windows XP and later versions. 

It has been re-designed for calculation of both standard 

bacterial and viral MPN values. All entries are saved in a 

default database and can be viewed to check for data entry 

errors using the View History selection under the Tools menu. 

Each program run can also be saved into Word, Excel, or text 

files for transfer to lab notebooks or to Laboratory Information 

Management Systems. 

12.2.4. Place the values obtained onto the Quantitation of Total Culturable 

Virus Data Sheet (section 17.4). 

12.2.5. Calculate the MPN per L value (ML) of the original water sample using 

equation 2 where S equals the Assay Sample Volume and D equals the 

Volume of Original Water Sample Assayed (the values for S and D can 

be found on the Virus Data Sheet). Record the value of ML onto the 

Virus Data Sheet (section 17.2). For MmL values of 0, calculate the 

sample detection limit rather than the ML value by dividing 1 by D. 

Report as equal to or less than the calculated detection limit. For samples 

where more than one cartridge filter or a prefilter was used, place the total 
METHOD 1615. Enterovirus and Norovirus occurrence in water

34 

MPN/L and Confidence Limits/L (from steps 12.2.7 and 12.2.9) values for 

all filters on the Virus Data Sheet and place the individual totals for each 

filter under “Other Comments.” 

D

M S M = m L

L Equation 2 

12.2.6. For example, if the sample described in the example in step 11.2.4.2, 

which has an Inoculum Volume equal to 0.833 mL, had 4 positive 

replicates, the MPN/mL value is 0.61 with 95% Confidence Limits of 0.12 

to 1.31. The MPN per L value then equals (0.61 MPN/mL x 8.33 mL)/500 

L = 0.0102. 

12.2.7. Calculate the lower 95% confidence limit per L value (CLL) for each 

water sample using equation 3 where CLlmL is the lower 95% confidence 

limit per milliliter from the Quantitation of Total Culturable Virus Data 

Sheet. Record the limit per L values on the Virus Data Sheet. 

D

CL S CL lmL

L  Equation 3 

12.2.8. Continuing with the example from step 12.2.5, CLL = (0.12 MPN/mL x 

8.33 mL)/500 L = 0.002. 

12.2.9. Calculate the upper 95% confidence limit per L value (CLU) using 

equation 4 where CLumL is the upper 95% confidence limit per milliliter 

from the Quantitation of Total Culturable Virus Data Sheet. Record 

the limit per L values on the Virus Data Sheet. 

D

CL S CL umL

U  Equation 4 

12.2.10. Continuing with the example from step 12.2.5, CLU = (1.31 MPN/mL x 

8.33 mL)/500 L = 0.0218. 

12.2.11. Calculate the total MPN value and the total 95% confidence limit values 

for each QC samples by multiplying the values per milliliter by S and 

dividing by 0.3. 

13. ENTEROVIRUS AND NOROVIRUS MOLECULAR ASSAY

The molecular assay uses RT-qPCR based upon standard curves to provide a quantitative 

estimate of enterovirus and norovirus genomic copies per liter in environmental and 

drinking waters. Only microliter volumes can be analyzed by RT-qPCR, so the procedure 

includes additional concentration of any viruses present in the sample beyond that required 

for culture. Surface and ground waters may contain substances that interfere with RTqPCR, so the assay uses an RNA extraction kit to reduce inhibition and a control to identify 

samples that are inhibitory to RT-qPCR. The assay uses primers and probes from the 

scientific literature (Table 4). The primer/probe sets are designed to detect many 

enteroviruses and noroviruses. Standard curves are prepared from Armored RNA®

reagents that contain the target sequence for the primer/probe sets. Armored RNA was 

chosen for standard curves because it is difficult to obtain high titered norovirus stocks. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

35 

13.1. PRELIMINARY PROCEDURES 

13.1.1. Purchase the primers and probes shown in Table 4. 

13.1.2. Prepare 100 μM stock solutions of each oligonucleotide primer (probes 

should come from the manufacturer at 100 μM solutions). 

13.1.2.1. Centrifuge the vials containing primers in a microcentrifuge for 

30 seconds. 

13.1.2.2. Dissolve the primers in a volume of molecular or PCR grade 

water (e.g., Roche Cat. No. 03 315 932 001) that equals the 

number of nanomoles of primer shipped (as identified on the 

specification sheet from the manufacturer) times 10 (in μl; e.g., 

if a primer contains 51.0 nmol; resuspend in 510 μl). 

13.1.2.3. Measure the absorbance at 260 nm. Calculate the total 

absorbance by multiplying the absorbance value by the volume 

used to resuspend the primer (and by any dilution required to 

obtain the absorbance reading). Confirm that the reading is the 

same as the total absorbance value recorded on the 

manufacturer’s specification sheet. Note: Probes should be 

quality checked using this same procedure. 

13.1.2.4. Aliquot the stock solutions into small quantities to avoid 

multiple freeze thaw cycles and store at -20oC. 

13.1.3. Prepare 10 μM primer and probe working solutions by diluting the stock 

solutions 1:10 (or by a dilution that compensates for any differences 

between the absorbance reading obtained in Section 13.1.2.3 and the 

manufacturer’s specification sheet) in molecular or PCR grade water. 

Aliquot stocks and store at -20oC. 

13.1.4. Note: Preparation of primers and probes must be performed in a clean 

room or other location to minimize the possibility of false positive 

reactions. A clean room or location is one in which molecular and 

microbiological procedures are not performed. 

13.2. TERTIARY CONCENTRATION OF SUBSAMPLE 2 

13.2.1. For each sample to be analyzed, label a Vivaspin 20 unit with the sample 

number, analyst’s initials and date, and fill the unit with PBS, 0.2% BSA 

(item 7.4.5). Soak at least 2 hours at room temperature or overnight at 

4oC. 

13.2.2. Discard the PBS, 0.2% BSA from the Vivaspin 20 unit and add an amount 

of the appropriate subsample 2 (see section11.2.4.4.2) equal to the Assay 

Sample Volume (S). 

13.2.3. Centrifuge at 3,000 x g with swinging buckets until the sample has been 

concentrated down to about 50 μL. 

