&EPA
United States
Environmental Protection
Agency
Method 1611: Enterococci in Water by
TaqMan® Quantitative Polymerase Chain
Reaction (qPCR) Assay
October 2012
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-12-008
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Acknowledgments
This method was developed under the direction of Rich Haugland, Kevin Oshima and Alfred P. Dufour of
the U.S. Environmental Protection Agency's (EPA) Human Exposure Research Division, National
Exposure Research Laboratory, Cincinnati, Ohio.
The following laboratories are gratefully acknowledged for their participation in the validation of this
method in marine and fresh waters:
Participant Laboratories
Single laboratory validation
• Mycometrics, LLC: King-Teh Lin and Pi-shiang Lai
• New York State Department of Health, Environmental Biology Laboratory: Ellen Braun-Howland
and Stacey Chmura
Marine ambient water multi-laboratory validation
• BioVir: Rick Danielson and Rosie Newton
• EPA Region 1 - New England Regional Laboratory: Jack Paar and Mark Doolittle
• EPA Region 10 Laboratory: Barry Pepich and Stephanie Harris
• Hampton Roads Sanitation District: Robin Parnell and Tiffany Elston
• King County Environmental: Eric Thompson and Joe Clark
• Mycometrics: King-Teh Lin and Jeff Cook
• NOAA - AOML/Ocean Chemistry Division: Chris Sinigalliano, David Wanless, and Maribeth
Gidley
• NOAA - NOS/Center for Coastal Environmental Health & Biomolecular Research: Janet Gooch
Moore and Chris Johnston
• Orange County Public Health Laboratory: Richard Alexander and Joe Guzman
• Texas A&M University - Corpus Christi: Joanna Mott, Ladonna Henson, Sarah Bortz, and Lindsey
Myers
• Virginia Institute of Marine Science: Howard Kator and Kimberly Reece
Fresh ambient water multi-laboratory validation
• BioVir: Rick Danielson and Rosie Newton
• Hampton Roads Sanitation District: Robin Parnell and Tiffany Elston
• King County Environmental: Eric Thompson and Joe Clark
• Orange County Public Health Laboratory: Richard Alexander and Joe Guzman
• Orange County Water District: Donald Phipps and Menu Leddy
• Scientific Methods Incorporated: Fu-Chih Hsu and Rebecca Wong
• Texas A&M University - Corpus Christi: Joanna Mott, Ladonna Henson, and Sarah Bortz
• Texas A&M University - College Station: Suresh Pillai, Palmy Jesudhasan, and Katherine McElhany
• University of Iowa Hygienic Lab: Nancy Hall, Greg Gingerich, Cathy Lord, and Lucy DesJardin
• Wisconsin State Lab of Hygiene: Sharon Kluender and Jeremy Olstadt
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Disclaimer
Neither the United States Government nor any of its employees, contractors, or their employees make any
warranty, expressed or implied, or assumes any legal liability or responsibility for any third party's use of
apparatus, product, or process discussed in this method, or represents that its use by such party would not
infringe on privately owned rights. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
Questions concerning this method or its application should be addressed to:
Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro. robin@epa. gov or OSTCWAMethods@epa.gov
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Introduction
Enterococci are commonly found in the feces of humans and other warm-blooded animals. Although
these organisms can be persistent in the environment, the presence of enterococci in water is an indication
of fecal pollution and the possible presence of enteric pathogens. Epidemiological studies have led to the
development of criteria for promulgating recreational water standards based on established relationships
between the measured density of enterococci colony forming units (CPU) in the water by culture based
methods and the risk of gastrointestinal illness (GI) associated with swimming in the water (References
17.3, 17.6, 17.7, and 17.8).
Method 1611 describes a quantitative polymerase chain reaction (qPCR) procedure for the detection of
DNA from enterococci bacteria in ambient water matrices based on the amplification and detection of a
specific region of the large subunit ribosomal RNA gene (IsrRNA, 23S rRNA) from these organisms.
The advantage of this method over currently accepted culture methods that require 24 - 48 hours to
obtain results is its relative rapidity. Results can be obtained by this method in 3 - 4 hours.
EPA conducted a single-laboratory validation (SLV) study of Method 1611 in marine and fresh water
matrices, to assess potential matrix issues across multiple fresh and marine water matrices from different
geographic regions across the U.S. During the SLV thirty-seven volunteer laboratories collected and
shipped recreational water (marine and/or fresh) samples to the two analytical laboratories for Method
1611 analyses. For all 25 (each) marine and fresh water matrices, no matrix inhibition was observed for
Method 1611. EPA then conducted multi-laboratory validation studies to assess method performance and
reproducibility and develop quantitative quality control (QC) acceptance criteria.
Results of the multi-laboratory validation studies enabled performance characterization of Method 1611
in the reference matrix phosphate buffered saline (PBS) and in marine and fresh water matrices. Results
also enabled the development of initial precision and recovery/ongoing precision and recovery (IPR/OPR)
and matrix spike (MS) QC acceptance criteria. In addition, results from marine water and fresh water
samples (5 different sources for each matrix type) spiked with commercially available target organism
cells were used to assess method reproducibility.
The highly variable recoveries observed during these studies should be taken into consideration when
analyzing results from Method 1611. It is recommended that analysts generate standard curves with each
new lot of qPCR reagents or primers and probes to demonstrate comparable performance with these new
reagents. In addition, weekly calibrator samples should be monitored to ensure that they are within the
acceptable range of the methods; if calibrator samples are not within the acceptable range, new calibrator
samples should be prepared and analyzed. Following these recommendations should increase analyst
proficiency and minimize variability of recoveries. In addition, care should be placed to verify the
accuracy of all reference materials used in the method (i.e. DNA standards, calibrator cells, control
DNA's and spike materials).
In Method 1611, water samples are filtered to collect enterococci on polycarbonate membrane filters.
Following filtration, total DNA is solubilized from the filter retentate using a bead beater. Enterococci
target DNA sequences present in the clarified homogenate are detected by the real time polymerase chain
reaction (PCR) technique using TaqMan® Universal master mix PCR reagent and the TaqMan® probe
system. The TaqMan® system signals the formation of PCR products by a process involving enzymatic
hydrolysis of a fluorogenically-labeled oligonucleotide probe when it hybridizes to the target sequence.
Method 1611 uses an arithmetic formula, referred to as the AACT comparative cycle threshold calculation
(AACT) method to calculate the ratio of Enterococcus IsrRNA gene target sequences (target sequences)
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recovered in total DNA extracts from water samples relative to those in similarly-prepared extracts of
calibrator samples containing a known quantity of Enterococcus cells. The AACT relative quantitation
method also uses measurements of a reference target sequence (from total salmon DNA, added in equal
amounts to both test and calibrator samples) to normalize these ratios for potential differences in DNA
recovery. The reference target sequence measurements are also used to signal inhibition or fluorescence
quenching of the PCR analysis caused by sample matrix components or possible technical error. These
ratios are converted to calibrator cell equivalents (CCEs) recovered from the test samples.
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Table of Contents
1.0 Scope and Application 1
2.0 Summary of Method 2
3.0 Definitions 2
4.0 Interferences 3
5.0 Safety 3
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 5
8.0 Sample Collection, Handling, and Storage 9
9.0 Quality Control 9
10.0 Calibration and Standardization of Method-Related Instruments 14
11.0 Procedure 14
12.0 Data Analysis and Calculations 25
13.0 Sample Spiking Procedure 27
14.0 Method Performance 33
15.0 Pollution Prevention 34
16.0 Waste Management 35
17.0 References 35
18.0 Acronyms 35
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List of Appendices
Appendix A: ABI StepOnePlus™, ABI 7900, and ABI 7500 Real-Time PCR System Operation
Appendix B: Cepheid Smart Cycler® Operation
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Method 1611: Enterococci in Water by TaqMan® Quantitative
Polymerase Chain Reaction (qPCR) Assay
October 2012
1.0 Scope and Application
1.1 Method 1611 describes a quantitative polymerase chain reaction (qPCR) procedure for the
measurement of large subunit ribosomal RNA (IsrRNA, 23 S rRNA) target gene sequences (target
sequences) from all known species of enterococci bacteria in water. This method is based on the
collection of enterococci on membrane filters, extraction of total DNA using a bead beater, and
detection of enterococci target sequences in the supernatant by real time polymerase chain
reaction (qPCR) using TaqMan® Universal master mix PCR reagent and the TaqMan® probe
system. The TaqMan® system signals the formation of PCR products by a process involving the
enzymatic hydrolysis of a labeled fluorogenic probe that hybridizes to the target sequence.
1.2 The Method 1611 test is recommended as a measure of ambient marine and fresh recreational
water quality. The significance of finding Enterococcus DNA target sequences in recreational
water samples stems from the association between the density of these sequences and the reported
occurrence of gastrointestinal (GI) illness associated with swimming observed in recent
epidemiological studies (References 17.4 and 17.10).
1.3 The highly variable recoveries observed during the validation studies should be taken into
consideration when analyzing results from Method 1611. Guidance is provided in section 9 of
this document on how laboratories can monitor the initial and ongoing performance of the method
in their hands. It is expected and has been observed that the performance of the method will
improve as laboratories gain additional experience with it through continued practice.
1.4 This method assumes the use of an Applied Biosystems (ABI) Real-Time PCR System as the
default platform. The Cepheid Smart Cycler® may also be used. The user should refer to the
platform specific instructions for these instruments in the appendices. Users should thoroughly
read the method in its entirety before preparation of reagents and commencement of the method
to identify differences in protocols for different platforms.
