EPA 600/R-15/130 I August 2015 I www.epa.gov/research
United States
Environmental Protection
Agency
Comparison of EPA Method 1615
RT-qPCR Assays in Standard and
Kit Formats
• *
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-15/130
August 2015
Comparison of EPA Method 1615 RT-qPCR
Assays in Standard and Kit Formats
by
G. Shay Fout,* Nichole E. Brinkman,* Jennifer L. Cashdollar,* Shannon M. Griffin,*
Asja Korajkic,* Brian R. McMinn,* Eunice A. Varughese,* Michael W. Ware,*
Eric R. Rhodes,* Ann C. Grimm, and Charles Li1"
*Biohazard Assessment Research Branch, Microbiological and Chemical Exposure
Assessment Research Division, National Exposure Research Laboratory, Office of
Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH
45268; +EMSL Analytical, Inc. 200 Route 130 North, Cinnaminson, NJ 08077
Microbiological and Chemical
Exposure Assessment Research Division
National Exposure Research Laboratory
Cincinnati, OH 45268
Cover photos:
Left: Prairie Du Sac, Wl Pump house, courtesy of Dr. Mark Borchardt
Middle: norovirus, courtesy of Fred P. Williams; Bar = 50 nanometers
Right: poliovirus, courtesy of Fred P. Williams; Bar = 50 nanometers
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Notice/Disclaimer Statement
This report covers research funded under the Office of Research and Development's Safe and
Sustainable Water Resources Program Task 5.3A, "Water Technology Innovation Cluster:
Develop sustainable processes for contaminant (including nutrient)," and through a Biological
Materials Transfer Agreement with EMSL Analytical, Inc. The research covered in the report
was conducted between August, 2014 and May, 2015 and completed as of June, 2015.
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Table of Contents
Notice/Disclaimer Statement 3
Table of Figures and Tables 5
Acronyms and Abbreviations 6
Acknowledgments 7
Abstract 8
1. Introduction 10
2. Materials and Methods 12
2.1 Samples 12
2.2 Sample Processing 13
2.3 RT (Standard Assay) 13
2.4 ^r(EMSL Kit Assay) 14
2.5 qPCR (Standard Assay) 14
2.6 qPCR (EMSL Kit Assay) 15
2.7 Inhibition Assay 15
2.8 Standard Curve 15
2.9 Genomic Copy Calculation 16
2.10 Statistical Analysis 16
2.11 Quality Assurance 16
3. Results and Discussion 18
3.1 Inhibition 18
3.2 Assay Performance 18
4. Conclusions 24
5. References 26
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Table of Figures and Tables
Figure 1. Norovirus Gil Amplification Plot for Standard Assay 22
Table 1. Samples Analyzed 12
Table 2. Primer/Probe Concentrations 14
Table 3. Standard Curve Genomic Copies 15
Table 4. Comparison of Mean Enterovirus Cq and Genomic Copy Values 20
Table 5. Comparison of Mean Norovirus GI Cq and Genomic Copy Values 20
Table 6. Comparison of Mean Norovirus Gil Cq and Genomic Copy Values 20
Table 7. Standard Curve Acceptance Criteriaa 21
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Acronyms and Abbreviations
cDNA complementary deoxyribonucleic acid
Cq Quantitative cycle
EPA U.S. Environmental Protection Agency
FCSV Final concentrated sample volume
GI, Gil Norovirus genogroup I and genogroup II
GC Genomic copies
GW Groundwater
kDa Kilodalton
MWCO Molecular weight cutoff
NIC No template control
NoV Norovirus
PCR Polymerase chain reaction
PE Primary effluent from a wastewater treatment plant
qPCR Quantitative PCR
RG Reagent grade water
RNA Ribonucleic acid
RT Reverse transcription
SE Secondary effluent from a wastewater treatment plant
UCMR Unregulated Contaminant Monitoring Rule
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Acknowledgments
The authors thank Dr. Sanjib Bhattacharyya, Deputy Laboratory Director, City of Milwaukee
Health Department, Milwaukee, WI, Dr. Frank W. Schaefer III, Decontamination and
Consequence Management Division, National Homeland Security Research Center, Cincinnati,
OH, Ms. Dawn King, Microbiologist, Microbiological and Chemical Exposure Assessment
Research Division (MCEARD), Cincinnati, OH, and Ms. Shawn Siefring, Biologist, MCEARD,
Cincinnati, OH for technical reviews of this report.