13.2.4. Add 1 mL of sterile 0.15 M sodium phosphate, pH 7-7.5, and repeat step 

13.2.3. Repeat one additional time. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

36 

13.2.5. Transfer the concentrate to a 1.5 ml microcentrifuge tube. Measure the 

volume and add 0.15 M sodium phosphate, pH 7-7.5 to bring the total 

volume to 0.4 mL. 

13.2.6. Immediately proceed to step 13.3 or hold at 4°C for no more than 72 

hours. Note: Freeze/thawing leads to norovirus losses. 

13.3. NUCLEIC ACID ISOLATION 

13.3.1. Preliminary procedures 

13.3.1.1. Add 310 μL of Buffer AVE (item 7.4.8) to a vial of carrier 

RNA (from item 7.4.7) to give a final concentration of 1 

μg/μL. 

13.3.1.2. Aliquot and store at -20°C. Prepare a sufficient number of 

aliquots so that each aliquot does not have to be frozen and 

thawed more than three times. 

13.3.1.3. Add carrier RNA to Buffer AVL (item 7.4.7; note: do not use 

the Buffer AL that comes with the Qiagen kit) to give a 

concentration of 0.027 μg/μL by adding, per sample, 5.6 μL of 

carrier RNA (from step 13.3.1.2) to 200 μL of Buffer AVL 

(e.g., 5.6 μL carrier RNA × number of samples + 200 μL 

Buffer AVL × number of samples). 

13.3.2. For each sample to be processed, label a 1.5 mL microcentrifuge tube and 

add 200 μL of the appropriate sample from section 13.2.5 (or from section 

12.1.4.1 for confirmation of culture positive samples) and vortex briefly to 

mix. Freeze any remaining tertiary concentrate at -70°C. 

13.3.3. Add 200 μL of Buffer AVL with carrier RNA from step 13.3.1.3. Vortex 

for 15 seconds. 

13.3.4. Incubate at 56°C for 10 minutes. 

13.3.5. Centrifuge at >5,000 rpm for 5 seconds in a microcentrifuge. 

13.3.6. Add 200 μL of ethanol. Vortex for 15 seconds and then centrifuge for 

about 5 seconds. 

13.3.7. Add the mixture to a QIAamp Mini Spin column, but avoid wetting the 

rim of the tube. Close the cap. 

13.3.8. Centrifuge at 6,000 x g for 1 minute. Check to determine if the sample 

has completely passed through the column. If it has not, centrifuge again 

for 1 minute at 10,000-20,000 x g or for longer times until the sample has 

completely passed through the column. 

13.3.9. Place the Mini Spin column into a clean 2 mL collection tube (item 7.4.9) 

and discard the collection tube containing the filtrate. 

13.3.10. Add 500 μL of Buffer AW1 without touching the tube rim. 

13.3.11. Centrifuge at 6,000 x g for 1 minute. Again, transfer the column to a clean 

collection tube and discard the tube containing the filtrate. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

37 

13.3.12. Add 500 μL of Buffer AW2 without touching the tube rim. 

13.3.13. Centrifuge at 20,000 x g for 3 minutes. Again, transfer the column to a 

clean collection tube and discard the tube containing the filtrate. 

13.3.14. Centrifuge at 20,000 x g for 1 minute. 

13.3.15. Add 40 units of RNasin to a clean microcentrifuge tube. Transfer the 

column from the collection tube to the 1.5 mL microcentrifuge tube and 

discard the collection tube. Alternatively, RNasin can be added to an 

amount of Buffer AE sufficient for the number of samples to be eluted at a 

concentration of 400 units/mL in place of adding it to the microcentrifuge 

tubes. 

13.3.16. Add 50 μL of Buffer AE to the column. Incubate at room temperature for 

1 minute and then centrifuge for 1 minute at 6,000 x g. 

13.3.17. Repeat step 13.3.16. Remove and discard the column. 

13.3.18. Proceed immediately to step 13.4 or prepare aliquots and store the RNA at 

-70°C until it can be assayed. 

13.4. REVERSE TRANSCRIPTION (RT) 

13.4.1. Preliminary procedures (to be performed in a clean room) 

13.4.1.1. Label PCR plates or tubes with appropriate sample numbers. 

13.4.1.2. Prepare RT master mix 1 and 2 using the guide in Table 5. The 

amounts shown for the volume per master mix can be scaled up 

or down according to the number of samples that need to be 

analyzed. 

13.4.1.3. Vortex the master mixes after the addition of all ingredients. 

13.4.1.4. Centrifuge at >5,000 rpm for 10 seconds in a microcentrifuge. 

13.4.2. Aliquot 16 μL of RT master mix 1 to the labeled PCR tubes or plate wells. 

13.4.2.1. Run every environmental water, QC, and PE sample in 

triplicate by adding 6.7 μL of the appropriate sample to each of 

the tubes or plate wells labeled for that sample. Note: It is not 

necessary to prepare 1:5 and 1:25 dilutions of the QC samples 

as done for the culture assay (section 12.1.2.2). See Figure 4 

for a schematic of the RT-qPCR process. 

13.4.2.2. Add 6.7 μL of molecular grade water to one or more tubes or 

plate wells as no template controls (NTC). Include at least one 

NTC for the replicates associated with every fourth water 

sample run on a plate; NTC controls must be distributed 

throughout the plate. Note: If any NTC control is positive, the 

cause of the false positive value should be investigated. After 

fixing the cause of the problem, all samples on the plate must 

be rerun. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

38 

13.4.2.3. Close the tubes or seal the plates and heat at 99°C for 4 

minutes, followed by quenching on ice or a hold temperature of 

4°C. 

13.4.3. Add 17.3 μL of RT mix 2 to each tube or well. 

13.4.4. Centrifuge at >5,000 rpm for 10 seconds in a microcentrifuge at 4°C. 

13.4.5. Run at 25°C for 15 minutes, 42°C for 60 minutes, 99°C for 5 minutes, and 

then hold at 4°C for up to 2 hours until PCR is performed. Note: Thermal 

cyclers from a number of different manufacturers can be used for this and 

the following real-time quantitative PCR step. Analysts must follow the 

manufacturers’ instructions for set-up, runs, and analysis for the 

instrument used. 