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Method 1611
2.0 Summary of Method
The method is initiated by filtering a water sample through a membrane filter. Following
filtration, the membrane containing the bacterial cells and DNA is placed in a microcentrifuge
tube with glass beads and buffer, and then agitated to extract the DNA into solution. The
supernatant is used for PCR amplification and detection of target sequences using the TaqMan8
Universal master mix PCR reagent and probe system.
3.0 Definitions
3.1 Enterococci: all species of the genus Enterococcus for which IsrRNA gene nucleotide sequences
were reported in the GenBank database (http://www.ncbi .nlm.nih/gov/Genbank) at the time of
method development.
3.2 Target sequence: A segment of the Enterococcus IsrRNA gene containing nucleotide sequences
that are homologous to both the primers and probe used in the Enterococcus qPCR assay and that
are common only to species within this genus.
3.3 Sample processing control (SPC) sequence (may also be referred to as reference sequence): A
segment of the ribosomal RNA gene operon, internal transcribed spacer region 2 of chum salmon,
Oncorhynchus keta (O. keta) and other salmon spp., containing nucleotide sequences that are
homologous to the primers and probe used in the SPC qPCR assay. SPC sequences are added as
part of a total salmon DNA solution in equal quantities to all water and calibrator sample filters
prior to extracting DNA from the samples. The purpose of this control is to determine, and, if
necessary, correct for different relative efficiencies of total DNA recovery from water and
calibrator sample extracts using the AACT comparative cycle threshold calculation method (AACT
method) as described in Section 12.3. This control is also used to signal potential co-extraction of
water sample components that interfere with the PCR analysis as described in Section 9.12.
3.4 DNA standard: A purified, RNA-free and spectrophotometrically quantified and characterized E.
faecalis strain ATCC® # 29212™ genomic DNA preparation. DNA standards are used to
generate standard curves for determination of performance characteristics of the qPCR assays and
instrument with different preparations of master mixes containing TaqMan® reagent, primers and
probe as described in Section 9.10. Also used for quantifying target sequences in calibrator
sample extracts as described in Section 12.3.
3.5 Calibrator sample: Samples containing constant added quantities of E. faecalis strain ATCC® #
29212™ cells and SPC sequences that are extracted and analyzed in the same manner as water
sample filtrates. Calibrator sample analysis results are used as positive controls for the
Enterococcus target sequence and SPC qPCR assays and as the basis for target sequence
quantification in water sample filtrates using the AACT comparative cycle threshold calculation
method (AACT method) as described in Section 12.3. Analysis results of these samples provide
corrections for potential method-related variations in Enterococcus cell lysis, target sequence
recovery and PCR efficiency.
3.6 AACT method: A calculation method derived by ABI (Reference 17.1) for calculating the ratios of
target sequences in two DNA samples (e.g., a calibrator and water filtrate sample) that normalizes
for differences in total DNA recovery from these samples using qPCR analysis CT values for a
reference (SPC) sequence that is initially present in equal quantities prior to DNA extraction.
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Method 1611
3.7 Amplification factor (AF): A measure of the average efficiency at which target or SPC sequences
are copied and detected by their respective primer and probe assays during each thermal cycle of
the qPCR reaction that is used in the AACT method. AF values can range from 1 (0% of
sequences copied and detected) to 2 (100% of sequences copied and detected) and are calculated
from a standard curve as described in Section 12.2. For an explanation of test inhibition, see
Section 9.12.
4.0 Interferences
Water samples containing colloidal or suspended particulate materials can clog the membrane
filter and prevent filtration. These materials can also interfere with DNA recovery and with the
qPCR analysis by inhibiting the enzymatic activity of the Taq DNA polymerase, and/or inhibiting
the annealing of the primer and probe oligonucleotides to sample target DNA, enzyme or
quenching of hydrolyzed probe fluorescence.
5.0 Safety
5.1 The analyst / technician must know and observe the normal safety procedures required in a
microbiology and/or molecular biology laboratory while preparing, using, and disposing of
cultures, reagents, and materials, and while operating sterilization equipment.
5.2 Where possible, facial masks should be worn to prevent sample contamination.
5.3 Mouth-pipetting is prohibited.
6.0 Equipment and Supplies
6.1 Separated, and dedicated workstations for reagent preparation and for sample preparation,
preferably with HEPA-filtered laminar flow hoods and an Ultraviolet (UV) light source, each
having separate supplies (e.g., pipettors, tips, gloves, etc.). Note: The same workstation may be
used for the entire procedure provided that it has been cleaned with bleach and UV sterilized as
specified in Section 11.6.1 between reagent and sample preparation. Under ideal conditions, two
dedicated workstations are used for sample preparation: one for preparing samples with high
target sequence DNA concentrations (e.g., DNA standards and calibrator samples) and one for
preparing samples with expected low target sequence DNA concentrations (e.g., filter blanks and
ambient water samples).
6.2 Balance capable of accuracy to 0.01 g
6.3 Extraction tubes: semi-conical, screw cap microcentriruge tubes, 2.0 mL (PGC 506-636 or
equivalent)
6.4 Glass beads, acid washed, 212 - 300 urn diameter (Sigma G-1277 or equivalent)
6.5 Autoclave, capable of achieving and maintaining 121°C [15 Ib pressure per square inch (PSI)] for
minimally 15 minutes (optional)
6.6 Workstation for water filtrations, preferably a HEPA-filtered laminar flow hood with a UV light
source. This can be the same as used for sample preparation (Section 6.1).
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Method 1611
6.7 Sterile bottles/containers for sample collection
6.8 Membrane filtration units (filter base and funnel) for 47 mm diameter filters, sterile glass, plastic
(Pall Gelman 4242 or equivalent), stainless steel, or disposable plastic (Nalgene CN 130-4045 or
CN 145-0045 or equivalent), cleaned and bleach treated (rinsed with 10% v/v bleach, then 3
rinses with reagent-grade water), covered with aluminum foil or Kraft paper, and autoclaved or
UV-sterilized if non-disposable.
6.9 Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source. In an emergency or
in the field, a hand pump or a syringe equipped with a check valve to prevent the return flow of
air can be used.
6.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing
6.11 Filter manifold to hold a number of filter bases
6.12 Flask for safety trap placed between the filter flask and the vacuum source
6.13 Forceps, straight or curved, with smooth tips to handle filters without damage, 2 pairs
6.14 Polycarbonate membrane filters, sterile, white, 47 mm diameter, with 0.45 \am pore size
(GE Osmonics Inc. K04CP04700 or equivalent)
6.15 Graduated cylinders, 100 - 1000 mL, cleaned and bleach treated (rinsed with 10% v/v bleach,
then 3 rinses with reagent-grade water), covered with aluminum foil or Kraft paper and
autoclaved or UV-sterilized.
6.16 Petri dishes, sterile, plastic or glass, 100 x 15 mm with loose fitting lids
6.17 Disposable loops, 1 and 10 uL
6.18 Permanent ink marking pen for labeling tubes
6.19 Visible wavelength spectrophotometer capable of measuring at 595 nm
6.20 Single or multi-place bead beater (Biospec Products Inc. 3110BX or equivalent)
6.21 Microcentrifuge capable of 12,000 x g
6.22 Micropipettors with 10, 20, 200 and 1000 uL capacity. Under ideal conditions, each workstation
should have a dedicated set of micropipettors (one micropipettor set for pipetting reagents not
containing cells or reference DNA and one set for reagents containing reference DNA and for test
samples). Ideally, micropipettors should be calibrated monthly.
6.23 Micropipettor tips with aerosol barrier for 10, 20, 200 and 1000 uL capacity micropipettors.
Note: All micropipetting should be done with aerosol barrier tips. The tips used for reagents not
containing DNA should be separate from those used for reagents containing DNA and test
samples. Each workstation should have a dedicated supply of tips.
6.24 Microcentrifuge tubes, low retention, clear, 1.7 mL (GENE MATE C-3228-1 or equivalent)
6.25 Test tube rack for microcentrifuge tubes, use a separate rack for each set of tubes
6.26 Conical centrifuge tubes, sterile, screw cap, 50 mL
6.27 Test tubes, screw cap, borosilicate glass, 16 x 125 mm
6.28 Pipet containers, stainless steel, aluminum or borosilicate glass, for glass pipets
6.29 Pipets, sterile, T.D. bacteriological or Mohr, disposable glass or plastic, of appropriate volume
(disposable pipets preferable)
6.30 Vortex mixer (ideally one for each work station)
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Method 1611
6.31 Dedicated lab coats for each work station
6.32 Disposable powder-free gloves for each work station
6.33 Refrigerator, 4°C (ideally one for reagents and one for DNA samples)
6.34 Freezer, -20°C or -80°C (ideally one for reagents and one for DNA samples)
6.35 Ice, crushed or cubes for temporary preservation of samples and reagents
6.36 Printer (optional)
6.37 Data archiving system (e.g., flash drive or other data storage system)
6.38 UV spectrophotometer capable of measuring wavelengths of 260 and 280 nm using small volume
capacity (e.g., 0.1 mL) cuvettes orNanoDrop® (ND-2000 spectrophotometer or equivalent)
capable of the same measurements at 2 uL sample volumes
6.39 ABI StepQnePlus™. ABI 7900. or ABI 7500
6.39.1 Optical 96 well PCR reaction tray (ABI N801-0560 or equivalent)
6.39.2 Optical adhesive PCR reaction tray tape (ABI 4311971 or equivalent) or Micro Amp™
caps (ABI N8010534 or equivalent)
6.39.3 ABI StepOnePlus™, ABI 7900, or ABI 7500 Real-Time PCR System
6.40 Cepheid (optional to ABI)
6.40.1 Smart Cycler® 25 uL PCR reaction tubes (Cepheid 900-0085 or equivalent)
6.40.2 Rack and microcentrifuge for Smart Cycler® PCR reaction tubes. Note: Racks and
microcentrifuge are provided with the Smart Cycler® thermocycler
6.40.3 Cepheid Smart Cycler® System Thermocycler
7.0 Reagents and Standards
Note: The E.faecalis stock culture (Section 7.8), Salmon DNA/extraction buffer (Section 7.12),
and DNA extraction tubes (Section 7.18), may be prepared in advance.
