Wednesday, April 13
1:20 p.m.-2:50 p.m.
Session 3:
Recreation Water Monitoring and
Implementation Challenges/Successes
33
-------
U.S. EPA's 2016 Recreational Waters Conference
Monitoring Beaches Statewide in
Michigan for E. coli with qPCR (USEPA
Draft Method C)
Shannon Briggs, PhD
Michigan Department of Environmental Quality
Abstract
In 2015, Michigan initiated a statewide,
rapid beach testing program by providing
10 laboratories with $500,000 worth of qPCR-
related equipment. In collaboration with
Michigan State University (MSU) and the U.S.
Environmental Protection Agency (EPA), labo-
ratory personnel are being trained to use the
EPA's Draft Method C: Escherichia coli in Water
by TaqMan Quantitative Polymerase Chain
Reaction (qPCR). The training effort includes
developing manuals containing standard oper-
ating procedures that can be easily followed by
laboratory staff. Michigan's qPCR network of
16 labs is connected with the MiqPCR listserv
hosted by MSU. Beaches will be posted sooner
and reopened faster because test results will
be available the same day. Monitoring results
are posted on Michigan's BeachGuard website
at http://www.deq.state.mi.us/beach/. During
the transition to qPCR methods, beaches will
be monitored using both the culture and qPCR
methods so that correlations between the two
methods can be determined, allowing for future
derivation of water quality standards for the
new method.
Biosketch
Dr. Shannon Briggs is a toxicologist for
the Water Resources Division of the Michigan
Department of Environmental Quality (DEQ).
She received her bachelor of science degree in
animal science and her doctorate in pharma-
cology and toxicology from Michigan State
University. She is a member of a planning
team that will host the 2016 Great Lakes Beach
Conference in Marquette, Michigan, October
5-7, 2016. Dr. Briggs assists local health depart-
ments with state and federal grants for monitor-
ing beaches across the State of Michigan. She is
leading a water quality initiative of the DEQ to
provide rapid testing equipment and training
for 10 new laboratories that will test beaches
using the U.S. Environmental Protection
Agency's draft Method C (i.e., qPCR method
for E. coli). Dr. Briggs is an active member, past
president, and cofounder of the Great Lakes
Beach Association.
34
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Day One: Session 3
11,000 inland lakes
77,000 river miles T
1,200 public beaches ¦
4 Great Lakes H
3,288 miles of coast
5.5 million acres of wetlands
4oinor5
MICHIGAN
yJlUililniiii,.,, i
¦ r.j J sjiSteji
Monitoring Michigan Beaches Statewide
for E. coli with QPCR
(USEPA Draft Method C, June 2015)
Shannon Briggs
briaass4@michiaan.uov
Michigan rtepartment of
Fnvironmental Quality
Michigan Public Health Code and
Public Beaches
• Monitoring is Voluntary for all 45 Health
Departments, however
• County Ordinance can require testing
• Health Officer has authority to test and close
• Requires signs at all public beaches
• Requires reporting if beach is tested
Beach Monitoring
"typical stats"
> 200 inland lake beaches monitored
• 200 Great Lakes beaches monitored
• 3.6% samples exceed WQS, n -186 samples
• 80% of beaches are open all season, n= 332
• 20% of beaches report action, n= 84 beaches
• Most actions are 1 to 2 days
Path to qPCR for Beach Testing
QPCR Methods
Year
Beaches
Kary B. Mullis
invents PCR
1985
1986 —
oERA Ambient
Water Quality
Criteria for
Bacleiia - 1988
2000 BEACH Act
Path to qPCR for Beach Testing
QPCR Methods
Year
Beaches ]
Dr. Joan Rose at
2003
Monitor Beaches
Michigan State
with local, state &
University
federal funds
Water Fellows
2005
Identify Impaired
Lectures &
Beaches
Discussion
Microbial Source
2007
Beach Sanitary
Tracking (MST)
Survey Tool
35
-------
Path to qPCR for Beach Testing
Year
QPCR & Beaches j
2010
MST at Impaired Beaches
2011
Training Manual & Video for Beach
Testing with QPCR
2012
Public Meeting for MST Results
U.S. EPA Rec Water Quality Criteria
Includes Enterococci QCPR values
U.S. EPA's 2016 Recreational Waters Conference
QPCR Lab
at Lake St. Clair
Metropark Beach
$100,000 for equipment
Park renovated office to lab ($50,000)
bja
ML . <\ \
-
$500,000 for 10 New Labs
• State of Michigan provided $500,000 to DEQ
for rapid beach testing equipment
• Only health departments have authority to
test beaches
• DEQ sent letters of invitation to 45 health
departments responsible for 83 counties
• 13 responses and description of lab capacity
Questions and Details
• Commitment & Expectations in Memorandum
of Understanding between DEQ and HDs
• 10 Health Departments signed MOUs
• MOU included equipment list with 50+ items
for each HD
• $30l< for Training and Support from MSU
just added $28,000 more
O G Existing qPCR Labs
O 10 New qPCR I ahs
36
-------
£3
Day One: Session 3
You(B3
Performing the qPCR Assay
for Enterococcus
qPCR Training Video
uploaded on Nov 79. 7(111
and 2011 using a rapid method (quantitative polymerase chain reaction) to assess beach water quality at
sites in Orange and Los Angeles Counties Tltts video was produced to tram laboratory statt to execute
Path to qPCR for Beach Testing
1 Year
QPCR & Beaches
2014
10 New QPCR Labs (15 total)
U.S. EPA draft Method C
(QPCR - E. coli)
2015
Equipment Ordered & Delivered
Samples filtered & frozen
Train the Trainer for QPCR
Path to qPCR for Beach Testing
Year QPCR & Beaches
2016 4-day Training on Draft Method C
Stockholm Water Prize awarded to
Dr. Joan Rose
37
-------
Jej*
U.S. EPA's 2016 Recreational Waters Conference
How would you rate your confidence in
your skills for each step of qPCR?
7 T
Pipetting Preparing Filtration DNIA Running DNA
Reagents Extraction StepOne Analysis
Step in qPCR Analysis Plus
Path to qPCR for Beach Testing
Year
QPCR & Beaches
2017
Samples tested and reviewed with
previous 2 years
2018 Continue sampling
Present equivalent QPCR results to
USEPA and Local Health Officers
2019
Beach status determined by
QPCR methods
Molecular Source Track Training?