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Abstract
EPA Method 1615 contains protocols for measuring enterovirus and norovirus by reverse
transcription quantitative polymerase chain reaction. A commercial kit based upon these
protocols was designed and compared to the method's standard approach. Reagent grade,
secondary effluent, and ground water samples seeded with primary effluent from a local
wastewater treatment plant were processed and analyzed for enterovirus and norovirus by both
formats. The kit format was easier to use and less labor intensive than the standard assay. The
two formats give similar results and it is concluded that either approach may be used for analysis
of water samples.
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1. Introduction
Human enteric viruses are found in waterbodies worldwide (Hewitt et al., 2013; Kishida et al.,
2012; Lee et al., 2014; Love et al., 2014; Maunula et al., 2012; Miagostovich et al., 2008;
Phanuwan et al., 2006; Sassoubre et al., 2012; Xagoraraki et al., 2007). They enter waterbodies
through wastewater plant discharges, septage drainage, combined sewer overflows, and other
point and non-point sources. Even though diluted upon entering natural waters, a sufficient
number of virus particles reach recreational sites and can cause disease. Viruses are thought to be
the primary etiological agent of disease at these sites (Seller et al., 2010), but virus monitoring is
challenging due to the lack of standard methods and to inherent uncertainty in measurements
caused by low virus concentrations. Method 1615, a standardized virus method was developed
by the EPA for detection of enteroviruses by both total culturable and molecular assays, and for
noroviruses by a molecular assay (Fout et al., 2014). This standardized method was designed for
use in the third monitoring round of the Unregulated Contaminant Monitoring Rule (UCMR),
with a focus upon groundwater monitoring.
Following the publication of Method 1615, the EPA Office of Science and Technology asked the
Office of Research and Development to revise the method for use in monitoring wastewater
effluents and recreational waters. In January 2011, EPA Administrator Lisa Jackson, and the
Small Business Administration Administrator Karen Mills, announced the formation of
Confluence (j\^iten^usterorg). Confluence is an organized network of federal/state/local
governments, universities, and companies in the Southwest Ohio, Southeast Indiana, and
Northern Kentucky region that are partnering together on water-related challenges. The EPA also
committed over five million dollars to support water technology innovation in the region. A
portion of these resources was set aside for a Water Cluster internal grant process to fund direct
interactions between the EPA and industry. A proposal developed in response to the Office of
Science and Technology's request to modify Method 1615 was accepted and funded through the
Water Cluster internal grant process. The portion of the proposal that dealt with private industry
was the development of a kit format for the method's molecular assay. This report summarizes
the development and testing of the kit, which has the potential to reduce interlaboratory and
intralaboratory variability and labor costs. The objective of the research was to determine
whether the kit provides equivalent results to the standard assay approach.
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2. Materials and Methods
2.1 Samples
To provide sufficient and equal virus concentrations for comparing the standard protocols and
kits prepared by EMSL Analytical, Inc. in different water matrices, primary effluent (PE) was
collected from a local wastewater treatment plant, mixed, divided into 700 mL aliquots, and the
aliquots were stored at -70°C. On the afternoon before samples were to be collected, one aliquot
of PE per sample was transferred from the -70°C freezer to a 4°C refrigerator. Just prior to use,
PE aliquots were thawed in a 37°C waterbath and the entire aliquot used for seeding.
Six samples each of reagent grade water (RG), secondary effluent (SE) from a local wastewater
treatment plant using an activated sludge process, and groundwater (GW) from a local drinking
water treatment plant were filtered through NanoCeram filters; the sample volumes collected are
shown in Table 1. For RG samples, 2.4 g of HEPES was added and the pH was adjusted to
between 7.0 and 7.5 prior to filtration. For each SE and GW sample, the majority of the volume
was filtered on site and the last nine liters of SE or GW collected and brought back to the
laboratory. Each of these portions was seeded with PE and then pumped through the NanoCeram
filter that had received the original sample via a peristaltic pump.