7.1 Purity of Reagents: Molecular-grade reagents and chemicals shall be used in all tests
7.2 Control Culture
• Enterococcus faecalis (E. faecalis) ATCC® #29212™ (ATCC, Manassas VA) or
• E. faecalis ATCC® # 29212™ (550 colony forming units [CPU]) BioBalls (bioMerieux Inc.,
Durham NC)
7.3 Sample Processing Control (SPC) DNA
Salmon testes DNA (Sigma D1626 or equivalent)
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Method 1611
7.4 Phosphate Buffered Saline (PBS)
7.4.1 Composition:
Monosodium phosphate (NaH2PO4) 0.58 g
Disodium phosphate (Na2HPO4) 2.50 g
Sodium chloride 8.50 g
Reagent-grade water 1.0 L
7.4.2 Dissolve reagents in 1 L of reagent-grade water in a flask and dispense in appropriate
amounts for dilutions in screw cap bottles or culture tubes, and/or into containers for use
as rinse water. Autoclave after preparation at 121°C (15 Ib PSI) for 15 minutes. Final
pH should be 7.4 ± 0.2.
7.5 Brain heart infusion broth (BHIB)
7.5.1 Composition:
Calf brains, infusion from 200.0 g 7.7 g
Beef heart, infusion from 250.0 g 9.8 g
Proteose peptone 10.0 g
Sodium chloride 5.0 g
Disodium phosphate (Na2HPO4) 2.5 g
Dextrose 2.0 g
Reagent-grade water 1.0 L
7.5.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
completely. Dispense 10 mL volumes in screw cap 16 x 125 mm tubes and autoclave at
121°C (15 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2.
7.6 Brain heart infusion agar (BHIA)
7.6.1 Composition:
BHI agar contains the same components as BHIB with the addition of 15.0 g agar per
liter of BHI broth.
7.6.2 Add agar to formula for BHIB provided above. Prepare as in Section 7.5.2. After
sterilization, dispense 12 - 15 mL into 100 x 15 mm petri dishes. Final pH should be 7.4
±0.2.
7.7 Sterile glycerol (used for preparation of E. faecalis stock culture as described in Section 7.8)
7.8 Preparation of E. faecalis (ATCC® # 29212™) stock culture
Rehydrate lyophilized E. faecalis per manufacturer's instructions (for ATCC® stocks, suspend in
5-6 mL of sterile BHIB and incubate at 37°C for 24 hours). Centrifuge to create pellet. Using a
sterile pipet, discard supernatant. Resuspend pellet in 10 mL of fresh sterile BHIB containing
15% glycerol and dispense in 1.5 mL aliquots in microcentrifuge tubes. Freeze at -20°C (short
term storage) or -80°C (long term storage). Note: Aliquots of suspension should be plated to
determine CPU concentration as described in Section 11.1. It is also advisable to verify the E.
faecalis culture as described, for example, in Section 15 of EPA Method 1600.
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Method 1611
7.9 PCR-grade water (OmniPur water from VWR EM-9610 or equivalent). Water must be
DNA/DNase free.
7.10 Isopropanol or ethanol, 95%, for flame-sterilization
7.11 AE Buffer, pH 9.0 (Qiagen 19077 or equivalent) Note: pH 8.0 is acceptable
Composition:
10 mM Tris-Cl (Tris-chloride)
0.5 mM EDTA (Ethylenediaminetetraacetic acid)
7.12 Salmon DNA/extraction buffer
7.12.1 Composition:
Stock Salmon testes DNA (10 ug/mL) (Section 7.3)
AE Buffer (Section 7.11)
7.12.2 Preparation of stock Salmon testes DNA: Using sterilized scissors and sterilized forceps,
cut a piece of DNA to weigh approximately 20 mg, and place in a sterile 50 mL conical
tube. After weighing, add 20 mL of PCR-grade water. Cap tightly, and resuspend by 2 -
4 hours of vigorous shaking (note: longer times may be required to fully suspend the
DNA - check for even consistency by repeatedly drawing up the suspension and
expelling with a sterile 25 ml pipet) . The concentration should be about 1 mg/mL.
Dilute a 1ml aliquot of this 1 mg/mL stock with 99 ml PCR-grade water to make 10
ug/mL working stock. Check absorbance (OD2eo) and calculate DNA concentration
using the assumption 50 ug/mL DNA is equal to 1 OD26o, (i.e. 10 ug/mL should equal ~
0.2 OD2eo.). If measured concentration is different than 10 ug/mL, adjust dilutions to
make 0.2 ug/mL Salmon DNA/extraction buffer in 7.12.3 accordingly. Dispense 1
mg/mL stock and 10 ug/mL working stock suspensions into aliquots (e.g. 1 ml) and
freeze at -20°C. Frozen aliquots of working stock should be thawed only once, but once
thawed can be stored in a refrigerator for several months.
7.12.3 Dilute Salmon testes DNA working stock with AE buffer to make 0.2 ug/mL Salmon
DNA/extraction buffer. Extraction buffer may be prepared in advance and stored at 4°C
for a maximum of 1 week.
Note: Determine the total volume of Salmon DNA/extraction buffer required for each day
or week by multiplying volume (600 uL) x total number of samples to be analyzed
including controls, water samples, and calibrator samples. For example, for 18 samples,
prepare enough Salmon/DNA extraction buffer for 24 extraction tubes: 3 tubes for water
sample filtration blank (method blank) and 3 tubes for calibrator samples). Note: At least
one method blank should be included with each sample batch. Additionally, prepare
excess volume to allow for accurate dispensing of 600 uL per tube, generally enough for
1 extra tube. Thus, in this example, prepare sufficient Salmon/DNA extraction buffer for
24 tubes plus one extra. The total volume needed is 600 uL x 25 tubes = 15,000 uL.
Dilute the Salmon testes DNA working stock 1:50, for atotal volume needed (15,000 uL)
+ 50 = 300 uL of 10 ug/mL Salmon testes DNA working stock. The AE buffer needed is
the difference between the total volume and the Salmon testes DNA working stock. For
this example, 15,000 uL - 300 uL = 14,700 uL AE buffer needed.
7.13 Bleach solution: 10% v/v bleach (or other reagent that hydrolyzes DNA), used for cleaning work
surfaces
7.14 Sterile water (used as rinse water for work surface after bleaching)
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Method 1611
7.15 TaqMan® Universal PCR master mix (ABI 4304437 or equivalent)
Composition:
AmpliTaq Gold® DNA Polymerase
AmpErase® UNO
dNTPs with dUTP
Passive Reference 1 (ROX™ fluorescent dye)
Optimized buffer components (KC1, Tris, EDTA, MgCl2)
7.16 Bovine serum albumin (BSA), fraction V powder (Sigma B-4287 or equivalent)
Dissolve in PCR-grade water to a concentration of 2 mg/mL.
7.17 Primer and probe sets: Primer and probe sets may be purchased from commercial sources.
Primers should be desalted, probes should be HPLC purified.
7.17.1 Enterococcus primer and probe set (Reference 17.5):
Forward primer: 5'-GAGAAATTCCAAACGAACTTG
Reverse primer: 5'-CAGTGCTCTACCTCCATCATT
TaqMan® probe: [6-FAM]-5'-TGGTTCTCTCCGAAATAGCTTTAGGGCTA-TAMRA
7.17.2 Salmon DNA primer and probe set (Reference 17.4):
Forward primer: 5'-GGTTTCCGCAGCTGGG
Reverse primer (Sketa 22): 5'-CCGAGCCGTCCTGGTC
TaqMan® probe: [6-FAM]-5'-AGTCGCAGGCGGCCACCGT-TAMRA
7.17.3 Preparation of primer/probes: Using a micropipettor with aerosol barrier tips, add PCR
grade water to the lyophilized primers and probe from the vendor to create stock
solutions of 500 uM primer and 100 uM probe and dissolve by extensive vortexing.
Pulse centrifuge to coalesce droplets. Store stock solutions at -20°C.
7.18 DNA extraction tubes
Note: It is recommended that tube preparation be performed in advance of water sampling and
DNA extraction procedures.
Prepare 1 tube for each sample, and 1 extra tube for every 6 samples (i.e., for method blank) and
a minimum of 3 tubes per week for calibrator samples. Weigh 0.3 ± 0.01 g of glass beads
(Section 6.4) and pour into extraction tube. Seal the tube tightly, checking to make sure there are
no beads on the O-ring of the tube. Check the tube for proper O-ring seating after the tube has
been closed. Autoclave tubes at 121°C (15 PSI) for 15 minutes. Note: Sterile extraction tubes
containing glass beads are available from Generite LLC., (50205-50 or equivalent).
7.19 Purified, RNA-free quantified and characterized E. faecalis genomic DNA preparations for use as
standards used to generate a standard curve (see Section 11.2 for preparation instructions).
7.20 RNase A 5 mg/mL (epicentre® MRNA092 or equivalent)
7.21 DNA extraction kit (Gene-Rite K102-02C-50 DNA-EZ® RW02 or equivalent)
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Method 1611
8.0 Sample Collection, Handling, and Storage
8.1 Sampling procedures are briefly described below. Adherence to sample preservation procedures
and holding time limits is critical to the production of valid data. Samples not collected
according to these procedures should not be analyzed. Note: It is recommended that when
possible that three samples be collected and analyzed from each sample location.