Path to qPCR for Beach Testing
Year
QPCR & Beaches
2016
Multi-lab Validation Study
2015 & 2016 Samples tested
Review Colilert & QPCR Results
jyj|
|
38
-------
Day One: Session 3
Report Data
DCa
BeacfiGuard' ,lon™' IAch^°*'9°'"omo t c°,»c,vo»,ort Incc I Ab°ut l*-¦
r quality campling roc ufe
Michigan Beaches
1??S Public Beaches
513 Private Beachec
Closures and Atfvlsorlco
No Current uosures or Acv.sor.es
QPCR is not the finish line
its just another tool in the tool box
Sleeping Bear Dunes Photo by Steve Keighly, Winner of the Instagram
Beach Photo Challenge for favorite beach to take a long walk.
Beach Sanitary Surveys
http://www2.epa.gov/beach-tech/beach-sanitary-surveys
Cf-S.I I I **>«<*« I nto9«4>
Related lupus Technical Resources about Beaches
Beach Sanitary Surveys
On this page:
• Bdikyiuund
• Marinp Btmrh Unitary ^iirypy
• other sanitary survey information
Background
A sanitary survey is a method of investigating the sources of fecal contamination to a water body. Sanitary
Beach Sanitary Surveys
Great Lakes Beach
Sanitary survey
User Manual
Canine Scent Tracking
Environmental Canine Services
I aura and Kama
39
-------
Remediation
landscaping
redesign slope of beach, groom beach
Great Lakes,
i Lanes fcteacn conference Adomonai into
Majcquette in October 5-7, 2016
Beach listservs
beachnet@great-lakes.net
MiQPCR@LIST.MSU.EDU
beachinfo@lists.epa.gov
40
-------
Day One: Session 3
Rapid Analyses of Water Quality at Five
Chicago Beaches, 2015
Abhilasha Shrestha
University of Illinois, School of Public Health
Abstract
In the summer of 2015, the Chicago Park
District (CPD) enhanced its beach monitoring
and notification through a pilot program of
rapid molecular testing of beach water. Water
samples were provided at approximately 8:30
a.m. 4 days per week to the University of Illinois
at Chicago School of Public Health (UIC SPH)
Water Research Laboratory. The results of the
rapid testing method, qPCR, were reported on
the same day by 1:00 p.m. The CPD used the
qPCR results to notify the public about mea-
sured bacterial concentrations. Previously, the
CPD posted notifications based on the most
probable number (MPN) of E. coli obtained from
overnight cultures.
Water samples from five Chicago beaches
were tested using the Enterococci qPCR. Similar
samples were set up for E. coli culture analy-
sis by a commercial laboratory on the same
days that UIC performed the qPCR test. The
CPD used the U.S. Environmental Protection
Agency's (EPA's) Beach Action Values (BAV) for
both the qPCR test results and the culture test.
Of the 270 qPCR tests, 23 exceeded EPA's
BAV, and of the 270 culture tests, 67 exceeded
the BAV. The results of E. coli culture test-
ing that became available on a given day (e.g.,
results that became available on a Thursday
from tests of beach water samples collected on
Wednesday) were frequently inconsistent with
the current qPCR results (from water samples
collected on Thursday). Our data suggest that
beach water notifications based on qPCR testing
presented a more accurate picture of same-day
water quality than the prior-day's culture test
results.
Biosketch
Ms. Abhilasha Shrestha is a doctoral stu-
dent in the Environmental and Occupational
Health Sciences Department at the University of
Illinois at Chicago School of Public Health (UIC
SPH). She earned her bachelor of science degree
in biology from the University of Minnesota-
Duluth and then worked as an aquatic toxicolo-
gist in a private laboratory in Minnesota for
more than 2 years. She completed her master's
degree from UIC SPH in 2013, focusing on
environmental and occupational health sci-
ences with a concentration in water quality and
health. Ms. Shrestha's research interests include
studying the use of different indicator targets/
genes for water quality assessment. In her dis-
sertation research, she is focusing on molecu-
lar methods for rapidly evaluating infectious
agents in surface water.
41
-------
U.S. EPA's 2016 Recreational Waters Conference
Rapid Analyses of Water Quality at Five Chicago Beaches, 2015
jjj |sa£
i im
Abhilasha Shrestha, PhD Student
Ira Heimler, Lathy Breitenbach, bamuel Uorevitch
U.S. EPA's Recreational Waters Conference
A|jiil 13,2016
University of Illinois at Chicago
School of Public I lealth
Chicago, IL
Overview
• Introduction
• Methods
v Beach Action Value (BAV)
• Results
> Data quality
> £ coli culture results
> Fntpracnrci qPCR results
> One day delay In E. coll results, and associations with qPCR results
> BAV exceeddiice after 0.5 iridi of idin
• Conclusion
• Future projects
• Acknowledgment
O ******
Introduction
• Chicago: 26 miles of public
beaches
• ~20 million visitors annually
• Chicago Park District: 27 beaches
• Point source discharges are rare
• Monitoring: Culture-based
methods such as Colilert®
• Prior-day culture poor
predictor of current conditions
• 2015: Pilot program with UIC
> 5 Chicago beaches
Methods
• qPC R at I lit", lah, Tuesrlay-Fririay, May - August 30, 7015
• Culture tests: Commercial laboratory, Colilert® method
• 1L samples, 2 transects each at 5 Chicago beaches (N-270)
• Delivered at approximately 8:30 AM
• Quantified for Enterococci DNA using the USEPA Method 1611 with
one modification
• Results reported to the CPL1 on the same day by 1:00 PM
Beach Action Value (BAV)
Entcrococct culiurablc
-Ui&tbiki&lL...
I k. coli - culturable
(fresh)''
I-Miniatcd I linos Rate
(NGI): 36 per 1,000
primary contact
rccrcaton
BAV
(Into n«r 100 ml.)
23 > cfu
jc
K*timatcd Illness Rate
(NCI): 32 per 1,000
primary contact
retreator*
BAV
(iHitipw 100 ml.)