Table 1. Samples Analyzed
Sample Source
RG#1
RG#2
RG#3
RG#4
RG#5
RG#6
SE#1
SE#2
SE#3
SE#4
SE#5
SE#6
GW#1
GW#2
GW#3
GW#4
GW#5
GW#6
Millipore Super Q Water
Millipore Super Q Water
Millipore Super Q Water
Millipore Super Q Water
Millipore Super Q Water
Millipore Super Q Water
Little Miami Wastewater Treatment Plant
Little Miami Wastewater Treatment Plant
Little Miami Wastewater Treatment Plant
Little Miami Wastewater Treatment Plant
Little Miami Wastewater Treatment Plant
Little Miami Wastewater Treatment Plant
Indian Hills Water Works
Indian Hills Water Works
Indian Hills Water Works
Indian Hills Water Works
Indian Hills Water Works
Indian Hills Water Works
Volume (L)
9
9
9
9
9
9
87
92
71
82
16
92
1662
1521
1660
1654
1520
1530
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2.2 Sample Processing
Samples were processed according to Method 1615 (Fout et al., 2014) using elution with beef
extract and secondary concentration by organic flocculation. Briefly, each NanoCeram filter
(Argonide, Sanford, FL) was eluted with 500 mL of 1.5% beef extract, desiccated powder
(Becton Dickinson, Franklin Lakes, NJ), 0.375% glycine, pH 9.0, twice using contact times of 1
min for the first elution and 15 min for the second. The combined eluate was adjusted to a pH of
3.5 and stirred for 30 min. The floe was collected by centrifugation at 2,500 xg for 15 min and
dissolved in 30 mL of 0.15 M sodium phosphate, pH 9.0. After removing undissolved material
by centrifugation at 4,000 xg for 10 min, the supernatant was filtered through a 0.2 micron
sterilizing filter to remove bacteria and eukaryotes. One third of the filtrate was concentrated
further by centrifugal ultrafiltration (Vivaspin 20 with 30 kDa MWCO, Sartorius-Stedim
Biotech, Bohemia, NY), resulting in a 0.4 mL final concentrate for each sample. RNA was
extracted from 0.2 mL of each final concentrate or from Buffer AE (Negative Extraction Control,
Qiagen, Valencia, CA) using the QIAamp DNA Blood Mini kit (Qiagen) according to the
manufacturer's instructions, except that Buffer AVL (Qiagen) was substituted for Buffer AL
provided in the kit. The RNA (100 jiL final volume) from each sample was stored at -70°C until
used for reverse transcription (RT) assays. On the day of the RT assay each sample was thawed
and diluted 1:5 and 1:25 in Buffer AE (Qiagen).
2.3 RT (Standard Assay)
Master mix RT1 containing 10 ng/|iL random primers (Promega, Madison, WI) and 2.5% (v/v)
hepatitis G Armored RNA (Asuragen, Austin, TX) and master mix RT2 containing 10 mM Tris,
50 mM KC1, pH 8.3, 3 mM MgCb, 0.8 mM deoxyribonucleotides (Promega), 10 mM
dithiothreitol (Promega), 0.5 units/|iL RNase Inhibitor (Promega), and 1.6 units/|iL Superscript
II reverse transcriptase (Life Technologies, Grand Island, NY) were prepared, with all
concentrations being relative to the final 40 jiL RT reaction volume. A volume of 16.5 jiL of
RT1 was added to wells of 96 well plates (Bio-Rad, Hercules, CA), followed by the addition of
6.7 jiL/well of samples or controls. Each sample was diluted 1:5 and 1:25 in Buffer AE and each
dilution and the undiluted sample was added to wells in triplicate. No template controls (NTC)
consisting of Buffer AE (Qiagen) were distributed throughout the plate. RNA from standard
curves, prepared as described in Section 2.8, was added in duplicate. Plates were then covered
with Microseal 'A' Film (Bio-Rad) and incubated at 99°C for 4 min followed by a 4°C hold time
in a PTC-200 thermal cycler (MJ Research, Waltham, MA). The Microseal film was removed
carefully and 16.8 jiL of RT2 was added per plate well. Plates were sealed again with Microseal
'F' film (Bio-Rad). Complementary DNA (cDNA) was prepared by incubation at 25 °C for 15
min, 42 °C for 60 min, and 99 °C for 5 min, followed by a 4 °C hold cycle in the PTC-200
thermal cycler (MJ Research). Plates were stored at or below -70°C until analyzed by qPCR.