8.2 Sampling Techniques
Samples are collected by hand or with a sampling device if the sampling site has difficult access
such as a dock, bridge or bank adjacent to a surface water. Composite samples should not be
collected, since such samples do not display the range of values found in individual samples. The
sampling depth for surface water samples should be 6 - 12 inches below the water surface.
Sample containers should be positioned such that the mouth of the container is pointed away from
the sampler or sample point. After removal of the container from the water, a small portion of the
sample should be discarded to provide head space for proper mixing before analyses.
8.3 Storage Temperature and Handling Conditions
Ice or refrigerate water samples at a temperature of <10°C during transit to the laboratory. Do
not freeze the samples. Use insulated containers to assure proper maintenance of storage
temperature. Ensure that sample bottles are tightly closed and are not completely immersed in
water during transit.
8.4 Holding Time Limitations
Examine samples as soon as possible after collection. Do not hold samples longer than 6 hours
between collection and initiation of filtration.
9.0 Quality Control
9.1 Each laboratory that uses Method 1611 is required to operate a formal quality assurance (QA)
program that addresses and documents instrument and equipment maintenance and performance,
reagent quality and performance, analyst training and certification, and records storage and
retrieval. Additional recommendations for QA and quality control (QC) procedures for
microbiological laboratories are provided in Reference 17.2.
9.2 The minimum analytical QC requirements for the analysis of samples using Method 1611 include
an initial demonstration of laboratory capability through performance of the initial precision and
recovery (IPR) analyses (Section 9.3), ongoing demonstration of laboratory capability through
performance of the ongoing precision and recovery (OPR) analysis (Section 9.4) and matrix spike
(MS) analysis (Section 9.5), and the routine analysis of positive control (Section 9.6), no template
controls (Section 9.7), method blanks (Section 9.8), media sterility checks (Section 9.9), DNA
standard curves (9.10), calibrator samples (9.11), and sample processing controls (9.12). For the
IPR, OPR and MS analyses, it is necessary to spike samples with either laboratory-prepared
spiking suspensions or BioBalls as described in Section 13.
9.3 Initial precision and recovery (IPR) — The IPR analyses are used to demonstrate acceptable
method performance (recovery and precision) and should be performed by each laboratory before
the method is used for monitoring field samples. An IPR should be performed by each analyst.
The IPR analyses are performed as follows:
9.3.1 Prepare four, 100 mL samples of PBS and spike each sample with the control culture, E.
faecalis ATCC® # 29212™ according to the spiking procedure in Section 13. Spiking
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Method 1611
with laboratory-prepared suspensions is described in Section 13.4 and spiking with a
commercially prepared product (e.g., BioBalls) is described in Section 13.7. Filter and
process each IPR sample according to the procedures in Section 11 and calculate the
calibrator cell equivalents (CCEs) in the samples according to Section 12. Note: prepare
calibrator samples from the same source of cells used to prepare the spike suspensions,
i.e. for samples spiked with laboratory-prepared cell suspensions, use the same
laboratory-prepared cell suspensions to prepare calibrator samples; for samples spiked
with BioBalls, use the same lot of BioBalls to prepare calibrator samples.
9.3.2 Calculate the percent recovery (R) for each IPR sample according to the Method 1611
Calculation Excel file using the appropriate spreadsheets "Laboratory-prepared" or
"BioBalls" for samples spiked with laboratory-prepared spiking suspensions or BioBalls,
respectively.
9.3.3 Using the percent recoveries of the four analyses, calculate the mean percent recovery
and the relative standard deviation (RSD) of the recoveries. The RSD is the standard
deviation divided by the mean, multiplied by 100.
9.3.4 Compare the mean recovery and RSD with the corresponding IPR criteria in Table 1,
below. If the mean and RSD for recovery of enterococci meet acceptance criteria, system
performance is acceptable and analysis of field samples may begin. If the mean recovery
or the RSD fall outside of the required range for recovery, system performance is
unacceptable. In this event, identify the problem by evaluating each step of the analytical
process, media, reagents, and controls, correct the problem and repeat the IPR analyses.
Table 1. Calculated Initial Precision and Recovery (IPR) Acceptance Criteria at
550 CPU
Spike Type
Lab-prepared
BioBall®
IPR Mean Recovery (%)
Detect - 2684%
Detect - 286%
IPR RSD
132
112
OPR Recovery (%)
Detect - 2846%
Detect - 325%
9.4 Ongoing precision and recovery (OPR) — To demonstrate ongoing control of the analytical
system, the laboratory should routinely process and analyze spiked PBS samples. The laboratory
should analyze one OPR sample after every 20 field and matrix spike samples or one per week
that samples are analyzed, whichever occurs more frequently. The OPR analysis is performed as
follows:
9.4.1
Prepare one, 100 mL sample of PBS and spike with E.faecalis ATCC® # 29212™
according to the spiking procedure in Section 13. Spiking with laboratory-prepared
suspensions is described in Section 13.4 and spiking with BioBalls is described in Section
13.7. Filter and process the OPR sample according to the procedures in Section 1 1 and
calculate the target sequences in the samples according to Section 12.
9.4.2 Calculate the percent recovery (R) for the OPR sample according to the Method 1611
Calculation Excel file using the appropriate spreadsheets "Laboratory-prepared" or
"BioBalls" for samples spiked with laboratory-prepared spiking suspensions or BioBalls,
respectively.
9.4.3 Compare the OPR result (percent recovery) with the corresponding OPR recovery criteria
in Table 1, above. If the OPR result meets the acceptance criteria for recovery, method
performance is acceptable and analysis of field samples may continue. If the OPR result
falls outside of the acceptance criteria, system performance is unacceptable. In this
10
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Method 1611
event, identify the problem by evaluating each step of the analytical process, reagents,
and controls, correct the problem and repeat the OPR analysis.
9.4.4 As part of the laboratory QA program, results for OPR and IPR samples should be
charted and updated records maintained in order to monitor ongoing method
performance. The laboratory should also develop a statement of accuracy for Method
1611 by calculating the average percent recovery (R) and the standard deviation of the
percent recovery (sr). Express the accuracy as a recovery interval from R - 2sr to R + 2sr.
9.5 Matrix spikes (MS) — MS analysis are performed to determine the effect of a particular matrix
on enterococci recoveries. The laboratory should analyze one MS sample when recreational
water samples are first received from a source from which the laboratory has not previously
analyzed samples. Subsequently, 5% of field samples (1 per 20) from a given source should
include a MS sample. MS samples must be accompanied by the analysis of an unspiked field
sample sequentially collected from the same sampling site. When possible, MS analyses should
also be accompanied by an OPR sample (Section 9.4), using the same spiking procedure
(laboratory-prepared spiking suspension or BioBalls). The MS analysis is performed as follows:
9.5.1 Prepare two, 100 mL field samples that were sequentially collected from the same site.
One sample will remain unspiked and will be analyzed to determine the background or
ambient concentration of enterococci for calculating MS recoveries (Section 9.5.3). The
other sample will serve as the MS sample and will be spiked with E. faecalis ATCC® #
29212™ according to the spiking procedure in Section 13.
9.5.2 Spike the MS sample volume(s) with a laboratory-prepared suspension as described in
Section 13.4 or with BioBalls as described in Section 13.7. Immediately filter and
analyze both the unspiked and spiked field samples according to the procedures in
Section 11.
9.5.3 For the MS sample, calculate the number of target sequences according to Section 12 and
adjust based on any background enterococci observed in the unspiked matrix sample.
9.5.4 Calculate the percent recovery (R) for the MS sample (adjusted based on ambient
enterococci in the unspiked sample) according to the Method 1611 Calculation Excel file
using the appropriate spreadsheets "Laboratory-prepared" or "BioBalls" for samples
spiked with laboratory-prepared spiking suspensions or BioBalls, respectively.
9.5.5 Compare the MS result (percent recovery) with the appropriate method performance
criteria in Table 2, below. If the MS recovery meets the acceptance criteria, system
performance is acceptable and analysis of field samples from this disinfected wastewater
source may continue. If the MS recovery is unacceptable and the OPR sample result
associated with this batch of samples is acceptable, a matrix interference may be causing
the poor results. If the MS recovery is unacceptable, all associated field data should be
flagged.
OO
9.5.6 Laboratories should record and maintain a control chart comparing MS recoveries for all
matrices to batch-specific and cumulative OPR sample results analyzed using Method
1611. These comparisons should help laboratories recognize matrix effects on method
recovery and may also help to recognize inconsistent or sporadic matrix effects from a
particular source.
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Method 1611
Table 2. Calculated Matrix Spike Precision and Recovery Acceptance Criteria at
550 CPU
9.6
Matrix
MW
FW
Spike Type
Lab-prepared
BioBall®
Lab-prepared
BioBall®
MS Recovery (%)
Detect -1198%
Detect -1123%
Detect - 3473%
Detect- 333%
MS RPD 1 (%)
160
191
110
354
Relative percent difference
Positive controls — The laboratory should analyze positive controls to ensure that the method is
performing properly. Fluorescence amplification growth curve (PCR amplification trace) with an
appropriate cycle threshold (CT) value during PCR indicates proper method performance. On an
ongoing basis, the laboratory should perform positive control analyses every day that samples are
analyzed. In addition, controls should be analyzed when new lots of reagents or filters are used.
9.6.1 Calibrator samples will serve as the positive control. Analyze as described in Section
11.0. Note: Calibrator samples contain the same amount of extraction buffer and starting
amount of Salmon DNA as the test samples; hence E. faecalis calibrator sample extracts
(Section 11.3) will be used as a positive control for both Enterococcus and SPC qPCR
assays.