60 cfti
iwcfa
Eruavcoccus spp
qPCR (fresh and mnnne)r 1,000 cce I 640 c
1,000 ccc I
" uM-auu«d UM1151 epa MmIumI il00(U v f ;>.\ 3002a) m MMb« wplvalMt im^kkM
cultivable euterocotci
6 E coli iuius EPA Method 1403 (U S EPA 2002b), ot my otlut Nine standard curve runs, each in triplicate, initially and every two weeks
v R4= 0.9957 (high accuracy)
1 Parameter
Standard
05% lower
05% upper 1
Mean
deviation
bound
bound
Slope
3.4945
0.0202
3.5345
3.4545
Intercept
38.2229
0.061
38.1122
38.3535
qPCR Precision/ Calibrators & sample processing controls (SPC)
> 55 calibrators
Sample processing control
23.00
026
1.11*
Enterococci cells
053
2.05*
qPCR Inhibition.
>- Of the 510 total beach samples, only two (0.37 %) exceeded the 3 CT unit
offset; other two (0.37%) had offsets in the 2-3 cycle range
42
-------
Day One: Session 3
E. coli culture (MPN/lOOmL geomean)
Enterococci qPCR (CCE/lOOmL geomean
383.1 (14.9 - >2420.0) 33(61.1) 14(25.9) 7(ia-°)
171 f, (1.(- ->9490 n) 16(^3.3) X13") '(3 7)
167.8 CS-fi ¦ >2420.0) 46(85-2) 7(13-0) 1(1.9)
i86.y (2.8 - iuzu./) 4i(y/.8) 11(20.4) i(i.y)
w*1
057.8 (6.0-01900) 37(68-0) 13(24-1) 4(7-4)
[All beaches
\ \ \ \ \ \
D*li
B««dl — L — RB — ^rt Note: 03 values were truncated at 24G0
liRilRIHSinBiBBil
Montrose
447-9
C27.1-569b.oj
49190-7)
5(9-3)
O.V'SHcc*
240.9
(14.2-1O77.G)
49(90-7)
5 (9-3)
SnnHl Shnro
3°6 0
0" 6-3136 s)
61 (91 1)
3(6 Q
Rainbow
501-9
(23.3-10385.8)
49(90.7)
5 (9-3)
(ATI hpartip«j
4&U-1
9*94
(2u.6-38m-3)
(10 5-1038S.8)
BBBHBH
5(9-3)
Time series graphs of daily measures of culture and qPCR
I -
Beach
Pearson's R
p-value
Montrose
0.78
<0001
6}rd Street
078
<¦0001
South Shore
060
<0001
Rainbow
0.29
0.035
calumet
O.tffl
<0001
One day delay in E. coli results, and associations with qPCR results
Beach management decisions based on today's qPCR results and the E. coli
results from yesterday's water sample were not associated, with the
exception of 63rd Street beach.
1 Pr ior day culture results frequently lead to the erroneous decisions when
rnmparpd tn the same day qPCR results as the gnld standard.
H¦¦¦¦¦¦¦¦J
| i 3 4
1 9
39 48
1 10
42 52
| ORl
44 fo.13,15.55)
BFr'^
347
ao a6 46
^3 30 53
nil nt»»(mn J »(.)
2 s /
16 29 45
10 34 52
OR O.T3 fO.13.4.17)
BAV exceedance after 0.5 inch of rain
• Odds of exceeding either the E. coli culture MPN BAV or the
Enterococci qPCR CCE BAV were increased
• Enterococci qPC'K: Odds ratio 4 2b (l.b9 - 11.43)
• E. coli Culture: Odds ratio 1.90 (0.85 - 4.24)
CCE <1,000
CCE> 1,000
Total
-0 0 inches past 24 hours
224 (93.3%)
16 (6.7%)
240 (100%)
i0.5 inches past 24 hours
23 (76.7%)
7 (23.3%)
30(100%)
Total
247
23
270
MPN<235
MPNi 235
Total
<0 5 inches past 24 hours
184 (7fi 7)
SB (23.3%)
?4O(10O%)
*0.5 Inches past 24 hours
19 (63.3%)
11 (36.7%)
30(100%)
Total
203
67
270
Conclusions
• Accurate, precise qPCR results can be available by 1.00 PM.
• Daily qPCR CCt values resulted In BAV exceedance less frequently
than the E. coli culture results (8.5% vs 24.8% of samples).
• Inhibition of the qPCR reaction was rare (<1% of samples).
• Results of £ coli testing (from prior day water samples) were not
consistently related to qPCR results.
• Beach management decisions should be based on same-day
rather than prior-day information.
• Hpavy precipitation tends to increase Fntprnrocci qPCR CCF
results significantly, and to a lesser degree, £ coli MPN.
43
-------
Jej*
U.S. EPA's 2016 Recreational Waters Conference
U
o
Q
>
u
oc
<
<
Q.
HI
Future Projects
• Archived filters
> Evaluate the concentration of a human-specific molecular
target like HF 183.
• Summer 2016
>• qPCR testing expanded to additional beaches, particularly
those that tend to have relatively frequent BAV exceedance
based on E. coli culture results.
v 9 beaches, 5 days a week, Wednesday- Sunday
> Goal; Earlier sample collection and results by noon.
THANK YOU
Acknowledgement
funding for this project was provided by the Chicago Park District.
, V)
111
g JO I
2 ^ Z h
5 Tia J
E
I gou«zm
Z Z U kiJ u 3
ilKOIlAL
KEEP
CALM
AND
RUN
qPCR
44
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S3
Day One: Session 3
Application of Rapid qPCR-Based Tests for
Enterococci (Method 1611) in Hawaiian Coastal
Waters
Marek Kirs, PhD
University of Hawaii
Abstract
To evaluate the applicability of the U.S.
Environmental Protection Agency's (EPS's)
enterococci qPCR method 1611 for beach waters
of Hawaii, a total of 127 water samples were
collected from 12 beaches on Oahu over a
10-month period. The samples were analyzed
using EPA methods for Enterolert®, 1600, and
1611. Clostridium perfringens, human-associated
Bacteroides, and human polyomaviruses
also were enumerated. Concentrations of
enterococci and C. perfringens varied from <
10 to 389 colony-forming units (CFU) 100ml-l
(Enterolert®), from < 1 to > 151 CFU 100ml-l
(1600), and from < 1 to 96 CFU 100ml-l (mCP).