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2.4 /?7(EMSL Kit Assay)
RT plates containing 16.5 jiL or master mix RT1 per well were prepared by EMSL Analytical,
Inc., Cinnaminson, NJ, covered with Microseal 'F' film (Bio-Rad, and frozen at -80°C. Master
mix RT2 sufficient for the number of wells to be tested was prepared and frozen at -80°C. The
RT plates and master mix were shipped to USEPA on dry ice and stored at -80°C until analyzed.
Samples were added to the RT plates and incubated as above. Following the denaturation step at
99°C, the master mix was added and cDNA produced as with the standard assay.
2.5 qPCR (Standard Assay)
A qPCR master mix consisting of 10 jiL per well of 2X LightCycler 480 Probes Master Mix (F.
Hoffmann-La Roche, Indianapolis, IN), 0.5 mM ROX reference dye (Life Technologies), and
primers and probes (Life Technologies) in the concentrations shown in Table 2 was prepared and
14 jiL added per Optical 96-well Fast Plate (Life Technologies) well. 6 jiL of cDNA from the
RT assays was added and plates were run in a StepOnePlus Real-Time PCR system (Life
Technologies) using the Quantitation - Standard Curve setup for TaqMan reagents and the
standard instrument run time. The instrument software was programmed to run for 1 cycle at 95
°C for 10 min, followed by 45 cycles of 95 °C for 15 sec, and 60 °C for 1 min. The values from
Table 3 for the standard curve (see Section 2.8) for the enterovirus and norovirus assays were
input into the thermal cycler software.
Table 2. Primer/Probe Concentrations
Primer/Probe Concentration Sequence (5' to 3')
(mM)
HepF
HepR
HepP
EntF
EntR
EntP
NoVGIBF
NoVGIBR
NovGIBP
NoVGIIF
NoVGIIR
NoVGIIP
500
500
100
300
900
100
500
900
250
500
900
250
CGGCCAAAAGGTGGTGGATG
CGACGAGCCTGACGTCGGG
6FAM-AGGTCCCTCTGGCGCTTGTGGCGAG-TAMRA
CCTCCGGCCCCTGAATG
ACCGGATGGCCAATCCAA
6FAM-CGGAACCGACTACTTTGGGTGTCCGT-TAMRA
CGCTGGATGCGNTTCCAT
CCTTAGACGCCATCATCATTTAC
6FAM-TGGACAGGAGAYCGCRATCT-TAMRA
ATGTTCAGRTGGATGAGRTTCTCWGA
TCGACGCCATCTTCATTCACA
6FAM-AGCACGTGGGAGGGCGATCG-TAMRA
a Abbreviations: Hep - hepatitis G assay; Ent - enterovirus assay; NoVGIB - norovirus
genogroup I assay; NoVGII - norovirus genogroup II assay; F - forward primer; R - reverse
primer; P - probe.
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Table 3. Standard Curve Genomic Copies
Standard Curve Genomic Copies per RT-qPCR Assay
Concentration
2.5 x 107
2.5 x 106
2.5 x 105
2.5 x 104
2.5 x 103
50,250
5,025
502.5
50.25
5.025
(1) Place the indicated genomic copy values in the standards section for the real time thermal
cycler used
2.6 qPCR (EMSL Kit Assay)
A qPCR master mix described in section 2.5 was prepared by EMSL Analytical, Inc., and 14 jiL
was added per Optical 96-well Fast Plate (Life Technologies) well. Plates were covered with
Microseal 'F' film, frozen at -80°C, shipped to EPA on dry ice, and stored at -80°C until used.
Plates were thawed just before use. Samples (6 jiL of cDNA) were added and run under the same
conditions and on the same StepOnePlus thermal cycler as the standard assay. For each assay
type, the EMSL kit assay was run in the morning and the standard assay in the afternoon of the
same day.