9.6.2 If the positive control fails to exhibit the appropriate fluorescence growth curve response,
check and/or replace the associated reagents, and reanalyze. If positive controls still fail
to exhibit the appropriate fluorescence growth curve response, prepare new calibrator
samples and reanalyze (see Section 9.11).
9.7 No template controls — The laboratory should analyze "No Template Controls" (NTCs) to
ensure that the PCR master mix reagents are not contaminated. On an ongoing basis, the
laboratory should perform NTC analyses every day that samples are analyzed. If greater than
one-third of the NTC reactions for a PCR master mix exhibit true positive logarithmic
amplification traces with CT values below 45 (not from chemical degradation of probe with linear
kinetics that exhibit rising baseline) or if any one NTC reaction has a CT value lower than 35, the
analyses should be repeated with new master mix working stock preparations.
9.8 Method blank (water sample filtration blank) — Filter a 50 mL volume of sterile PBS before
beginning the sample filtrations. Remove the funnel from the filtration unit. Using two sterile or
flame-sterilized forceps, fold the filter on the base of the filtration unit and place it in an
extraction tube with glass beads as described in Section 7.18. Extract as in Section 11.5. The
absence of a fluorescence amplification growth curve during PCR analysis of these samples
(reported as "0" on Smart Cycler® and "undetermined" on ABI instruments) indicates the absence
of contaminant target DNA (see Data Quality Acceptance, below). Prepare at least one method
blank filter for every PCR run (e.g., minimally one method blank per 96 well plate or Smart
Cycler run).
9.9 Media sterility check — The laboratory should test media sterility by incubating one
uninoculated unit (tube or plate) from each batch of medium (BHIA/BHIB) as appropriate and
observing for growth. Absence of growth indicates media sterility. On an ongoing basis, the
laboratory should perform media sterility checks when preparing new E. faecalis cell stocks.
9.10 DNA standards and standard curves -Purified, RNA-free and spectrophotometrically
quantified E. faecalis genomic DNA should be prepared as described in Section 11.2. Based on
reported values for its size, the weight of a single E. faecalis genome can be estimated to be ~3.6
fg and there are four IsrRNA gene copies per genome in this species (see the rRNA operon
12
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Method 1611
database at http://rrndb.cme.msu.edu). The concentration of IsrRNA gene copies per uL in the
standard E. faecalis genomic DNA preparation can be determined from this information and from
its spectrophotometrically determined total DNA concentration by the formula:
„ . , Total DNA concentration (fg/uL) 4 IsrRNA gene copies
Concentration of IsrRNA \&^> & v
X
gene copies per uL =
3.6fg/genome genome
A composite standard curve should be generated from multiple analyses of serial dilutions of this
DNA standard using the Enterococcus primer and probe assay and subjected to linear regression
analysis as described in Section 12.2. From that point on, it is recommended that additional
standard curves be generated from triplicate analyses of these same diluted standard samples with
each new lot of TaqMan® master mix reagents or primers and probes to demonstrate comparable
performance by these new reagents. The r2 values from regressions of these curves should ideally
be 0.99 or greater. Comparable performance is assessed by their slopes and y-intercepts which
should be consistent with those from the initial composite analyses (e.g., within the 95%
confidence range of the average values). Note: The 95% confidence ranges for these parameters
in the initial composite standard curve can be generated using the Regression Analysis Tool
which can be accessed from the "Data Analysis" selection under the "Tools" menu in Excel.
Subsequent regressions can be performed by plotting the data using the Chart Wizard in Excel
and using the "add trend line" selection in the Chart menu and "display equation on chart"
selection under Options to obtain slope and y-intercept values as illustrated in Section 12.2.
In the event that the slope value from a subsequent standard curve regression is outside of the
acceptance range, the diluted standards should be re-analyzed. If this difference persists, new
working stocks of the reagents should be prepared and the same procedure repeated. If the
differences still persist, the amplification factor values used for calculations of target cell
numbers, as described in Section 12, should be modified based on the new slope values. If the
slopes are within acceptance range but y-intercepts are not within acceptance range of this
previous average, new aliquots of serial dilutions of the DNA standard should be analyzed or
prepared and analyzed as described in Section 11.2 and above.
9.11 Calibrator samples — The cell concentration of each cultured E. faecalis stock suspension used
for the preparation of calibrator sample extracts should be determined as described in Section
11.1. A minimum of nine calibrator sample extracts should initially be prepared from three
different freezer-stored aliquots of these stocks as described in Section 11.3. Dilutions of each of
these calibrator sample extracts equivalent to the anticipated dilutions of the test samples used for
analysis (e.g., 1-, 5- and/or 25-fold) should be analyzed with the Enterococcus primer and probe
assay. The average and standard deviation of the CT values from these composite analyses should
be determined. From that point on, a minimum of three fresh calibrator sample extracts should be
prepared from an additional frozen aliquot of the same stock cell suspension at least weekly and
preferably daily before analyses of each batch of test samples. Dilutions of each new calibrator
sample extract equivalent to the initial composite dilutions (e.g., 1-, 5- and/or 25-fold) should be
analyzed using the Enterococcus primer and probe assay. The average CT value from these
analyses should not be significantly different from the laboratory's average values from analyses
of the initial calibrator sample extracts from the same stock cell suspension (i.e., not greater than
three standard deviations). If these results are not within this acceptance range, new calibrator
extracts should be prepared from another frozen aliquot of the same stock cell suspension and
analyzed in the same manner as described above. If the results are still not within the acceptance
range, the reagents should be checked by the generation of a standard curve as described in
Section 9.10.
13
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Method 1611
9.12 Salmon DNA SPC sequence analyses — The Salmon DNA SPC is used as a DNA isolation
efficiency control. While not essential, it is good practice to routinely prepare and analyze
standard curves from serial dilutions of Salmon DNA working stocks in a manner similar to that
described for the Enterococcus DNA standards in Section 9.10. At this time rRNA gene operon
copy numbers per genome have not been reported in the literature for the salmon species O. keta.
Therefore log-transformed total DNA concentration values or dilution factor values can be
substituted for target sequence copy numbers as the x-axis values in these plots and regression
analyses.
In general, target DNA concentrations in test samples can be calculated as described in Section
12. However, the Salmon DNA PCR assay results for each test sample's 5-fold dilution should
be within 3 CT units of the mean of the 5-fold diluted calibrator (and/or method blank) Salmon
DNA sample results. Higher CT values may indicate significant PCR inhibition or poor DNA
recovery possibly due to physical, chemical, or enzymatic degradation. Repeat the Enterococcus
and Salmon DNA PCR assays of any samples whose 5-fold dilution exhibits a Salmon DNA PCR
assay CT value greater than 3 CT units higher than the mean of the calibrator sample results using
a 5-fold higher dilution (net dilution: 25-fold) of the extracts. The Enterococcus PCR result from
assaying the original 5-fold dilution of the sample can be accepted if its Salmon DNA assay CT
value is lower than that of the corresponding 25-fold dilution of the sample. This pattern of
results is indicative of poor recovery of total DNA in the extract rather than PCR inhibition. The
poor DNA recovery is compensated for by the calculation method. Contrarily, if the Salmon
PCR assay CT value of the 25-fold dilution of the sample is lower than that of the 5-fold dilution
of the sample, then the Enterococcus PCR assay result from the 25-fold dilution of the sample is
considered more accurate. However the Enterococcus PCR results should be reported as
questionable if the Salmon DNA assay's result is still not within 3 CT units of the mean CT result
of the 25-fold dilution of the three calibrators.
10.0 Calibration and Standardization of Method-Related Instruments
10.1 Check temperatures in incubators twice daily with a minimum of 4 hours between each reading to
ensure operation within stated limits.
10.2 Check thermometers at least annually against a NIST certified thermometer or one that meets the
requirements of NIST Monograph SP 250 -23. Check columns for breaks.
10.3 The spectrophotometer should be calibrated each day of use using OD calibration standards
between 0.01 - 0.5. Follow manufacturer instructions for calibration.
10.4 Micropipettors should be calibrated at a minimum annually, ideally monthly, and tested for
accuracy on a weekly basis. Follow manufacturer instructions for calibration.
10.5 Follow manufacturer instructions for calibration of real-time PCR instruments.
11.0 Procedure
Note: E. faecalis cell suspensions (Section 11.1) and E. faecalis DNA standards (Section 11.2)
may be prepared in advance. Calibrator samples (Section 11.3) should be prepared at least
weekly.
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Method 1611
11.1 Preparation of E. faecalis cell suspensions for DNA standards and calibrator samples
11.1.1 Thaw an E. faecalis (ATCC® # 29212™) stock culture (Section 7.8) and streak for
isolation on BHIA plates. Incubate plates at 37°C ± 0.5°C for 24 ± 2 hours.
11.1.2 Pick an isolated colony of E. faecalis from BHIA plates and suspend in 1 mL of sterile
PBS and vortex.
11.1.3 Use 10 uL of the 1 mL suspension of E. faecalis to inoculate a 10 mL BHIB tube. Place
the inoculated tube and one uninoculated tube (sterility check) on a shaker set at 250 rpm
and incubate at 37°C ± 0.5°C for 24 ± 2 hours. Note: It is advisable to verify that the
selected colony is Enterococcus as described, for example, in Section 15 of EPA Method
1600.
11.1.4 After incubation, centrifuge the BHIB containing E. faecalis for 5 minutes at 6000 x g.
11.1.5 Aspirate the supernatant and resuspend the cell pellet in 10 mL PBS.
11.1.6 Repeat the two previous steps twice and suspend the final E. faecalis pellet in 5 mL of
sterile PBS. Label the tube as E. faecalis undiluted stock cell suspension, noting cell
concentration after determination with one of the following steps (11.1.7).