13.4.6. Centrifuge at >5,000 rpm for 10 seconds in a microcentrifuge. 

13.4.7. Proceed immediately to step 13.5 or store reverse transcribed samples at -

70°C until they can be processed. 

13.5. REAL-TIME QUANTITATIVE PCR (qPCR) 

13.5.1. Preliminary procedures 

13.5.1.1. Label PCR plates or tubes with appropriate sample numbers. 

13.5.1.1.1. Note: each sample will require 15 plate wells or 

tubes (i.e., 3 RT replicates times 5 PCR assays; 

see Figure 4). 

13.5.1.1.2. To save reagents, the HGV inhibition control 

assay may be run before all other qPCR assays 

(see section 13.5.8). 

13.5.1.2. Prepare PCR master mixes using the guides in Tables 6-10. 

The amounts shown for the volume per master mix can be 

scaled up or down according to the number of samples that 

need to be analyzed. 

13.5.1.3. Vortex the master mix after the addition of all ingredients. 

13.5.1.4. Centrifuge at >5,000 rpm for 10 seconds in a microcentrifuge. 

13.5.1.5. Dispense 14 μL of the appropriate mix to the labeled plates or 

tubes. 

13.5.2. Add 6 μL of the appropriate sample to each tube (see Figure 4). 

13.5.3. Run on a thermal cycler with a setting of 1 cycle at 95°C for 10 minutes 

followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. 

13.5.4. Analyze the results of each run to calculate the Genome Copy (GC) 

numbers of unknown samples according to the instructions of the 

manufacturer of the thermal cycler used based upon the standard curve 

samples described in step 13.5.9. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

39 

13.5.5. Record the GC values on the Molecular Virus Results Data Sheet

(section 17.6) of each replicate and mean and standard deviation of the 

three replicates for each sample. Include non-detects (zeros) in the 

calculation of the mean 

13.5.6. Calculate the Genomic Copies per L (GCL) for each sample using 

equation 5 and the mean GC value from step 13.5.4, where DF equals the 

reciprocal of any dilution performed to compensate for inhibition (see step 

13.5.8; e.g., 5 and 25, or 1 for undiluted samples) and D equals the 

Volume of Original Water Sample Assayed (see step11.2.4.2). 

D

GC DF GCL

   199 Equation 5

For example, if the PCR assay from the sample example described in step 

11.2.4.2 detects 15 genomic copies in a 1:5 dilution, then the number of 

genomic copies per 100 L is 100 x ((15 x 199 x 5)/500 L) =2985. Note: 

199 is the total dilution factor for the volume reductions that occur in steps 

13.2 through 13.5. 

13.5.6.1. Record the GCL value on the Molecular Virus Results Data 

Sheet (section 17.6). 

13.5.6.2. For water samples with a mean value of zero, report the 

Genomic Copies per L as less than or equal to the detection 

limit (i.e., ≤1/D). 

13.5.7. Calculate the genomic copies of QC samples by multiplying the mean 

genomic copy value by 199 and dividing by 0.3. 

13.5.8. Inhibition Control 

13.5.8.1. Hepatitis G Armored RNA is used as an inhibition control 

rather than specific enterovirus and norovirus to reduce the 

possibility of cross-contamination that could occur between 

seeded and unseeded samples with the actual virus groups 

being tested.

13.5.8.2. Before running any water samples, run ten replicate RT and 

hepatitis G qPCR assays to determine the mean hepatitis G Cq 

value and standard deviation.

13.5.8.2.1. Process a volume of FCSV from at least two 

negative QC controls (step 11.2.4.6) equal to the 

Assay Sample Volume (S) using steps 13.2 to 

13.3. Use 6.7 μL of the RNA from one of these 

samples; hereafter designated “negative FCSV” 

for five of the replicates and 6.7 μL from the other 

sample for the other five replicates. 

13.5.8.2.2. Calculate the mean quantitation cycle (Cq) value 

and standard deviations. Record the results on the 

Molecular Virus Results Data Sheet (section 
METHOD 1615. Enterovirus and Norovirus occurrence in water

40 

17.6). The mean value should be between 25 and 

32 Cq units. The standard deviation of the mean 

should be <0.3 units. 

13.5.8.2.3. Note: If the mean value is not between 25 and 32 

Cq units, readjust the amount of Hepatitis G 

Armored RNA added to RT Master Mix 1 (see 

Table 5) and repeat step 13.5.8.2 until the value is 

within the acceptable range. 

13.5.8.3. Compare the hepatitis G Cq values obtained with all samples 

against the value calculated in step 13.5.8.2. If the value in the 

unknown samples is more than 1 Cq value higher than that 

calculated in step 13.5.8.2 dilute the unknown sample 1:5 and 

1:25 in molecular grade (nuclease-free) water.

13.5.8.4. Re-run the sample along with the 1:5 and 1:25 dilution.

13.5.8.5. Because some samples will demonstrate some level of 

inhibition, the hepatitis G assay can be run with all samples 

prior to doing the other qPCR assays as stated in step 

13.5.1.1.2. All other assays can then be run without dilution 

for samples giving no inhibition and with the appropriate 

dilution for all other samples.

13.5.8.6. Calculate the sample concentration using the highest dilution 

for which the hepatitis G Cq values are within 1 unit of the 

value calculated in step 13.5.8.2. If the inhibition control fails 

again and the sample Cq value is lower than 38, re-run the 

sample at higher five-fold dilutions. If any sample run at the 

higher dilution fail the inhibition control again or if any 

unknown samples are below the detection limit (e.g., Cq values 

of 45 or higher), list the sample as a potential false negative 

sample on the Molecular Virus Results Data Sheet (section 

17.6)

13.5.9. Preparation of standard curves. Note: Analysts must run new standard 

curves (a single standard curve for each Armored RNA standard) with 

every set of unknown samples run together or use calibrators (see step 

13.5.10) with stored standard curves, if the thermal cycler allows standard 

curves to be stored.

13.5.9.1. Dilute enterovirus, norovirus GI and norovirus GII Armored 

RNA in negative FCSV to give a concentration of 2.5×106

particles/250 μL (i.e., 1×107

/mL) based upon the concentration 

of the Armored RNA lots supplied. Note: unless specified 

otherwise by EPA, transcribed RNA from plasmids containing 

the appropriate viral sequence or poliovirus, norovirus GI, and 

norovirus GII stocks may be substituted for the Armored RNA.
METHOD 1615. Enterovirus and Norovirus occurrence in water

41 

13.5.9.1.1. Transcribed RNA must be titered using standard 

methods, e.g., see reference (32), and genomic 

copies per mL substituted for particles per mL 

above.