Four samples (3.1%) analyzed using Enterolert,
and two samples (1.6%) using method 1600
exceeded the EPA-recommended statistical
threshold value (STV) of 130 CFU 100ml-l, while
C. perfringens concentrations exceeded 50 CFU
100ml-l in a single sample (0.8%), indicating
generally good water quality at the beaches
studied. In the samples exceeding the STV,
human-associated Bacteroides was detected in
a single sample, while human polyomaviruses
were not detected. Importantly, 88 samples
(69.3%) tested using method 1611 could not
be quantified because of the PCR inference.
After those samples were diluted in molecular
grade water (1:10), the majority of the samples
(85 samples, 66.9%) remained compromised
by the PCR inference. In contrast, for an addi-
tional set of monthly samples (n=39) collected
at three sites from the brackish Ala Wai Canal,
only a single sample was compromised (2.5%).
Although good agreement existed between the
methods for enterococci when samples were not
compromised, our data indicate serious short-
comings for the recommended qPCR method
1611 for enterococci enumeration for Hawaiian
beaches. New technology that alleviates inhibi-
tion issues for qPCR is being evaluated.
Biosketch
Dr. Marek Kirs is an assistant researcher
at the Water Resources Research Center of the
University of Hawaii. He received his bach-
elor of science degree from Tartu University in
Estonia, his master of science degree from the
University of Edinburgh in the UK, and his
doctorate from the University of Rhode Island.
He also has completed postdoctoral training at
the University of North Carolina at Chapel Hill.
More recently, Dr. Kirs worked at the Cawthron
Institute in New Zealand, where he was
involved in establishing microbial source track-
ing services and lead microbial water quality
research and consultancy projects. His research
focuses on a wide range of microbial water
quality and related public health issues.
45
-------
U.S. EPA's 2016 Recreational Waters Conference
Application of rapid qPCR-based
tests for enterococci (Method 1611)
in Hawaiian coastal waters
Marek Kirs, Denene Blackwood, Rachel Noble,
Philip Moravcik
April 13, 201G
U.S. EPA's 2016 Recreational Water Conference, New Orleans
©
INIVCRS1TY ||,||
-------
Water quality
Enterococci
Method 1600(mEI)
Water quality
Enterococci
Enterolerf l600(mEl)
Water quality
Clostridium perfringens
Water quality
Human-associated Bacteroides; human polyomaviruses
Water quality
Summary
• Fnterocncri and C pprfringpns indicated good water quality
on the beaches studied
• 3.1% of the samples exceeded STV for enterococci by Enterolert®
• 1.6% of the samples exceeded STV for enterococci by method 1600
• Only a single sample exceeded both , the STV for enterococci and
threshold level for C. perfringens
• Human sewage was not conclusively identified as the contamination
source in any of Ihe coastal samples based on the markers
Rapid Method Application (1611)
PCR Inference (inhibition)
PCR inference can be caused by:
• Mechanical blocking of the enzyme, template
• Physical and chem. modification of the enzyme, template
• Binding and chelating of other chemicals necessary in PCR
• Other ..(see Schraderetal, 2012, J. Appl. Microbiology 113:1014-1026)
PCR inference results in:
• Severe underestimate of bacterial concentrations
• False negatives
47
-------
U.S. EPA's 2016 Recreational Waters Conference
Rapid Method (1611)
PCR Inference (inhibition)
PCR inference was measured:
23 3 cycles)
Rapid Method (1611)
PCR Inference
27 3
63.7 , m 100.0
63 6 tjj Tin Total of 69.3% samples
compromised
& ¦v/
Sc. - 81.8
8LS
''' .
% compromised samples0 45.5" 81.° 455
(ACtsketa >3.3 cycles)
0% samples extraaed using
commercial kit compromised
(AC^iO.61)
(mixed cellulose filters)
Rapid Method (1611)
PCR Inference 1:10 diluted
'\ . -27.3
bi. 7 100.0
03 G it,. 72.7
- '
% compromised samples
(AC^ 23 3 cVcles)
67.7% samples
still compromisec
27.3 81.8
Rapid Method (1611)
Ala Wai
Site
Description
n
Salinity
Compromised
(PPt)
(%i
A
Coastal
13
34.9
7G.9
B
Canal
13
27.6
7.7
C
Canal
13
23.4
0
D
Stream
13
6.8
0
¦ — - _
Rapid Method (1611*)
Comparisons with cultivation based methods (combined set)
1A-0 90
R2=0M7
i!:
::
jB* '
to- 10>
Cnterolert
• *'
! *
n=32
IA=0.884
R*=o a?o
Rapid Method (1611*)
Beach management decisions (combined set)
Method 1600
%
Close
Open
tnterolert*
Clnsp
68
5
Open
7
20
Method 1611
%
Close
open
Enterolert®
Close-
11
33
Open
0
56
Method 1611
%
Close
open
Method
1600
Close
40
9
Open
11
40
maam
48
-------
Day One: Session 3
Rapid Method (1611)
Summary
Good water quality of the beaches sampled
PCR inhibitors can compromise application of
rapid qPCR based methods in Hawaiian coastal waters
There was good agreement between enterococci concentration
estimates as well as beach management decisions based on
all three methods
Rapid accurate methods are highly desired in HI
(number of beach goers, distances, impact)
Rapid Method (1611)
Future plans
A study funded by the Sea Grant College Program/NOAA:
1) identify cause,
2)tmuhleshnnt, arid
3)secondary assay needed
Coral sand?