11.1.7 Determine calibrator sample cell concentrations based on one of two the options
indicated below.
Option 1: Spectrophotometric absorbance - Remove three 0.1 mL aliquots of
undiluted cell suspension and dilute each with 0.9 mL of PBS (10"1 dilution). Read
absorbance at 595 nm in spectrophotometer against PBS blank (readings should
range from 0.05 to 0.3 OD). Calculate cells/mL (Y) in undiluted cell suspension
using the formula from the standard curve shown below where X is the average 595
nm spectrophotometer reading. Note: Cell concentration (spectrophotometric
determination) of undiluted E. faecalis cell suspension should be at least ~109
CFU/mL in order to obtain adequate DNA yield for accurate UV spectrophotometric
reading.
Y = (l x 109 cells/mLxX) 70.19
Option 2: plating - Note: PBS and BHIA plates should be prepared in advance if this
option is chosen. For enumeration of the E. faecalis undiluted cell suspension, dilute
and inoculate according to the following.
A) Mix the E. faecalis undiluted cell suspension by vigorously shaking the 5 mL
tube a minimum of 25 times. Use a sterile pipette to transfer 1.0 mL of the
undiluted cell suspension to 99 mL of sterile PBS, cap, and mix by vigorously
shaking the bottle a minimum of 25 times. This is cell suspension dilution "A".
A 1.0 mL volume of dilution "A" is 10"2 mL of the original undiluted cell
suspension.
B) Use a sterile pipette to transfer 11.0 mL of cell suspension dilution "A" to 99 mL
of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25
times. This is cell suspension dilution "B". A 1.0 mL volume of dilution "B" is
10"3 mL of the original undiluted cell suspension.
C) Use a sterile pipette to transfer 11.0 mL of cell suspension dilution "B" to 99
mL of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of
25 times. This is cell suspension dilution "C". A 1.0 mL volume of dilution "C"
is 10"4 mL of the original undiluted cell suspension.
15
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Method 1611
D) Use a sterile pipette to transfer 11.0 mL of cell suspension dilution "C" to 99 mL
of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25
times. This is cell suspension dilution "D". A 1.0 mL volume of dilution "D" is
10"5 mL of the original undiluted cell suspension.
E) Use a sterile pipette to transfer 11.0 mL of cell suspension dilution "D" to 99 mL
of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25
times. This is cell suspension dilution "E". A 1.0 mL volume of dilution "E" is
10"6 mL of the original undiluted cell suspension.
F) Prepare BHIA (Section 7.6). Ensure that agar surface is dry. Note: To ensure
that the agar surface is dry prior to use, plates should be made several days in
advance and stored inverted at room temperature or dried using a laminar-flow
hood.
G) Each of the following should be conducted in triplicate, resulting in the
evaluation of nine spread plates:
• Pipet 0.1 mL of dilution "C" onto surface of BHIA plate [10"5 mL (0.00001)
of the original cell suspension].
• Pipet 0.1 mL of dilution "D" onto surface of BHIA plate [10"6 mL (0.000001)
of the original cell suspension].
• Pipet 0.1 mL of dilution "E" onto surface of BHIA plate [ 10"7 mL
(0.0000001) of the original cell suspension].
H) For each spread plate, use a sterile bent glass rod or spreader to distribute
inoculum over surface of medium by rotating the dish by hand or on a rotating
turntable.
I) Allow inoculum to absorb into the medium completely.
J) Invert plates and incubate at 35°C ± 0.5°C for 24 ± 4 hours.
K) Count and record number of colonies per plate. Refer to the equation and Table
3 below for calculation of undiluted cell suspension concentration.
CFUn
ue
Vi + V2 + ... + Vn
Where:
diiuted = E. faecalis CFU/mL in undiluted cell suspension
CPU = number of colony forming units from BHIA plates yielding
counts within the ideal range of 30 to 300 CPU per plate
V = volume of undiluted sample in each BHIA plate yielding
counts within the ideal range of 30 to 300 CPU per plate
n = number of plates with counts within the ideal range
16
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Method 1611
Table 3. Example Calculations of E. faecalis Undiluted Cell Suspension Concentration
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from
BHIA plates
10"5mL plates
275, 250, 301
TNTCb, TNTC,
TNTC
10"6mL plates
30, 10, 5
TNTC, 299, TNTC
10"7 mL plates
0, 0, 0
12, 109, 32
E. faecalis CPU / mL in undiluted
suspension3
(275+250+30) /(10-5+10-5+10-6) =
5557 (2.1 x10'S) = 26,428,571 =
2.6x107CFU/mL
(299+1 09+32) /(10'6+10-7+10-7) =
440 / (1 .2 x 1 0'6) =366,666,667 =
3.7x108CFU/mL
Cell concentration is calculated using all plates yielding counts within the ideal range of 30 to 300 CPU per plate
3 Too numerous to count
11.1.8 Divide remainder of undiluted cell suspension (approximately 5 mL) into 6 x 0.5 mL
aliquots for DNA standard preparations and 100 - 200 x 0.01 mL (10 (iL) aliquots for
calibrator samples and freeze at -20°C. Note: Cell suspension should be stirred while
aliquoting. It is also recommended that separate micropipettor tips be used for each 10 (iL
aliquot transfer and that the volumes in each tip be checked visually for consistency.
11.2 Preparation of E. faecalis genomic DNA standards
11.2.1 Remove two 0.5 mL undiluted E. faecalis cell suspensions (Section 11.1.8) from freezer
and thaw completely.
11.2.2 Transfer cell suspensions to extraction tubes with glass beads.
11.2.3 Tightly close the tubes, making sure that the O-rings are seated properly.
11.2.4 Place the tubes in bead beater and shake for 60 seconds at the maximum rate (5000 rpm).
11.2.5 Remove the tubes from the mini bead beater and centrifuge at 12,000 x g for one minute
to pellet the glass beads and debris.
11.2.6 Using a 200 uL micropipettor, transfer 350 uL of the supernatants to sterile 1.7 mL
microcentrifuge tubes. Recover supernatants without disrupting the glass beads at the
tube bottom.
11.2.7 Centrifuge crude supernatants at 12,000 x g for 5 minutes and transfer 300 uL of clarified
supernatant to clean, labeled 1.7 mL low retention microcentrifuge tubes, taking care not
to disturb the pellet.
11.2.8 Add 1 uL of 5 ug/uL RNase A solution to each clarified supernatant, mix by vortexing
and incubate at 37°C for 1 hour.
Note: Steps 11.2.9 - 11.2.15 may be substituted with an optional method if a DNA
purification procedure is chosen other than the DNA-EZ purification kit. In such a
case, manufacturer instructions should be followed rather than these steps. Continue
onward from step 11.2.16.
11.2.9 Add 0.6 mL of binding buffer solution from a DNA-EZ purification kit to each of the
RNase A-treated extracts and mix by vortexing. Note: In general, a minimum
concentration of 5 x 1Q8 cells is required for this step.
17
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Method 1611
11.2.10 If using the DNA-EZ purification kit, perform the following steps. Insert one
DNAsure™ column from the DNA-EZ purification kit into a collection tube (provided
with kit) for each of the two extracts. Transfer the extract and binding buffer mixtures
from Section 11.2.9 to a DNAsure™ column and collection tube assembly and
centrifuge for 1 minute at 12,000 x g.
11.2.11 Transfer each of the DNAsure™ columns to new collection tubes. Discard previous
collection tubes and collected liquid.
11.2.12 Add 500 uL EZ-Wash Buffer from the DNA-EZ purification kit to each of the
DNAsure™ columns and centrifuge at 12,000 x g for 1 minute. Discard the liquid in
the collection tube.
11.2.13 Repeat step 11.2.12.
11.2.14 Transfer each of the DNAsure™ columns to a clean, labeled 1.7 mL low retention
microcentrifuge tube and add 50 uL of DNA elution buffer to each column.
Centrifuge for 30 seconds at 12,000 x g. Repeat this procedure again to obtain a total
DNA eluate volume of-100 uL from each column.
11.2.15 Pool the two eluates to make a total volume of approximately 200 uL.
11.2.16 Transfer the entire purified DNA eluate volume from each column to a clean and
sterile microcuvette for UV spectrophotometer and read absorbance at 260 and 280
nM. (Note: The cuvette should be blanked with DNA elution buffer before reading
sample). If necessary, the sample may be diluted with elution buffer to reach the
minimum volume that can be accurately read by the spectrophotometer (see
manufacturer's recommendation), however, this may reduce the DNA concentration to
a level that cannot be accurately read by the spectrophotometer. If available, readings
can be taken of 2 uL aliquots of the sample with aNanoDrop™ Spectrophotometer.
11.2.17 Sample is acceptable as a standard if ratio of OD26o/OD28o readings is > to 1.75.
11.2.18 Record UV spectrophotometer results.
11.2.19 Calculate total DNA concentration in sample by formula:
OD26o reading x 50 ng/uL DNA/OD260
11.2.20 Transfer sample back to labeled 1.7 mL non-retentive microcentrifuge tube and store
at -20°C.
11.2.21 Using total DNA concentration (Section 11.2.19), calculate E. faecalis PCR target
sequence concentration (TSC) in purified extract (Section 9.10).
Example.
OD260 = 0.7271 Total DNA concentration = OD260 Reading x 50 ng/(iL
0.7271 x 50 = 36.36 ng/^L = 36.36 x 106 fg/jiL = 3.636 x 107 fg/jiL
3.636 x 107 fg/uL 4 IsrRNA gene copies . „ . ,„? • , T
,,,,, &^ x 6 v = 4.04 x 10'gene copies/uL
3.6fg/genome genome
11.2.22 Record UV spectrophotometer results and all calculations.
18
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Method 1611
11.2.23 In appropriately labeled 1.7 mL low retention microcentrifuge tubes, perform serial
dilutions using AE buffer to prepare DNA standards of 4 x 103 TSC/5 uL, 4 x 102
TSC/5 uL, 100 TSC/5 uL and 40 TSC/5 uL (see below).