2.7 Inhibition Assay
The hepatitis G PCR assay was run before the enterovirus and norovirus assays. The mean
quantitative cycle (Cq) value of each sample dilution was compared to the mean value of NTC
and negative extraction controls. The Cq value is the cycle at which the fluorescence of a PCR
assay crosses the threshold that defines a positive reaction. Samples that exhibited a mean Cq
value greater than 1 Cq unit higher than the mean of the uninhibited NTC/negative extraction
controls was considered inhibited and not used, except as indicated in the Results section. The
undiluted sample or the first dilution that was uninhibited was used for subsequent enterovirus
and norovirus assays.
2.8 Standard Curve
Armored RNA EPA-1615 (Asuragen, Austin, TX; custom order) containing the enterovirus and
norovirus regions amplified by the primer sets used (Brinkman et al., 2013) was diluted to 2.5 x
108 genomic copies/mL, divided into 250 jiL aliquots, and stored at -20°C. One aliquot was
removed from the freezer and thawed. Five serial dilutions were prepared giving concentrations
of 2.5 x 107 to 2.5 x 103 genomic copies/mL. RNA from each dilution was extracted and
analyzed in the same manner as samples. Table 3 gives the final genomic copy number for each
qPCR assay (assuming that the percent loss during extraction is the same for both samples and
standards). To be acceptable each standard curve must have an R2 value of >0.97, a percent
efficiency of 80 to 115%, and an overall standard deviation of <1.0.
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2.9 Genomic Copy Calculation
The number of genomic copies for each virus was calculated using Equation 1:
GC = GCPCR X 199 X Equation 1
Where GCpCT is the quantity calculated by the real time thermal cycler software based upon the
standard curve, 199 is the factor that correct for the assay volumes used for RNA extraction, RT,
and qPCR, FCSVis the volume of filtered concentrate after organic flocculation, and S is the
volume of FCSVused for centrifugal ultrafiltration.
2.10 Statistical Analysis
Cq and quantity values (calculated from the standard curve) were analyzed first for normality
and equal variance (Sigma Plot version 13.0, Systat Software, San Jose, CA). Many comparisons
between the values from the standard and EMSL kit assays were not normal, even when log-
transformed. ANOVA on Ranks with Dunn's or Tukey tests (Sigma Plot) was used for
comparison of multiple parameters. Finally, the Mann-Whitney Rank U Test (Sigma Plot) was
used to determine statistical significance of comparisons of the standard method with the EMSL
kit on a per virus basis, considering either each matrix individually or all matrices combined.
2.11 Quality Assurance
EMSL Analytical, Inc. tested each master mix used for kit preparation for activity using EPA-
1615 and hepatitis G Armored RNAs as controls. All mixes receiving the Armored RNAs were
positive by RT-qPCR for all primer/probe sets and all no template controls were negative. EPA's
quality assurance guidelines (Sen et al., 2004) were followed for analysis of samples performed
in EPA laboratories. This included the use of a dedicated laboratories and rigorous work flow
requirements. Negative extraction and no template controls were included as required by EPA
Method 1615. These controls consisted of Buffer AE (Qiagen) in place of sample for RNA
extraction or of RNA for RT-qPCR.
The six reagent grade water samples were processed and RNA extracted prior to collection of the
other sample types. Two of the reagent grade water samples were analyzed by RT-qPCR to
determine if the PE seed contained detectible quantities of each virus type. Cq values for each
virus were sufficient to proceed with the remaining sample matrices (data not shown). The two
reagent grade samples were analyzed again during analysis of all samples.
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3. Results and Discussion
3.1 Inhibition
No difference in the level of PCR inhibition was observed between the standard and EMSL kit
assays for reagent grade and secondary effluent samples (data not shown). In contrast, one
additional dilution was required to remove inhibition of groundwater samples when assayed by
the standard procedure than when assayed using the EMSL kit format. Secondary effluents were
inhibited even at a 1:25 dilution with both formats. The number of available EMSL RT plates for
the kit format was limiting. As a result, higher sample dilutions could not be investigated and the
1:25 sample dilutions were used in subsequent virus assays. For groundwater samples the
dilutions for subsequent assays were chosen based upon the standard assay to provide direct
comparisons of Cq values and genomic copy numbers. The reason for the difference in inhibition
between assay types is unclear, but may be related to storage temperature. In support of this
assumption the mean Cq value for the hepatitis G PCR assay for all 18 samples tested was 1.8 ±
0.8 Cq units higher for the standard assays than for the EMSL kit assays. Both assay formats
used the same reagent lots, but reagents for the standard assay were stored at -20°C while those
for the EMSL kit were stored at -80°C. It is likely that lower stability of enzymes or
oligonucleotide primers and probes at -20°C led to greater sensitivity to inhibition.