Acknowledgements
Contributors:
Dr. Roger Fujioka
Dr. Valerie lody Harwnnd
Dr. Mayee Wong
Ms. Martina Frycova
Clean Water Branch {HI DOH)
Funding:
National Institute of Water Resources (USGS) and
start up
49
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U.S. EPA's 2016 Recreational Waters Conference
Multi-Laboratory Survey of U.S. EPA
Enterococci qPCR Methods Acceptability for
Analyses of U.S. Coastal and Inland Waters
Richard Haugland, PhD
U.S. Environmental Protection Agency, Office of Research and Development
Abstract
The U.S. Environmental Protection Agency
(EPA) offers two similar quantitative poly-
merase chain reaction (qPCR) methods, method
1611 and method 1609, for the rapid estimation
of enterococci fecal indicator bacteria densities
in recreational surface waters. Water quality
monitoring results from either of these methods
can be compared with 2012 EPA Recreational
Water Quality Criteria (RWQC) values for
site-specific notification programs if the meth-
ods are demonstrated to meet performance
acceptability guidelines at the site. Current
site acceptability guidelines that are available
from EPA recommend a maximum frequency
of 10% of samples that can exhibit excessive
sample matrix interference to the EPA methods
as assessed by results and acceptance criteria of
the sample processing and/or amplification con-
trol assays prescribed in the methods. Here we
report the results of a multi-laboratory survey
of 22 different marine, Great Lakes, inland lake,
and river or stream sites from across the U.S. for
their potential acceptability in implementing
methods 1611 and 1609 based on these guide-
lines. Combined laboratory results from 20 and
16 of these sites were found to meet the guide-
lines using methods 1609 and 1611, respectively.
The benefits of augmenting the control assay
results with qPCR analysis estimates of recover-
ies of target sequences from enterococci that are
spiked into the test samples also are presented.
Results from the analyses in this study indi-
cated that the recommended protocol in method
1609 provided the greatest assurance (>98%) of
preventing excessively underestimated entero-
cocci densities (< 50% recovery) caused by
matrix interference in samples meeting control
assay results acceptance criteria.
Biosketch
Dr. Richard Haugland is a microbiologist
in the Environmental Methods & Measurements
Division of the National Exposure Research
Laboratory. He received his bachelor of science
degree in biology from Muskingum College,
Ohio, and his doctorate in developmental
biology from the Ohio State University. His
past research has addressed diverse prob-
lems including biodegradation of hazardous
chemicals in the environment, assessment of the
microbiological quality of indoor environments,
detection of biothreat agents for homeland
defense, and most recently, monitoring ambient
water quality using bacterial indicators of fecal
pollution. Since joining the U.S. Environmental
Protection Agency (EPA) in 1991, Dr. Haugland
has authored or coauthored more than 60 pub-
lications and has received a number of awards
for his work, including the EPA bronze and gold
medals.
50
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Day One: Session 3
Multi-laboratory survey of U.S. EPA
enterococci qPCR methods
acceptability for analyses of U.S.
coastal and inland waters
RichardHaugland, Shawn Siefring, Manju Varma Kevin H
Oshima, Mono Sh'aganesait, YipingCao, Mcir cktn Raith,
John Griffith, Stephen B. Wetsberg, Rachel T. Noble, A.
I)i>t>ene. Blackwood, Julie K inzelman, Tamam A nan eva,
RcbeccaN. Bushon, Erin A. Stelzor, Valaric J. Norwood,
Kuirinu V. Ov/xlon, Chiitlupher Siiiigulliunu
SEFW,
Study background
• QPCR methods can provide rapid (same day)
estimates of fecal indicator bacteria (FIB) densities in
recreational waters.
• Enterococci FIB densities determined by qPCR have
been found in a series of epidemiological studies.
(U.S.EPA NEEAR studies and others) to correlate with
bather gastrointestinal illness rates.
• Based on these observations, qPCR density values for
enterococci are provided In the U.S.EPA (EPA), 2012
Recreational Water Quality Criteria (RWQC).
oEPA
Study background
~ 2012 RWQC further indicates that: "overall testing of the
qPCR method with different types of ambient waters, and by
different laboratories, remains limited and (EPA) anticipates that
there may he situations at some locations where the performance
of the qPCR method may be inconsistent'.
~ For this reason, the RWQC suggests that: "states
evaluate the qPCR method with respect to laboratory
performance and sample interference in their prospective waters
prior to developing new or revised standards relying on this
method
SEPA
Study background
~ EPA has provided guidelines for determining acceptability of
qPCR method performance at prospective sites based on
Ihe peicenlaye of samples passing Ihe uonLiol assay
acceptance criteria specified in the EPA methods:
(http://www2.epa.gov/cwa methods/other clean water act
test-methods-microbioloaicalV
* EPA offers two methods (Method 1611 and Method 1609)
that can be evaluated at prospective sites for their ability to
meet these performance acceptability guidelines.
SERA
Study objectives
~ Use the two EPA methods and different EPA-recommended
or alternative method permutations to:
1. Determine the percentage of samples passing EPA Method
specified and alternative control analysis acceptance criteria
from a variety of different water body types based on analyses
of shaied samples by multiple labs.
2. Evaluate the reliability of the controls in identifying accurate
sample analyses based on estimated recoveries of target gene
sequences from spiked enterococci in these water sample
matrices.
Site Water body type
River or Stream (RS) Racine, W
White Sands Beach
Fischer Park Beach
Quarry I alreParlr
Cabrillo teach
Dohenv Beath
Study sites
Buckeye lake, Ohio
Lake Carroll, Florida
Browns Lake. Wisconsin
Kwer or stream (Kbj Near mouth, uncmnati, umo
River or Stream (RS) Riverfront Park.Tamra. Florida
Inland Loire (IL) Buckeye Lolic, Ohio
interna lake (il)
Inland Lake(lL)
Inland Lake (IL)
Inland I al» (II)
i*eat Lakes |t.ij
Atlantic Ocean (AO) Wilmington North Caroline
51
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U.S. EPA's 2016 Recreational Waters Conference
S-EPA
Study design
I ahoratnry 1* 11S FPft. National Fvprmirp R«parrh I ahnratnry. finrinnati. f»H
Laboratory 2: Southern California Coastal Wotcr Research Project, Costa Mesa, CA
LoUuioloi v 3. Cily i>r Rounc Heallli Dcyoi liuciil, Racine, Wl
LaDoratory 4: uepartment of biology, university of bouth Honda, lampa. n
Laboratory 5: U.S. Geological Survey. Columbus, OH
lahorafmvfi'lnrtitiitpnf Marinp Sripnr« I lniwpr<:itvnf Nnrth farnlina at Thanfl Hill MnrphpaH fitv Nf
^m (i i rn a in ens. l- i
sx CZ] h q m Q Q
From "Millb-laboratory survey of qPCR enterococc* analysis method performance in U.S. coastal and inland surface
waieisHauoland."byR. A. Hauoland.S-Sefrmo M Vaima. KH Gshima.M. Si/aoanesan. Y. Cao M Ralh J Griffith. S.B.