Note: Perform initial dilution of purified and quantified DNA as required to achieve a
concentration of 2 x 108 TSC/5 uL or 2 x 107 TSC/5 uL (as appropriate) prior to
initiating serial dilutions.
11.2.23.1 Perform 10-fold serial dilutions of the 2 x 108 or 107 TSC/5 uL stock
(10 uL : 90 uL) with AE buffer to achieve 2 x io7 TSC/5 uL; 2 x 106
TSC/5 uL; and 2 x IO5 TSC/5 uL
11.2.23.2 Perform 5-fold dilution of 2 x IO5 TSC /5 uL stock (100 uL : 400 uL)
with AE buffer to achieve 4 x IO4 TSC/5 uL stock
11.2.23.3 Perform successive 10-fold serial dilutions of the 4 x IO4 TSC/5 uL stock
(100 uL : 900 uL) to achieve 4 x io3 TSC/5 uL; 4 x IO2 TSC/5 uL stocks
11.2.23.4 Perform a 4-fold dilution of 4 x 102TSC/5 uL stock (100 uL : 300 uL) to
achieve 100 TSC/5 uL stock
11.2.23.5 Perform a final 2.5-fold dilution of the 100 TSC/5 uL stock (100 uL :
150 uL) to achieve 40 TSC/5 uL stock
Note: It is recommended that DNA standards be stored and manipulated
in a different room than that used for routine sample processing in order
to minimize the potential for laboratory contamination.
11.2.24 Store DNA standards in refrigerator at 4°C. For long term storage, freeze aliquots at -
20°C or -80°C
11.2.25 Generate DNA standard curves using the 40, 100, 400, and 4000 TSC/5 uL dilutions (3
replicates each) according to Section 11.7 (ABI) or 11.8 (Cepheid), as appropriate.
11.2.26 qPCR analyses of these diluted standards using the Enterococcus primer and probe
assay should initially be performed at least six separate times in triplicate. From that
point on, it is recommended that additional standard curves be generated from triplicate
analyses of these same diluted standard samples with each new lot of TaqMan® master
mix reagents or primers and probes to demonstrate comparable performance by these
new reagents.
11.3 Extraction ofE.faecalis calibrator samples
11.3.1 A minimum of three calibrator extracts should be prepared during each week of
analysis.
Note: To prevent contamination of water sample filtrates and filter blanks, this
procedure should be performed at a different time, and, if possible, in a different work
station than the procedures in Sections 11.1 and 11.2 above and Section 11.5 below.
11.3.2 Remove one tube containing a 10 uL aliquot of E. faecalis undiluted stock cell
suspension (Section 11.1.8) from the freezer and allow to thaw completely on ice.
Note: If using BioBalls for calibrators add a single BioBall®to each of 3, 1.0 mL sterile
PBS blanks, filter (Section 11.4), and extract according to Section 11.5.
11.3.3 While cell stock is thawing, using sterile (or flame-sterilized) forceps, place one
polycarbonate filter (Section 6.14) in an extraction tube with glass beads. Prepare one
filter for each sample to be extracted in this manner.
19
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Method 1611
11.3.4 Dispense 590 \\L of Salmon DNA/extraction buffer (Section 7.12) into three
extraction tubes with glass beads and filters. Prepare one tube for each of the three
calibrator samples to be extracted in this manner. Label tubes appropriately.
11.3.5 When E. faecalis suspension has thawed, transfer 990 uL AE buffer (Section 7.11) to
the 10 uL E. faecalis stock cell tube and mix thoroughly by vortexing. Pulse
microcentrifuge tube briefly (1 - 2 sec.) to coalesce droplets in tube.
11.3.6 Immediately after vortexing the E. faecalis suspension, spot 10 uL onto the
polycarbonate filter in the extraction tube from step 11.3.4.
Note: For the blank extraction tube, spot 10 uL AE buffer onto the polycarbonate filter
11.3.7 Tightly close the tube, making sure that the O-ring is seated properly and label tubes
appropriately.
11.3.8 Repeat Sections 11.3.6 and 11.3.7 for the other two filters to prepare three calibrator
samples with E. faecalis.
11.3.9 Place the tubes in the mini bead beater and shake for 60 seconds at the maximum rate
(5000 rpm).
11.3.10 Remove the tubes from the mini bead beater and centrifuge at 12,000 x g for one
minute to pellet the glass beads and debris.
11.3.11 Using a 200 uL micropipettor, transfer the crude supernatant to the corresponding
labeled sterile 1.7 mL microcentrifuge tube. Transfer 400 uL of supernatant without
disrupting the debris pellet or glass beads at the tube bottom. Note: The filter will
normally remain intact during the bead beating and centrifugation process. Generally,
400 uL of supernatant can be easily collected. Collect an absolute minimum of 100
uL of supernatant. Record volume of supernatant collected.
11.3.12 Centrifuge at 12,000 x g for 5 minutes and transfer clarified supernatant to a clean,
labeled 1.7 mL tube, taking care not to disturb the pellet. Note: Cell pellet may not be
visible in calibrator samples.
11.3.13 Label the tubes as undiluted or IX E. faecalis calibrator extracts. Label additional 1.7
mL tubes for 5- and 25-fold dilutions. In appropriately labeled tubes, using a
micropipettor, add a 50 uL aliquot of each IX E. faecalis calibrator extracts and dilute
each with 200 uL AE buffer (Section 7.11) to make 5-fold dilutions. In appropriately
labeled tubes using a micropipettor, add a 50 uL aliquot of each 5-fold dilution and
dilute each with 200 uL AE buffer to make 25-fold dilutions. Store all diluted and
undiluted extracts in refrigerator.
11.3.14 If the extracts are not analyzed immediately, refrigerate.
11.4 Water sample filtration
Note: It is required that minimally one water sample filtration blank (method blank) be analyzed
for every qPCR run of water samples (Section 9.8)
11.4.1 Place a polycarbonate filter (Section 6.14) on the filter base (shiny side "up" facing
you), and attach the funnel to the base so that the membrane filter is now held between
the funnel and the base.
11.4.2 Shake the sample bottle vigorously 25 times to distribute the bacteria uniformly, and
measure 100 mL of sample into the funnel.
11.4.3 Filter 100 mL of water sample. After filtering the sample, rinse the sides of the funnel
with 20 - 30 mL of sterile PBS (Section 7.4) and continue filtration until all liquid has
20
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Method 1611
been pulled through the filter. Turn off the vacuum and remove the funnel from the
filter base.
11.4.4 Label an extraction tube containing glass beads (Section 6.4) to identify water sample.
Leaving the filter on the filtration unit base, fold filter into a cylinder with the sample
side facing inward, being careful to handle the filter only on the edges, where the filter
has not been exposed to the sample. Insert the rolled filter into the labeled extraction
tube with glass beads. Prepare one filter for each sample filtered in this manner.
11.4.5 Cap the extraction tube. Refrigerate.
11.5 DNA extraction of water sample filter extracts and method blanks
11.5.1 Using a 1000 uL micropipettor, dispense 590 uL of the Salmon DNA/extraction buffer
(Section 7.12) into each labeled extraction tube with glass beads containing water
sample or method blank filters from Section 11.3.4. Extract the method blank last.
11.5.2 Tightly close the tubes, making sure that the O-ring is seated properly.
11.5.3 Place the tubes in the mini bead beater and shake for 60 seconds at the maximum rate
(5000 rpm).
11.5.4 Remove the tubes from the mini bead beater and centrifuge at 12,000 x g for 1 minute
to pellet the glass beads and debris. Note: To help prevent contamination, a new pair
of gloves may be donned for this step, and steps 11.5.6and 11.5.7.
11.5.5 Using the 200 uL micropipettor, transfer 400 uL of the supernatant to a corresponding
labeled sterile 1.7 mL microcentrifuge tube, taking care not to aspirate glass beads or
sample debris (pellet). Note: The filter will normally remain intact during the bead
beating and centrifugation process. Generally, 400 uL of supernatant can easily be
collected. Collect an absolute minimum of 100 uL of supernatant. Recover the
method blank supernatant last.
11.5.6 Centrifuge crude supernatant from Section 11.5.5 for 5 minutes at 12,000 x g.
Transfer 350 uL of the clarified supernatant to another 1.7 mL tube, taking care not to
disturb the pellet. Note: Pellet may not be visible in water samples. Recover the
method blank supernatant last.
11.5.7 Label the tubes from Section 11.5.6 as undiluted or IX water sample extracts with
sample identification. These are the water sample filter extracts. Also label tubes for
method blanks. Label additional 1.7 mL tubes for 5-fold dilutions. In appropriately
labeled tubes, using a micropipettor, add a 50 uL aliquot of each IX water sample
extract and dilute each with 200 uL AE buffer (Section 7.11) to make 5-fold dilutions.
Dilute the method blank supernatant last.
11.5.8 Optional: For samples where the concentration of target DNA is anticipated to be
high, an additional 25-fold dilution can be done In appropriately labeled tubes using a
micropipettor, add a 50 uL aliquot of each 5-fold dilution and dilute each with 200 uL
of AE buffer to make 25-fold dilutions. Dilute the method blank supernatant last.
11.5.9 Store all diluted and undiluted extracts in refrigerator.
11.5.10 Note: Use of 5-fold diluted samples for analysis is currently recommended if only one
dilution can be analyzed. Analyses of undiluted water sample extracts have been
observed to cause a significantly higher incidence of PCR inhibition while analyses of
25-fold dilutions may unnecessarily sacrifice sensitivity.