13.5.9.1.2. Virus stocks must be titered using RT-qPCR on 

endpoint serial dilutions and ten replicates per 

dilution for each stock. The MPN calculator must 

then be used to obtain the titer of the stock and the 

MPN value substituted for particles per mL 

above. If the efficiency of the standard curve is in 

the acceptable range (see step 13.5.9.7), genome 

copies per mL can be substituted for MPN/mL.

13.5.9.2. Prepare 6 ten-fold dilutions to give concentrations of 1×106, 

1×105

, 1×104, 1×103, 1×102, 1×101 and 1 × 100 particles/mL. 

Prepare by serially adding 25 μL of Armored RNA to 225 μL 

of negative FCSV to create each dilution with vortexing for 5-

15 seconds before proceeding to the next higher dilution.

13.5.9.3. Run 200 μL of each dilution separately through steps 13.3-

13.5.4 using the volumes described in the steps and only the 

specific primers/probe for the Armored RNA standard. This 

will result in six standards with 100

 to 105 particles per assay.

13.5.9.4. Calculate the standard curve slope and R2 values for each 

standard curve by plotting Cq values against the log of the 

concentration for each point or, if available, by using the slope 

and R2

 values determined by the qPCR instrument.

13.5.9.5. Calculate the mean of the slopes and standard deviation of the 

mean for the three runs and the mean of the R2

 values. Note: 

An acceptable standard curve will have a mean R2

 value >0.97 

and a standard deviation of <0.25. Standard deviations of 0.25 

or higher represent errors in preparing dilutions or in pipetting.

13.5.9.6. Calculate the percent amplification efficiency using equation 6. 

Record the results on the Molecular Virus Protocol Data 

Sheet (section 17.5).

% 100 (10 1) 1     slope Efficiency Equation 6 

13.5.9.7. For example, the ideal efficiency occurs when the slope equals 

-3.32, where the % Efficiency = 100 x (10-1/-3.33 – 1) = 100 x 

(2.0-1) = 100. Note: An acceptable standard curve will have an 

amplification efficiency of 80-110%.

13.5.9.8. Standard curves that meet the criteria specified in steps 

13.5.9.5 and 13.5.9.6 above must be used to calculate genomic 

copies of unknown samples in step 13.5.4.

13.5.10. Preparation of calibrators and standard curves for use with calibrators
METHOD 1615. Enterovirus and Norovirus occurrence in water

42 

13.5.10.1. Stored standard curves must be used with calibrators. The 

stored standard curves must be based upon the mean of three 

runs with each set of standards. New standard curve sets must 

be generated as described above every eighth sample batch (see 

section 3) or every two months, whichever comes first, or 

anytime a calibrator fails twice in a row to meet acceptance 

criteria.

13.5.10.2. To prepare calibrators for each virus standard, choose the 

dilution from the standard curve that gives the Cq value closest 

to, but not greater than 32. Note: calibrators must be run with 

all unknown samples if stored standard curves are used, but 

may be run even if standard curves are run with each test as an 

additional quality check.

13.5.10.3. Prepare the dilution corresponding to the chosen value in 

negative FCSV and extract the RNA as described in step 13.3. 

Prepare a sufficient number of dilutions to last for the entire 

study, taking into consideration that each 200 μL extraction 

will yield sufficient material for about 20 runs. Aliquot into 

single run batches and store at -70°C.

13.5.10.4. Run a set of at least ten calibrators from each Armored RNA 

standard. Calculate the mean Cq value and standard 

deviations. Record the mean and standard deviation values as 

the Target Value on the Molecular Virus Results Data Sheet

(section 17.6). The standard deviation must be less than 0.25 

units.

13.5.10.5. Run all calibrators with every set of unknown samples. Accept 

a run if the value of the calibrator for each corresponding PCR 

assay falls within 1.0 Cq units of the calibrators’ mean values. 

Record the observed value for each target virus as the Actual 

Value on the Molecular Virus Results Data Sheet (section 

17.6).

13.5.10.6. Reject sample sets run in a PCR assay where the calibrator for 

that assay falls outside the acceptance criteria. Repeat the run 

once upon failure. If the assay fails again, generate a new 

standard curve or take steps to determine the cause of the 

failure.

14. METHOD PERFORMANCE

14.1. CULTURABLE ASSAY 

14.1.1. This method is subject to a number of biases that reduce its precision and 

accuracy. The isoelectric point of the virus particle affects its ability to 

bind to, and be eluted from, electropositive filters. The isoelectric point 

can vary significantly across virus species and even within members of the 

same species. Other capsid and matrix related characteristics and 
METHOD 1615. Enterovirus and Norovirus occurrence in water

43 

substances could affect virus recovery at various stages of the method. 

The passage number of the BGM cell line and the media used to passage 

and maintain cells is known to affect the ability of viruses to replicate in 

cells. The performance characteristics given below are based upon Sabin 

poliovirus type 3 and may not be reflective of other viruses that are 

detected by this method. The best performance data for the method comes 

from the performance evaluation (PE) samples that were analyzed during 

the ICR as described (19). In total, 12 laboratories with 25 ICR-approved 

analysts analyzed 828 PE samples, consisting of low (<300 MPN per 

filter), medium (300-1,500 MPN per filter) and high (>1,500 MPN per 

filter) virus levels. The mean interlaboratory recovery was 56% with a 

coefficient of variation (CV) of 92%, a false negative rate of 1.3%, and a 

false positive rate of 1.1%. The highest mean recoveries values (71%) 

were obtained with the PE samples containing the low virus levels. Table 

11 gives the mean recovery and CV value ranges for individual analysts 

and intralaboratory variation. Although Method 1615 uses a different 

electropositive filter than the ICR study, both filters have been shown to 

give the same recoveries in a single (24) and a four laboratory validation 

study (unpublished data). 

14.1.2. The detection limit of the culture method is 0.01 MPN/L for surface water 

and 0.002 MPN/L for groundwater. 