WfeishfirO.RT Nnhlfi.AD RhrVwnnd..1 Krophian.T Anan'pwa.RN Fkishfm V.I Hsrwnnd.KV Rnrrlnn anilC
Rinigalliann. Tfllfi../ Micratonl Method* fflp|> 1U-I?5
5-EPA
Sample analysis methods and
permutations
Method
PCR Master
Extract Dilution
Calculation
Mix Reagent
Analyses
Models
EPA Method
Universal
.Sx-diluted extracts
Delta-Delta Ct
1611
Master Mix
(recommended in
(recommended in
Method], undiluted extract
Method) & Delta Ct
data collected but not
recommended in Method
EPA Method
Environmental
Undiluted extracts
Delta-Delta Ct
1609
Master Mix
(recommended in Method)
(recommended In
& 5x-dlluted extracts
Method) & Delta Ct
(optional in Method)
—
SEPA Matrix interference control
analyses & acceptance criteria
Control Analysis
Acceptance Criterion
Reference
Salmon DNA sample
processing control (SPC)
assay
lebl bdtnple Cl within'3
units of positive control
samples
EPA Melhudb 1611
&1G09
Competitive Internal
Amplification Control
(IAC) assay
test sample Ct within 1.5
units of negative control
samples
EPA Method 1600
& updated Method
1611
Enterococcus assay Ct
shift across undiluted - 5x
sample extract dilutions
test sample Ct shift within
2.32 ±1 units
Caoctal., 2012*
HI *J. flppl. Microbiol. 1tl 66-7?
SEPA
Spike recovery estimations
• Spiked test matrix (SIM) samples: ~1UA4 t. taecalis (hnt) cells
added to filters containing water sample retentates.
• Spiked control matrix (SCM) samples: same number of Hnt cells
added to clean filters.
• Ratios ot total hnt target sequences recovered from SIM/SCM
samples calculated by Delta & Delta-Delta Ct formulas:
• ACt ratio = AI~A(-(a - c))
• AACt ratio = AFA(-((a - b) - (c - d)))
• where AF = amplification factor (amp efficiency + 1), a - mean STM
sample Ent Ct, b = mean STM sample SPC Ct, c = mean SCM
sample hnt Ct. d = mean S( ;m sample SPC: C :t
- Ratios converted to STM/SCM recovery percentages
• The same analyses and calculations weie perluimed fur
corresponding unspiked samples and recoveries subtracted from
the spiked sample recoveries to determine net spike recoveries.
• Net recoveries within 50 200% were considered as acceptable
SERA
Overall results
Method
Total analyses
(sample extract , .
dilution) |N>
percent of
analycec
passing Sft &
IAC control
assay cntena
Percent of
analyses patting
Enterococcus
assay Cl shift
criterion (Cao et
dl>
Percent of act Percent of flflCt
net recovery net recovery
analyses within analyses within
50-200% 50-200%
recovery ranee recovery range
STM/SCM STM/SCM
1611 (lx)
Not determined
)t determined 8/%
84%*
xbll (SX) II8 34%
* Peicenlaye leUuteii by a yiuup of sample analyses Dial would nul meet current QC triletia
&EFA
Summary of site acceptability
analyses based on current
control assay criteria
Method
(extract
dilution)
Sample
analysis
acceptability
criterion
Sites passiny
EPA guidelines
(> 90% earn pie
analyses pass
criteria)
Sites passiny ui
approaching EPA
Guidelines (5 80%
sample analyses
pass criteria)
Method 1609
(undiluted)
SPC and IAC
assay controls
14/22(64%)
18/22 (82%)
Method 1609
(undiluted)
Enterococcus
assay Ct shift
13/22(59%)
17/22 (77%)
Method 1H09
(5x-diiuted)
vSK: and IAC
assay controls
A) m («1%)
¦/?!/? (100%)
Method 1611
(5x-diluted)
SPC and IAC
assay controls
16/22(73%)
21/22 (95%)
¦a
52
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Day One: Session 3
SEPA
Great Lakes,
Lake Michigan site
y North Beach, Racine, Wisconsin
E9Aft*" M
/ mai.oI »« * Analyses % ACT net % MCT net
Method ... labs ' . nutans rprrwcrv rpriMMV
(extract doi Passings & Cnterococcu' anal » analye"
MjZ.,1 Analvses :Z. lACcontrol
a«avrntwia ^
1609 (IX) S9 1 « O » O » M
11
1609 (5x) 93 O ®
KllM << « O
¦a
o Passes CPA site guide# nes (s90%)
O Approochoc EPA cito guidolinoc (80 00%), furthor anatyooc warrantod?
© FailsFPA site (jiiflfJinpc; (< Sfl1*.)
AEPA
Pacific Ocean sites
4o.~v
Newport Dunes Beach, Newport, California
Analyses
% Analyses
% ALi net % MCI net
dilution) Anal''ses
analyse,,
|M*>imS SPC a
ial contra
RltMWlYriK
analvw analyw
1C09 (lx) 32
2
76 O
09 O
75
1«W M 40
« O
M
1611 (5x1 50
3
86 O
90 84
Doheny Beach, Dana Point, California
(attract Tl*al
dilution) Aria v™'
analyses
% Analyses
Dassine SPC &
IAC control
assay Ct shift
analyses analyses
within 50- within 50-
200% 20O9S
1609 (lx) 31
2
ioo m
ioo m
90 100
1609 (bXJ 3b
3
1U0 Q }
9/ 9/
1#11 (SX) 34
3
100 ©
"
AEPA
Environmental Protection
Atlantic Ocean sites
, Jockey
s Ridge Beach, Outer Banks, North Carolina
(ora
% Analyses
Total ' na«ine SPC ft
Analyse) ^ lACcontrol
tnterococcus analyses 'analyses
dilution)
0 " analyses a ' Uj™,j0
assay Q shift within 50- within 50-
1609 (lx)
20 2 100
95 © 85 100
16U9 (bx|
24 I 1UU
92 1U0
1S11 (SX)
24 2 100 ©
»
- South Naes Head Beach, Outer Banks, North Carolina
Method
iotal .s passing iKL &
Analyses s lACcontrol
"" v*a assay criteria
% Analyses KACTnet KMCTnet
1009 (lx)
21 2 B1 ©
80 © 07
1
1G09 (5x)
Ittl (Ex)
100 ©
2 100 ©
02 100
Midwest inland lake sites
Tischer Park Beach, Browns Lake, Wisconsin
1609 (lx) 33 2 91 C J 85 O 70
1609 (bXj 21 2 1U0 © ill
mi 1S*1 21 2 100 O "
Quarry Lake Park Beach, Racine, Wisconsin
« Analyses % ACT ne
sum ""i its I-*"-* s"™ s™
Analyses ® IAC control . . ^
SEPA
Midwest inland lake sites
Rrnnk«: Rpath, Riirkpyp lakp, fpntral Ohin
MAid % Analyses %ACTnet «AACTne
»ni if Mwcn zmz nzir
(extract ""S lACcontrol 0rQ"""' ^T.00'
Uiluliuii) alidpo assay Ctshrft within 50- within 50-
cntenon 20U%
Crystal Beach, Buckeye Lake, Central Ohio
53
-------
S-ER<\ Spike Recovery Estimates
Samples passing SPC & IAC criteria Samples failing SPC & IAC criteria
> MSJ!