3.2 Assay Performance
The performance of the standard and EMSL kit assays were compared by examining the overall
number of detects versus non-detects, difference in mean Cq values between the assay types, and
by the derived virus quantities. Tables 4 (enterovirus), 5 (norovirus GI), and 6 (norovirus Gil)
show the number of detects, the mean Cq values, and quantities for each matrix.
The number of detects and non-detects and Cq values are a function of initial virus
concentrations in the PE seed and the dilution required for removal of inhibition. The
concentrations were very low for the SE samples, due to the use of 1:25-fold dilutions and the
presence of PCR inhibitors, and for the norovirus Gil assays. As a result of these low
concentrations, Cq values for these assays were in the 39-40 range. Although these values are
high, they represent true positive results. Each result checked individually demonstrated typical
qPCR profiles well separated from negative samples. Figure 1 shows the profile for the NoVGII
standard assays and the degree of separation between positive and negative samples that was
seen with all assays.
All samples from each matrix type analyzed for norovirus GI produced positive results with both
the standard and EMSL kit assays (Table 5). In contrast, the standard enterovirus and norovirus
Gil assays showed 7% and 20% more non-detections than the EMSL kit assay, respectively.
Each of the enterovirus and norovirus assays resulted in a higher Cq value in the standard assay
(overall difference of 0.6±1.0 Cq units) with the greatest difference being the norovirus Gil assay
(Tables 4 and 6). Both the Cq differences and the numbers of non-detections may be related to
storage conditions or shelf life as above. Small decreases in enzyme or primer/probe
concentrations would increase the number of cycles required for a sample to become positive
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(e.g., higher Cq values) and the number of non-detections of samples with very low starting virus
genome concentrations. Differences in Cq values were tested for significance using the Kruskall-
Wallis Anova on Ranks to compare data across all matrices for each virus assay and by the
Mann-Whitney Rank Sum Test for comparing the standard versus EMSL kit results for each
individual matrix. While it appears that the EMSL kit assays were better than the standard assays
on the basis of detection rates and Cq values, the differences are not significant by the Kruskal-
Wallace test; however, the differences between the standard and EMSL kit results for the reagent
grade water matrix and for all matrices combined were significant by the Mann-Whitney Rank
Test (Table 6).
The genomic quantity values shown in Tables 4-6 are calculated using Equation 1, which gives
the number of genomic copies present in the PE seed added to each sample. These values were
based on standard curves that met the acceptance criteria for both the standard and EMSL kits
assays (Table 7). Although the Cq values of the samples were higher for the standard assays than
for the EMSL kit assays, it was expected that a similar difference would have been observed
with the standard curves. This would have normalized the differences, however, the genomic
copy quantity values generally were higher for the standard than the EMSL assay. The higher
values resulted from a greater Cq (1.3±0.6) difference between the standard curves for the
standard assay and the EMSL kit assays versus the Cq difference in samples analyzed by the two
formats. As above, the differences are not significant when compared using the Kruskal-Wallace
test, but there were significant differences between most enterovirus assays (Table 4) and half of
the norovirus GI assays (Table 5) by the Mann-Whitney Test.
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Table 4. Comparison of Mean Enterovirus Cq and Genomic Copy Values
Sample Standard Assay Standard Assay Standard Assay EMSL Kit EMSL Kit EMSL Kit
Cq±S.D.a LogGC±S.D. Nb Cq±S.D. Log GC ± S.D. n
RG
SE
GW
Overall
36.46 ± 1.07
40.01 ±0.43
36.02 ± 1.48
36.85 ± 1.85
6.75±0.48T
6.79 ±0.14
7.76±0.31T
7.18±0.62T
18
7
18
43
35.98± 1.07
39.62 ±0.9
35.78± 1.21
36.74 ± 1.94
6.44±0.45T
6.49 ±0.31
7.36±0.23T
6.80±0.56T
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11
18
47
a S.D. = standard deviation; GC = genomic copies
b Each sample was analyzed in triplicate, resulting in an n of 6 x 3 = 18 when all replicates produced positive values.
t The comparisons indicated by this symbol are statistically significant at P <0.05.