14.1.3. Acceptance limits of PE and QC samples is set for this method at a mean 

recovery of 35 to 150% with a CV of less than or equal to 70% based upon 

the 90% confidence limits of the data from the ICR PE analyses. 

14.2. MOLECULAR PROCEDURE 

14.2.1. The molecular procedure is subject to the same bias as the culturable 

procedure in terms of virus adsorption and recovery from the 

electropositive filters and secondary concentration procedures. Additional 

bias can occur during tertiary concentration, RNA elution and RT-qPCR. 

The method was tested using seven groundwater samples from five 

different wells with a range of physicochemical characteristics. In 

addition to bias from matrix effects, these tests may have had additional 

bias because they were performed as described in section 8.6. The seven 

groundwater samples gave a mean recovery of 26% with a recovery range 

of 5-60% and a CV of 73%. These same samples were also tested for 

norovirus recovery using Murine norovirus and Murine norovirus specific 

primers and probe with the Method 1615 protocols. Mean recovery of 

Murine norovirus was 35% with a recovery range of 7-63% and a CV of 

69%. 

14.2.2. As with the culture method, the acceptance criteria for the molecular 

procedure is based upon the variation among PE samples seeded with 

Sabin poliovirus type 3. The acceptance criteria for the molecular 

procedure is a mean recovery of 20 to 150% with a CV of less than or 

equal to 75% based upon genomic copies detected by the molecular 
METHOD 1615. Enterovirus and Norovirus occurrence in water

44 

procedure and the genomic copies seeded onto the PE samples, unless 

specified otherwise by EPA for specific applications. 

14.2.3. The detection limit of the molecular method based upon the overall 

detection limit of the RT-qPCR assay and the volume of water sample 

assayed. The detection limit for the poliovirus assay is about 2 and 0.4 

genomic copies/L for surface water and groundwater, respectively. The 

detection limit can be increased by running more than three RT-qPCR 

replicates from each sample. 

14.3. PERFORMANCE RECORD 

The laboratory should maintain a record of the performance of QC and PE samples 

for both the cultural and molecular portions of this method and measure each 

individual performance against the ongoing laboratory values and the performance 

data given in this section. This should be done by continuously updating the mean 

recovery and coefficient of variation values with the addition of each new QC and 

PE sample. 

15. STERILIZATION AND DISINFECTION

15.1. GENERAL GUIDELINES 

15.1.1. Use aseptic techniques for handling test waters, eluates and cell cultures. 

15.1.2. Sterilize apparatus and containers that will be exposed to test waters and 

all solutions that will be added to test waters unless otherwise indicated. 

Thoroughly clean all items before final sterilization using laboratory 

standard operating procedures. 

15.1.3. Sterilize all contaminated materials before discarding. 

15.1.4. Disinfect all spills and splatters. 

15.2. STERILIZATION TECHNIQUES 

15.2.1. Solutions 

15.2.1.1. Sterilize all solutions, except those used for cleansing, standard 

buffers, HCl, NaOH, and disinfectants by autoclaving them at 

121°C for at least 15 minutes. 

15.2.1.2. HCl, NaOH, and disinfectants used are self-sterilizing. When 

autoclaving buffered beef extract, use a vessel large enough to 

accommodate foaming. 

15.2.2. Autoclavable glassware, plasticware, and equipment 

15.2.2.1. General considerations: Add sufficient dH2O to all vessels to 

be autoclaved to equal about 1-2% of the vessel’s rated 

volume. Water speeds the sterilization process by enhancing 

the transfer of heat. Place large vessels on their sides in the 

autoclave, if possible, to facilitate the displacement of air in the 

vessels by flowing steam. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

45 

15.2.2.2. Cover the openings into autoclavable glassware, plasticware, 

and equipment loosely with aluminum foil before autoclaving. 

Autoclave at 121°C for at least 30 minutes. 

15.2.2.3. Glassware may also be sterilized in a dry heat oven at a 

temperature of 170°C for at least 1 hour. 

15.2.2.4. Sterilize stainless steel vessels (dispensing pressure vessel) in 

an autoclave at 121°C for at least 30 minutes. Vent-relief 

valves on vessels so equipped must be open during autoclaving 

and closed immediately when vessels are removed from 

autoclave. 

15.2.2.5. Pre-sterilize 1MDS filters and prefilters by wrapping the filters 

in Kraft paper and autoclaving at 121°C for 30 minutes. 

Warning: Do not autoclave the NanoCeram filters specified in 

item 6.1.2.4. These filters are sterilized by the manufacturer 

and have housings that cannot be autoclaved. 

15.2.3. Sterilize instruments, such as scissors and forceps, by immersing them in 

95% ethanol and flaming them between uses. 

15.2.4. Sodium hypochlorite sterilization - Sterilize pumps, plasticware (filter 

housings) and tubing that cannot withstand autoclaving, and vessels that 

are too large for the autoclave with sodium hypochlorite (item 7.1.4). 

Ten-inch cartridge prefilters, but not NanoCeram or 1MDS filters, may be 

presterilized with sodium hypochlorite as an alternative to autoclaving. 

Filter apparatus modules should be disinfected after use by sterilization 

and then cleaned according to laboratory standard operating procedures 

before final sterilization. 

15.2.4.1. Sterilize the items that cannot be autoclaved by recirculating or 

immersing the items in 0.525% sodium hypochlorite for 30 

minutes. pH electrodes should be sterilized with 0.525% 

hypochlorite for at least 5 minutes. 

15.2.4.2. Drain the hypochlorite from the objects being sterilized and 

rinse in sterile water. 

15.2.4.3. Dechlorinate by recirculating or immersing the items in a 

solution containing 50 mL of 1M sodium thiosulfate per liter of 

sterile dH2O. . Ensure that the sodium hypochlorite (step 

15.2.4.1) and sodium thiosulfate (step 15.2.4.3) solutions come 

in full contact with all surfaces when performing this 

procedure. 

15.2.4.4. Cover the apparatus module ends and the injector port(s) with 

sterile aluminum foil. 