U.S. EPA's 2016 Recreational Waters Conference
SERA
Summary
uslnq data from all labs, 20 out of 22 of the sights passed the current EPA site
acceptability guidelines based on Method 1609 analyses of either undiluted or
diluted sample extracts 1fi sites passed hased Method 1fi11 analyses nf diluted
sample exliads.
Ayieemenl un bile acceptability by different labs was 76% (seveidl facluis may be
involved in the differences)
The current controls were generally, but not always, accurate in predicting
acceptable (50-200%) spike recoveries.
Enterococcus assay ct shift and spike recovery results from delta ct analyses
suggested that some of the samples (e.g. Buckeye Lake) interfered with the
analyses.
Delta delta Ct analyses suggested that use of SPC assay results in the calculation
model was effedive ill adjusliny reuuvety estimates lu the acceptable lanye in
many of these interfering samples.
Method 1609 vwth undiluted extracts passed the control assay criteria at a lower
rate but. when outside the accepted spike recovery range, the delta delta Ct
estimates from these analyses were nearly always high rather than low.
Method 1609 (and 1611) with diluted extracts passed the CPA control assay
criteria at a higher rate but delta delta Ct recovery estimates were below 50% in a
higher number of sample analyses passing these controls.
oEPA Possible future guidelines for site
Environment! Protection evaluations (based on this study)
Use Method 1609 with undiluted extracts
Current EPA guidelines
are met:
AACt Spike recoveries
> sn% a < ?nn%-
Current EPA guidelines
are met (& AACt spike
recoveries > 50%):
yes
yes
no
i
Determine spike recoveries
no
i
Use Method 1609 (or 1611) with diluted extracts
yes no
li I
Acceptable Site Site not acceptable
54
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S3
Day One: Session 3
Towards Field-Portable Instrumentation for
Real-Time Water Quality Monitoring Using
Digital Droplet PCR
Kevan Yamahara, PhD
Monterey Bay Aquarium Research Institute
Abstract
The release of the 2012 Recreational Water
Quality Criteria allows beach managers to
utilize quantitative PCR (qPCR) for routine
water quality monitoring. While methods used
to assess water quality have advanced, tech-
niques for automating the process have lagged;
few technologies exist that fully automate the
water quality monitoring process from sample
collection to delivery of quantitative results.
The Environmental Sample Processor (ESP) is
one tool that may enable researchers and beach
managers to monitor beach water quality in an
autonomous manner. Current development of
the ESP system is designed to allow for in-situ
sample collection, sample lysis, and continuous
flow digital droplet PCR (ddPCR) to quantify
the Enterococci 23rDNA gene and other source
tracking targets. Processes performed using the
new ESP system, including sample collection,
DNA extraction, and ddPCR quantification, are
shown to be equivalent to traditional laboratory
methods using real-time qPCR for quantifica-
tion of enterococci. Quantification of enterococci
gDNA by the continuous flow ddPCR instru-
ment developed during the course of this proj-
ect is positively correlated with quantifications
using the BioRad ddPCR instrument (slope =
0.72, R2 = 0.99, p=0.0001). The evolving ESP/
ddPCR technology may provide a new plat-
form for conducting water quality monitoring
tests that can be packaged in a portable, field-
deployable unit, reducing sample handling and
complex assay standardization associated with
traditional qPCR.
Biosketch
Dr. Kevan Yamahara is a research special-
ist at the Monterey Bay Aquarium Research
Institute (MBARI) in Moss Landing, California.
He earned his doctorate in environmental
engineering and science at Stanford University,
where his dissertation focused on the fate and
transport of fecal indicators and pathogens in
California beach sands. At MBARI, he focuses
on developing new technologies for biologi-
cal monitoring of the marine environment.
Dr. Yamahara is currently developing field-
portable instrumentation for monitoring fecal
indicators and source-tracking markers and
autonomous vehicle instrumentation to detect
environmental DNA of marine phytoplankton
and vertebrates.
55
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S3
U.S. EPA's 2016 Recreational Waters Conference
Ul
u
o
Q
HI
>
u
at
<
<
a
Ul
Towards Field-
Portable
Instrumentation for
Real-Time Water
Quality Monitoring
Kevan Yamahara, Andrew Hatch, Joshua
Steele, Cody Youngbull, John Griffith,
Christopher Scholin
WARNING
BEACH CLOStD
Santa Cruz Wharf, CA, Deployment
~ P. australis ~ P.multicorioe ~ p muHiteriesMelics
O Pseudo-nltzschla spp # P a
A FnlRmnnr.r.iiR © Human finrtarinriAs
Yamahara etal. 2015. Lett Appl Microbiol
Proof of Concept
Proof of Concept
0E5P
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Collection
1
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ahara etal. 2015. Lett Appl Microbiol
Sample
Collection
5-nn*
.uu
Data
Uploaded
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|
Outline
_| WARNINb
¦ BEACH CLOSFR
arngmmm
¦: : : .
• Environmental Sample
Processor (ESP)
1
v
tT•*' isf- ' "•
k a*.-;.. .