Table 5. Comparison of Mean Norovirus GI Cq and Genomic Copy Values
Sample
RG
SE
GW
Overall
Standard
Cq±S
36.35 ±
34.17±
33.31 ±
33.81 ±
Assay
.D. a
0.98
1.18
1.20
1.19
t The comparisons indicated by
Table 6.
Sample
RG
SE
GW
Overall
Comparison
Standard
Cq±S
39.24 ±
39.65 ±
38.58±
39.05 ±
of Mean
Assay
.D. a
1.16^
1.17
1.42
1.33?
Standard
LogGC
4.60 ±
5.88±
Assay
±S.D.
0.51
0.34
5.79±0.25T
5.43 ± 0.70T
Standard
n
18
18
18
54
Assay
this symbol are statistically significant
Norovirus
Standard
LogGC
4.22 ±
5.16±
5.12 ±
4.80 ±
GUCq
Assay
±S.D.
0.58
0.33
.030
0.63
EMSL Kit
Cq±S.D.
34.28 ±0.98
33.44 ± 1.39
33.20 ± 1.22
33.64± 1.27
atP<0.05.
EMSL Kit
Log GC ± S.D.
4.33 ±
5.80±
5.22 ±
5.22 ±
0.37
0.39
0.21^
0.72T
EMSL
n
18
18
18
54
Kit
and Genomic Copy Values
Standard
n
13
9
17
39
Assay
EMSL Kit
Cq±S.D.
38.28 ± 1.44T
38.32± 1.46
35.78± 1.21
38.06 ± 1.42T
EMSL Kit
Log GC ± S.D.
4.15±
5.34±
5.17±
4.87 ±
0.53
0.37
0.23
0.66
EMSL
n
17
15
18
50
Kit
The comparisons indicated by this symbol are statistically significant at P <0.05.
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Table 7. Standard Curve Acceptance Criteria a
Criteria
Slope
% Efficiency
R2
Overall S.D.
Standard Assay
Enterovirus
-3.85
82
0.99
0.5
Standard Assay
GIB
-3.26
103
1.0
0.5
Standard Assay EMSL Kit
Gil
-3.72
86
.998
0.2
Enterovirus
-3.11
109.5
1.0
0.1
EMSL Kit
GIB
-3.39
97
0.99
0.5
EMSL Kit
Gil
-3.73
85
1.0
0.4
a Acceptable values: % efficiency of 80-110%, R2 >0.97, and overall standard deviations < 0.5.
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Figure 1. Norovirus Gil Amplification Plot for Standard Assay
Amplification Plot
Plot Settings \_
Plot Type: ARn vs Cycle -r ] Graph Type: [tog -r] Plot Color: Well
Save current settings as the default
Amplification Plot
Q-
0.01
0.001
0.0001
0.00001
0.000001
10 12 11 16 18 20 22 21 28 28 30 32 34 36 39 40
Options
Targst:
Threshold: h/jAuto
0.2413S5
0 Auto Baseline
Show: [7] Threshold—LJ Baseline Start] Well Target^ Baseline End: Well •Target^
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4. Conclusions
It was expected that the standard assay would outperform the EMSL kit. The kit required the RT
and PCR enzymes to be frozen under ionic conditions that had not been tested for stability and at
-80°C rather than the manufacturers' recommend storage at -20°C. In addition, the use of the
EMSL kit requires thawing and refreezing the enzymes which could have led to loss of activity.
It is concluded that the conditions used in preparation of the kit do not adversely affect
enzymatic activity.
Although there is a statistically significant difference between the standard and EMSL kits for
some assays, the differences are very minor and not consistent. The inconsistency stem from the
fact that the EMSL kits statistically outperforms the standard assay when Cq values and non-
detections are considered while the standard assay is best when genomic quantities are being
evaluated. It is concluded that the differences lack biological significance and therefore, either
format may be used. Further testing is needed to determine the useable shelf life of the kits.
The EMSL kits were easier to use and less labor intensive than the standard assay. For large
studies within a single laboratory or across multiple laboratories the use of the kit should reduce
analyst error as well as intralaboratory and interlaboratory variability.
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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