15.2.4.5. Place the injector module and tubing into a sterile bag or 

wrapping in such a way that they may be removed without 

contaminating them. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

46 

15.2.5. Contaminated materials 

15.2.5.1. Autoclave contaminated materials for at least 30 minutes at 

121°C. Be sure that steam can enter contaminated materials 

freely. 

15.2.5.2. Many commercial disinfectants do not adequately kill enteric 

viruses. To ensure thorough disinfection, disinfect spills and 

other contamination on surfaces with either a solution of 0.5% 

iodine (item 7.2.2) or 0.525% sodium hypochlorite. The iodine 

solution has the advantage of drying more rapidly on surfaces 

than sodium hypochlorite, but may stain some surfaces. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

47 

16. TABLES AND FIGURES

Table 1. Viruses Detected by EPA Method 1615

Virus Genus or Species Detected by TCVAa Detected by qPCR 

Human enterovirus A Some serotypes Yes 

Human enterovirus B Most serotypes Yes 

Human enterovirus C Some serotypes Yes 

Human enterovirus D Some serotypes Yes 

Norovirus genogroup I and II No Many genotypes 

Mammalian orthoreovirus Yes No 

a

 TCVA – Total Cultural Virus Assay (see section 12) 

Table 2. Specified and Recommended Sample Volumes 

Water Type Flow Ratea

(L/min) 

Sampling duration 

(hours) 

Sample Volume 

(liters)b,c

Sewage effluent 10 0.2 120d

Surface 10 0.6 360e

Finished/Groundwater 10 3.0 1,800f

Finished/Groundwater 4 16±2 ≤4,320g

a

 Poliovirus retention is independent of flow rates between 4 and 20 L/minute for 

NanoCeram filters, but a constant flow rate, such as describe here, should be used for any 

single study (24). EPA may specify alternative flow rates for specific studies. 

b

 Consistent sample volumes should be used for any single study. EPA may specify 

alternative sample volumes for specific studies. 

c

 Turbidity and other factors may affect the volume collected during any sampling event. 

The sample duration must be increased to meet the specified or recommended volume 

during these situations. As an alternative, two cartridge filter modules may be used to 

obtain the specified volume. 

d

 This is a recommended value for final sewage effluents. There is no recommended 

volume for raw sewage. 

e

 The minimum specified volume is 300 L for surface waters. 

f

 The minimum specified volume is 1,500 L for treated tap or untreated groundwater. 

g

 For convenience, samples may be collected by starting the sampling at the end of a work 

day and stopping it in the morning of the next day. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

48 

Table 3. MPN Program Default Settings for MPN Calculator 

Item Default Setting 

Data Entry Mode Keyboard 

Dilution type Standard 5-Fold Serial 

Approximation type Cornish & Fisher Limits 

Confidence Level 95% 

Number of Dilutions 1 (or 3 or actual number of dilutions) 

Number of Tubes per dilution 10 

Inoculum volume (mL) The Inoculum Volume used (see section 

11.2.4.5) 

Table 4. Primers and TaqMan® Probes for Virus Detection by RT-qPCR 

Virus 

group Primer pairsa,b TaqMan Probec,d Reference 

Enterovirus 

CCTCCGGCCCCTGAATG 

ACCGGATGGCCAATCCAA 

CGGAACCGACTACTTTGGGTGTCCGT 

De Leon et al. (17) 

Monpoeho et 

al.(30)

Norovirus

GIA

GCCATGTTCCGITGGATG 

TCCTTAGACGCCATCATCAT 

TGTGGACAGGAGATCGCAATCTC Jothikumar et al. 

(23)

Norovirus 

GIB

CGCTGGATGCGNTTCCAT 

CCTTAGACGCCATCATCATTTAC 

TGGACAGGAGAYCGCRATCT Butot et al. (12) 

Norovirus 

GII

ATGTTCAGRTGGATGAGRTTCTCWGA 

TCGACGCCATCTTCATTCACA 

AGCACGTGGGAGGGGATCG Butot et al. (12) 

Hepatitis G 

CGGCCAAAAGGTGGTGGATG 

CGACGAGCCTGACGTCGGG 

AGGTCCCTCTGGCGCTTGTGGCGAG Schlueter et al. 

(35)

a

 EPA may specify additional or alternative primer sets for specific applications. Degenerate 

bases in primers and probes are as follows: N equals a mixture of all four nucleotides; R 

equals A + G; Y equals T + C; and W equals A + T. 

b

 The top primer in each set is the forward primer and the bottom primer is the reverse primer. 

All primer and probe sequences are 5’ to 3’. 

c

 Probes are labeled on the 5’-end with 6-FAM and with TAMRA on the 3’-end. 

d

 Primers and probes are designated in Tables 6-10 by the first three letters of the virus name 

followed by F, R, or P for forward, reverse, and probe. GIA, GIB, or GII are also added to the 

norovirus designations. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

49 

Table 5. RT Master Mix 1 and 2 

Ingredient Volume per 

Reaction (μL)a

Final Concentration Volume per 

Master Mix (μL)b

RT Mix 1 

Random Primer 0.8 10 ng/μL (c. 5.6 μM) 77.6 

Hepatitis Gc

 1 97 

PCR grade water 14.2 1377.4 

Total 16 1552 

RT Mix 2

10X PCR Buffer II 4 10 mM tris, pH 8.3, 

50 mM KCL 388 

25 mM MgCl2 4.8 3 mM 465.6 

10 mM dNTPs 3.2 0.8 mM 310.4 

100 mM DTT 4 10 mM 388 

RNase Inhibitord

 1 0.5 units/μL 97 

SuperScript II RT 0.3 1.6 units/μL 29.1 

Total 17.3 1678.1 

a

 The volumes given are for 40 μL RT assays. 

b

 The volumes shown should be multiplied by the number of reactions to be performed plus 

an additional 0.5-1 reaction to account for losses during transfer of the master mix to tubes 

or plates. The amounts shown are sufficient for a 96 well PCR plate. 

c

 Hepatitis G Armored RNA is supplied as an untitered stock. The amount to use should be 

determined for each lot as described in section 13.5.8.2. 