• Proof of concept study
for water quality
monitoring
• New sensor
development
2nd Generation Environmental Sample
Processor (ESP)
Collection
Concentration
Extraction
Detection
56
-------
Day One: Session 3
Proof of Concept
¦ I1"1 1 1
Quantification of BOTH fecal indicators and harmful
algae from the same sample
Sample to Results in ~ 4 hours
I imitations nf si7P and portability
The Next Conceptual Idea
\-Vy * r '*
j.
wf|'i 1
Instrument to survey a number of locations and
to determine "hot spots"
The Next Conceptual Idea
Instrument that allows for tracking sources of pollution
Instrument Design Criteria
GD,
Samplp f nllprtinn/
Processing
£3 u SMJ
MP
¦
41
Analyte Detection
V;.-.
^ I ¦ #
• Tracking sources of contamination requires mobility
- Engineering design for a hand-carry instrument
- Modular design - separate sample collection and detection
Sample Collection/Processing
Sample Collection/ ¦ MP::
Processing
ii
SSE JH, %
:l r
r 1 k ,J'
3rd Generation ESP Solution
Rotating Section
^Tump Heads ^aste x Analytical Modules
and Ambient Valves
\
Filter housing
seawaierour
Same engineering concepts, different form factor
57
-------
U.S. EPA's 2016 Recreational Waters Conference
Prototype 3rd Generation ESP
3rrl Generation (3G) ESP technology
- Sample Collection and Processing
• Preservation and In-situ Lysis
- Digital PCR (ddPCR) or Surface Plasmon Resonance (SPR)
ESP DNA Extraction Comparison
>
3
iff
O ,0
qPCR ddPCR qPCR
8ead-beating Lysis esp I
ddPCR
s Method
Analyte Detection
Analyte Detection
ASU Droplet Digital PCR Module
Partition a normal PCR reaction with many
DNA templates into many individual PCR reactions
Digital readout of positive and negative reactions
provides an absolute quantification
10 110 1
110 0 10
0 0 1 0 0 1
0 10 0 10
0 10 111
Droplet Digital PCR Module
Partition a normal PCR reaction with many
DNA templates into many individual PCR reactions
Diyiial readout of positive and negative reatlioitb
provide an absolute quantification
10 110 1
1 10 0 10
0 0 1 0 0 1
0 10 0 10
0 10 111
Potitlv* fiw tiniK
Nrgtm* Rwilom
Partitioning to 1-nL Reactions Racetrack
Thermotytler
Digital Positive & Negative
Droplets
ddPCR Quantification of Enterococcus
3 10* -
r - 0.99
oearson
p = 0.0001
r
Q.
o ,
U 10
c
o
2
£ io21
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BioRad ddPCR Concentration (Copics/uL)
58
-------
ddPCR Quantification of HF183
Wo»=°-97
p = 0.002
Qubit Fluorometric Concentration (Copies/uL)
Conclusions and Next Steps
The challenge of portable biological sensors for
water quality monitoring ib surriple acquisition
and processing for downstream analyses
Modular designs may allow for greater flexibility
for detecting/quantifying intended targets (e.g.
ddPCR tor DNA, cELISA for toxins)
- New analytical detection methods are being
developed all the time
Field sampling trials will begin later this year in
2016
Day One: Session 3
Quantification of Environmental
Samples Using ESP Methods
Entorocoeci Concentration (Copfo/uL)
Sample l
Acknowledgements
Sesp
Chris Seholin
James Birrh
Douglas Pargetr
Scull Jeiiben
Drent Roman
Chris Preston
Roman Marin
Rill Ussier
MBARI Technicians
the David
Luc!lcPackard
FOUNDATION
MCll' School of Earth
rw & Space Extlohation
Cody Youngbull
Andrew Hatch
Andrew Larson
Tathagata Ray
Kelly Lmtecum
Greg Wells
Steve welsberg
John Giiffilli
Joshua Steele
Blythc Layton
&, , V MBARI sjS MOORE
M-mtwey Bay Aquarium 1 v ,VV-,\L'
:h Institute
FOUNDATION
59
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J.
U.S. EPA's 2016 Recreational Waters Conference
Question & Answer Session
Question 1
(Unknown): How long does it take for a digital droplet?
Answer 1
Kevan Yamahara: It's about the same time as for the qPCR [quantitative polymerase chain
reaction] system; we could reduce the number of cycles so we are looking into that.
Comment 1
(Unknown): Rumor was that it takes 5 hours for results with digital qPCR.
Comment 1 (follow-up)
Kevan Yamahara: No, it is probably less than an hour.
Question 2
(Unknown): How do you keep the integrity of the sample once you launch it? When the sample goes
from point A to point B, how do you make sure the second site doesn't have the carryover from the first
site?
Answer 2
Kevan Yamahara: We have looked at how to flush the system out. We let it sit for 15 to 20 min-
utes, then flush it with a solution, and are working on a handoff system between cartridge
handling (based on bleach or other solution).
Answer 2 (follow-up)
John Griffith: We work closely with EPA. It's not ready for prime time, but in the upcoming
year it will be comparable to regular qPCR. We'll communicate with EPA as usual.
Question 3
Steve Weisberg: For Shannon Briggs. I find this session to be gratifying. I took a look back at prior
beach conferences. I looked back at the needs back then, then how we started developing the newer
technologies to respond to those needs, then how we started getting more specific, then getting into
application and learning from the challenges. It is great to see the transition from concept and method-
ology to the application. But, what is next? You put effort and resources into training these laboratories
in qPCR, but who is watching you? Shannon, you invested a lot in this equipment, and it could be
replaced in a few years. Was this a good time to make the investment?
Answer 3
Shannon Briggs: Yes things have evolved. The certification process has changed. We're not
near drinking water yet; we discussed this last night. The site-specific document that came
out in 2014 is a bit of a guidance that proves we are doing something right. But it's a day-by-
day thing. Kevan's stuff looks very promising. This thing landed on us by chance—the con-
nection started because of a public meeting. But, yes, I have 5 years to make it work.
Question 4
Suzanne Young: For the extraction methods for DNA, is everyone using kits?
Answer 4
Abhilasha Shrestha: It was a crude extraction for us.
Answer 4 (follow-up)
Kevan Yamahara: Ours was crude with a DNA sequence. We used a gene extraction kit.
60
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