d

 The amount shown is for a stock concentration of 20 unit/μL. The manufacturer’s stock 

concentration varies between 20 and 40 units/μL. Amounts need to be adjusted for stocks 

with higher concentrations. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

50 

Table 6. PCR Master Mix for Enterovirus Assay 

Ingredient Volume per 

Reaction (μL)a

Final Concentration Volume per Master 

Mix (μL)b

2X LightCycler 480 

Probes Master Mixc 10 Proprietary 970 

ROX reference dyed 0.4 0.5 mM 38.8 

PCR grade water 1 97 

EntF 0.6 300 nM 58.2 

EntR 1.8 900 nM 174.6 

EntP 0.2 100 nM 19.4 

Total 14 1358 

a

 The volumes given are for using 6 μL of cDNA from Step 13.4.7 in a qPCR assay using a total 

qPCR volume of 20 μL. 

b

 The volumes shown should be multiplied by the number of reactions to be performed plus an 

additional 0.5-1 reaction to account for losses during transfer of the master mix to tubes or 

plates. The amounts shown are sufficient for a 96 well PCR plate. 

c

 10X PCR Buffer II (2 μL/reaction), 25 mM MgCl2 (5 μL/reaction), and AmpliTaq Gold (0.2 

μL/reaction) can be substituted for the LightCycler 480 Probe Master mix. 

d

 This reagent is necessary for use with Applied Biosystems and similar instruments. It should 

be substituted with PCR grade water for use with the LightCycler and similar instruments. 

Table 7. PCR Master Mix for Norovirus GIA Assay 

Ingredient Volume per 

Reaction (μL)a

Final Concentration Volume per Master 

Mix (μL) 

2X LightCycler 480 

Probes Master Mix 10 Proprietary 970 

ROX reference dye 0.4 0.5 mM 38.8 

PCR grade water 1.4 135.8 

NorGIAF 1 500 nM 97 

NorGIAR 1 500 nM 97 

NorGIAP 0.2 100 nM 19.4 

Total 14 1358 

a

 See table 6 for legends. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

51 

Table 8. PCR Master Mix for Norovirus GIB Assay 

Ingredient Volume per 

Reaction (μL)a

Final Concentration Volume per Master 

Mix (μL) 

2X LightCycler 480 

Probes Master Mix 10 Proprietary 970 

ROX reference dye 0.4 0.5 mM 38.8 

PCR grade water 0.3 29.1 

NorGIBF 1 500 nM 97 

NorGIBR 1.8 900 nM 174.6 

NorGIBP 0.5 250 nM 48.5 

Total 14 1358 

a See table 6 for legends. 

Table 9. PCR Master Mix for Norovirus GII Assay 

Ingredient Volume per 

Reaction (μL)a

Final Concentration Volume per Master 

Mix (μL) 

2X LightCycler 480 

Probes Master Mix 10 Proprietary 970 

ROX reference dye 0.4 0.5 mM 38.8 

PCR grade water 0.3 29.1 

NorGIF 1 500 nM 97 

NorGIR 1.8 900 nM 174.6 

NorGIP 0.5 250 nM 48.5 

Total 14 1358 

a

 See table 6 for legends. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

52 

Table 10. PCR Master Mix for Hepatitis G Assay 

Ingredient Volume per 

Reaction (μL)a

Final Concentration Volume per Master 

Mix (μL) 

2X LightCycler 480 

Probes Master Mix 10 Proprietary 970 

ROX reference dye 0.4 0.5 mM 38.8 

PCR grade water 1.4 135.8 

HepF 1 500 nM 97 

HepR 1 500 nM 97 

HepP 0.2 100 nM 19.4 

Total 14 1358 

a

 See table 6 for legends. 

Table 11. Mean Recovery and Coefficient of Variation Range 

Variation Type Mean Recovery Range CV Range

Individual Analysts 36-98 34-157 

Intralaboratory 36-85 58-131 
METHOD 1615. Enterovirus and Norovirus occurrence in water

53 

Figure 1. Uninfected BGM Cells 

Figure 2. Early CPE from Poliovirus 
METHOD 1615. Enterovirus and Norovirus occurrence in water

54 

Figure 3. Sample Filtration Apparatus 

Figure 4. Elution of an Electropositive Filter with Beef Extract
METHOD 1615. Enterovirus and Norovirus occurrence in water

55 

Figure 5. RT-qPCR Schematic 

Each sample is reverse transcribed in triplicate (RT1, RT2, RT3) using 6.7 μL of extracted 

sample RNA for each RT assay in a 40 μL assay volume. Five qPCR assays (EV PCR, NoV 

GIA PCR, NoV GIB PCR, NoV GII PCR, and HGV PCR) are run from each of the triplicate RT 

reactions using 6 μL of cDNA for each qPCR assay. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

56 

17. DATA SHEETS

17.1. SAMPLE DATA SHEET 

Sample Data Sheet 

Sample Number/ID 

Utility Name 

Site Address 

City, State 

Sampler’s Namea 

Water Type Surface 

waters 

 Treated 

Surface or 

Groundwaters 

 Untreated 

Groundwater 

 Other (specify in 

comments section 

Location Treatment 

Plant/Pumping 

Station 

 Distribution 

system 

 Other (specify in comments 

section) 

Start of Sampling Event End of Sampling Event 

Date 

Time 

Totalizer Reading (L) 

Flow Rate (L/minute) 

Total Sample Volume (L) 

Water Parameter Readings 

Water Temperature 

pH 

Turbidity (NTU) 

Free Chlorine (mg/L) 

Quality Controls 

Flow meter model and serial number: 

Totalizer model and serial number: 

Date of last flow meter/totalizer calibration: 

Metering pump model and serial number 

Temperature meter model and serial number: 

pH meter model and serial number 

Turbidity meter model and serial number 

Chlorine test meter model and serial number 

Metering pump flow rate 

QC check performed 

 Yes 

Comments: 

a

 If any other individuals assist the sampler, include their name in the comments section and add 

the initials of the person who performed measurements after the recorded value. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

57 

17.2. VIRUS DATA SHEET 

VIRUS DATA SHEET 

Sample Number/ID: Sample Date: 

Sample Arrival Date: Hold Time Met (Y/N) 

Analytical Laboratory Name and ID: 

Analytical Laboratory Address: 

City: State: ZIP: 

Analyst Name (Please print or type): 

Sample Batch Number: 

Date Eluted: Time: 

Eluate Volume Recovered: L

Date Concentrated: Time: 

Centrifugation Speed (step 11.2.3.6): x g

Final Concentrated Sample Volume (FCSV): mL

Volume Of Original Water Sample Assayed (D)b: L

Assay Sample Volume (S): mL

Inoculum Volume: mL

Final Inoculation Volume (If Used): mL

Dates Assayed By CPE: 1st Passage 2nd Passage 

3rd Passage 

(If necessary) 

Subsample 1a: 

95% Confidence Limits/L 

MPN/Lc: Lower: Upper: 

Comments: 

Did a heavy floc form during the organic flocculation step? Yes___ No___ 

Was the floc difficult to dissolve? Yes___ No___ 

Other comments: 

Analyst Signature: 

a

 Use a separate Virus Data Sheet if it is necessary to assay an additional subsample 

b

 e.g., 100 L of surface water or 500 L of finished or ground waters 

c

 Value calculated from the Quantitation of Total Culturable Virus form as described in the Virus 

Quantitation section. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

58 

17.3. TOTAL CULTURABLE VIRUS DATA SHEET 

TOTAL CULTURABLE VIRUS DATA SHEET 

Sample Number: 

Incubator Model and Serial Number: 

Passage Sample 

Type Confirmeda Total Number of Replicates 

Inoculated Without CPE With CPE 

1st 

Neg. Cont. 

Pos. Cont. 

Undiluted 

1:5 Dil. 

1:25 Dil. 

1:125 Dil. 

2ndb

Neg. Cont. 

Pos. Cont. 

Undiluted 

1:5 Dil. 

1:25 Dil. 

1:125 Dil. 

3rdc 

Neg. Cont. 

Pos. Cont. 

Undiluted 

1:5 Dil. 

1:25 Dil. 

1:125 Dil. 

a Place a check (√) next to the negative controls and dilutions that were confirmed. 

b A portion of medium from each 1st passage vessel, including negative controls, must be 

passaged again for confirmation. The terms "Undiluted," "1:5 Dilution" and "1:25 Dilution" 

under the 2nd and 3rd Passage headings refer to the original sample dilutions for the 1st passage. c

 Samples that were negative on the first passage and positive on the 2nd passage must be 

passaged a third time for confirmation. If a third passage is required, negative controls must be 

passaged again. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

59 

17.4. QUANTITATION OF TOTAL CULTURABLE VIRUS DATA SHEET 

QUANTITATION OF TOTAL CULTURABLE VIRUS DATA SHEET 

SAMPLE NUMBER: 

Sample 

Number 

Replicates 

Inoculated 

Number 

with CPEa MPN/mLb

95% Confidence 

Limits/mL 

Lower Upper 

Undiluted 

 1:5 Dilution 

1:25 Dilution 

1:125 Dilution 

a The number of flasks with confirmed CPE from the second passage (or third passage, if 

necessary). 

b The MPN/mL and 95% Confidence Limit values must be obtained using the computer 

program supplied by the U.S. EPA. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

60 

17.5. MOLECULAR VIRUS PROTOCOL DATA SHEET 

MOLECULAR VIRUS PROTOCOL DATA SHEET 

Sample Number: 

Analytical Laboratory Name/ID: 

Analytical Laboratory Address: 

City: State: ZIP: 

Analyst Name (Please print or type): 

Subsample Number: 

Sample Batch Number: 

Tertiary Concentration Date: Time: Initials:a

Concentrator Cat. No/Lot No.: 

Assay Sample Volume Concentrated: bmL 

Final Tertiary Concentrated Volume: μL 

RNA Extraction Date: Time: Initials: 

RNA Extraction Kit Cat. No./Lot No.: 

Amount Used For RNA Extraction: μL 

RNA Extract Final Volume: μL 

Reverse Transcription (RT) Step 

Master Mixes Prepared Date: Time: Initials: 

RNA Extract Volume Used For RT: μL 

RT Samples Run: Date: Time: Initials: 

Thermal Cycler Used:c

PCR Step 

Master Mixes Prepared: Date: Time: Initials: 

Volume Of RT Used For PCR: μL 

PCR Samples Run Date: Time: Initials: 

Thermal Cycler/Real Time Instrument Used:c

Standard Curves 

Enterovirus Lot #d Eff.:e R2 SDf

Norovirus GI Lot # Eff. R2 SD 

Norovirus GII Lot # Eff. R2 SD 

a

 Record the initials of the analyst at the time this procedure is performed. 

b

 The Assay Sample Volume should be the same as recorded on the Virus Data Sheet. c

 Record the make and model of thermal cycler. 

d

 Assign a new lot number to each standard curve run. 

e

 Percent efficiency 

f

 Record the largest standard deviation among the different concentrations of the standard curve lot. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

61 

17.6. MOLECULAR VIRUS RESULTS DATA SHEET 

MOLECULAR VIRUS RESULTS DATA SHEET 

Sample Number: 

Analytical Laboratory Name/ID: 

Analytical Laboratory Address: 

City: State: ZIP: 

Analyst Name (Please print or type): 

RT-qPCR Results: 

All No Template Controls Negative: Yes Noa

Sample Type Target Value (Sd) b Actual Valuea,c

Inhibitor Control 

Enterovirus Calibrator 

Norovirus GI Calibrator 

Norovirus GII Calibrator 

Run Number:d 

If Required, Dilution Used In Calibration Of Sample Concentration: 

Replicatee 1 2 3 Mean (SD)b

Genomic Copy Values 

Genomic Copies per L:f 

a

 If any no template controls are positive or the inhibition control or calibrator falls outside 

specification limits, the samples must be re-run; however, each run should be recorded on this 

data sheet. 

b

 Record the mean and the standard deviation values for the sample type. For water samples, 

include non-detects (zeros) in the calculation of the mean. c

 Record the actual Cq value obtained for the sample type 

d

 A serial record identification of samples that have to be re-run. 

e

 If more than three replicates are used, record the data from the additional replicates onto 

another Molecular Virus Results Data Sheet. 

f

 Calculate the Genomic Copies per L using Equation 5. For water samples with a mean value of 

zero, report the Genomic Copies per L as less than or equal to the detection limit. 
METHOD 1615. Enterovirus and Norovirus occurrence in water

62 

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