11.5.11 Refrigerate the extracts if they are not analyzed immediately. Water sample filter
extracts should be analyzed the same day that they are prepared.
21
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Method 1611
11.6 Preparation of qPCR assay mix
11.6.1 To minimize environmental DNA contamination, routinely treat all work surfaces with a
10% bleach solution, allowing the bleach to contact the work surface for a minimum of
15 minutes prior to rinsing with sterile water. If available, turn on UV light for 15
minutes. After decontamination, discard gloves and replace with a new pair.
11.6.2 Remove primers and probe stocks from the freezer and verify that they have been diluted
to solutions of 500 uM primer and 100 uM probe.
11.6.3 Prepare working stocks ofEnterococcus, and Salmon DNA (Sketa 22) primer/probe
mixes by adding 10 uL of each Enterococcus or Salmon DNA (Sketa 22) primer stock
and 4 uL of respective probe stock to 676 uL of PCR-grade water, and vortex. Pulse
centrifuge to create a pellet. Use a micropipettor with aerosol barrier tips for all liquid
transfers. Transfer aliquots of working stocks for single day use to separate tubes and
store at 4°C.
11.6.4 Using a micropipettor, prepare assay mix of the Enterococcus and Salmon DNA (Sketa
22) reactions in separate, sterile, labeled 1.7 mL microcentrifuge tubes as described in
Table 4.
Table 4. PCR Assay Mix Composition
Reagent
Sterile H2O
BSA
TaqMan® master mix
Primer/probe working stock
solution
Volume/Sample (multiply by # samples to be analyzed
per day)
1.5|JL
2.5 pi
12.5 pi
3.5 pi
Note: This will give a final concentration of 1 uM of each primer and 80 nM of probe in the
reactions. Prepare sufficient quantity of assay mix for the number of samples to be analyzed per
day including calibrators and negative controls plus at least two extra samples. Prepare assay
mixes each day before handling DNA samples.
11.6.5 Vortex the assay mix working stocks; then pulse microcentrifuge to coalesce droplets.
Return the primer/probe working stocks and other reagents to the refrigerator.
11.7 ABI StepOnePlus™. ABI 7900. and ABI 7500 (all non-Fast mode) qPCR assay preparation
(Reference 17.1)
Transfer 20 uL of either the master mix containing Enterococcus primers and probe or the master
mix containing the Salmon DNA primers and probe to the wells of a 96 well PCR reaction tray
equal to number of samples to be analyzed including calibrator and negative control samples.
(Note: The same tip can be used for pipetting multiple aliquots of the same assay mix as long as
no contact is made with anything else).
Example: For the analysis of 18 recreational water samples, 47 wells will require the addition of
assay mix with Enterococcus primers and probe as follows: 18 samples, two replicates each (36),
3 method blanks, one replicate each (3), 3 calibrators, 2 replicates each (6) and 1 no template
control (NTC), 2 replicates (2) = 47 wells. In addition, 47 wells will require the addition of assay
mix with Salmon DNA assay primers and probe as follows: 18 samples, two replicates each (36),
22
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Method 1611
3 method blanks, one replicate each (3), 3 calibrators, 2 replicates each (6) and 1 NTC, 2
replicates (2) = 47 wells. Thus a total of 94 wells are used for 18 recreational water samples.
11.7.1 Transfer 20 uL of either the master mix containing Enterococcus primers and probe or
the master mix containing the Salmon DNA primers and probe to wells of a 96 well
PCR reaction tray equal to number of samples to be analyzed including calibrator and
negative control samples. Pipet into the center of the wells, taking care to not touch
the well walls with the pipet tip. (Note: The same tip can be used for pipetting
multiple aliquots of the same assay mix as long as it doesn't make contact with
anything else).
11.7.2 When all wells are loaded transfer to the PCR preparation station used for handling
DNA samples (Section 6.1). Note: All aliquoting of assay mixes to reaction trays
must be performed each day before handling of DNA samples.
11.7.3 DNA standards - Transfer 5 uL each concentration of the undiluted DNA extracts of
(Section 11.2.24) to separate wells of the PCR reaction tray containing Enterococcus
assay mix. Note: Record positions of each concentration. See Section 11.2.26
regarding the frequency of DNA standard analyses.
11.7.4 Water samples - Transfer 5 uL each of the diluted (or undiluted) DNA extracts of
method blanks and water samples (Section 11.5.7), and corresponding dilutions of
calibrator samples (Section 11.3.13), to separate wells of the PCR reaction tray
containing Enterococcus assay mix. Note: Record positions of each sample.
11.7.5 Transfer 5 uL each of the diluted (or undiluted) DNA extracts of method blanks and
water samples (Section 11.5.7), and corresponding diluted calibrator samples (Section
11.3.13) to separate wells of the PCR reaction tray containing Salmon DNA assay
mix. Record positions of each sample.
11.7.6 Transfer 5 uL aliquots of AE buffer to wells of PCR reaction tray containing
Enterococcus master mix that are designated as NTCs. Record positions of these
samples.
11.7.7 Tightly cap wells of PCR reaction tray containing samples or cover tray and seal
tightly with optical adhesive. Note: 96 well plates should be freshly prepared just prior
to analyses. Do not prepare and refrigerate multiple plates prior to analyses, as signal
degradation may occur.
11.7.8 Run reactions in ABI StepOnePlus™, ABI 7900, or ABI 7500 (all in non-Fast mode)
Real-Time PCR Systems. For platform-specific operation see Appendix A. Note: The
ABI 7500 Fast may be used but it should be run in the 9600 emulation mode.
11.8 Smart Cycler® qPCR assay preparation
11.8.1 Label 25 uL Smart Cycler® tubes with sample identifiers and assay mix type (see
Section 11.8.8 for examples) or order tubes in rack by sample number and label rack
with assay mix type. It is recommended that the unloaded open Smart Cycler® tubes
be irradiated under UV light in a PCR cabinet for 15 minutes. Using a micropipettor,
add 20 uL of the Enterococcus assay mix (Section 11.6.4) to labeled tubes. Avoid
generating air bubbles, as they may interfere with subsequent movement of the liquid
into the lower reaction chamber. The same tip can be used for pipetting multiple
aliquots of the same assay mix as long as it doesn't make contact with anything else.
Repeat procedure for Salmon DNA (Sketa 22) assay mix.
11.8.2 Add 5 uL of AE buffer to NTC tubes and close tubes tightly.
23
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Method 1611
1 1 .8.3 Close the other PCR tubes loosely and transfer to refrigerator or directly to the PCR
preparation station used for handling DNA samples (Section 6.1). Note: All aliquoting
of assay mixes to reaction tubes must be performed each day before handling of DNA
samples.
1 1 .8.4 DNA standards - Transfer 5 uL of each concentration of the undiluted DNA extracts
(Section 1 1 .2.24), to tubes containing Enterococcus mix. Close each tube tightly after
adding sample. Label the tube tops as appropriate. See Section 1 1.2.26 regarding the
frequency of DNA standard analyses.
1 1 .8.5 Water samples - Transfer 5 uL each of the diluted (or undiluted) DNA extracts of
method blanks and water samples (Section 1 1.5.7), and then corresponding dilutions
of calibrator samples (Section 1 1.3.13) to tubes containing Enterococcus and Salmon
DNA (Sketa 22) mixes. Close each tube tightly after adding sample. Load the method
blank last. Label the tube tops as appropriate.
When all Smart Cycler® tubes have been loaded, place them in a Smart Cycler®
centrifuge, and spin for 2-4 seconds.
Inspect each tube to verify that the sample has properly filled the lower reaction
chamber. A small concave meniscus may be visible at the top of the lower chamber,
but no air bubbles should be present. (If the lower chamber has not been properly
filled, carefully open and reclose the tube, and re-centrifuge). Transfer the tubes to the
thermocycler.
11 .8.8 For platform-specific operation see Appendix B.
1 1 .8.9 Suggested sample analysis sequence for Smart Cycler®
Example: For analyses on a single 16 position Smart Cycler®, calibrator samples and
water samples will need to be analyzed in separate runs and a maximum of 6 water
samples (or 2 replicates of 3 samples) can be analyzed per run, as described in Tables
5 and 6, below.
Table 5. Calibrator PCR Run - 14 Samples
1 1 .8.6
1 1 .8.7
Sample Description1
3 Calibrators * 2 replicates (1-, 5-, or 25-fold dilution)
3 Calibrators * 2 replicates (1-, 5-, or 25-fold dilution)
No template controls (reagent blanks)
Quantity
6
6
2
PCR Assay Master Mix
Enterococcus
Salmon DNA
Enterococcus
Diluted equivalently to the water samples
Table 6. Water Sample PCR Run -14 Samples
Sample Description1
Water samples (1-, 5-, or 25-fold dilution)
Method blank (1-, 5-, or 25-fold dilution)
Water samples (1-, 5-, or 25-fold dilution)
Method blank (1- 5-, or 25-fold dilution)
Quantity
6
1
6
1
PCR Assay Master Mix
Enterococcus
Enterococcus
Salmon DNA
Salmon DNA
Use of 5-fold diluted samples for analysis is currently recommended if only one dilution can be analyzed.
Analyses of undiluted water sample extracts have been observed to cause a significantly higher incidence of
PCR inhibition while 25-fold dilutions analyses may unnecessarily sacrifice sensitivity.
24
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Method 1611
12.0 Data Analysis and Calculations
12.1 Overview: This section describes a method for determining the ratio of the target sequence
quantities recovered from a test (water filtrate) sample compared to those recovered from
identically extracted calibrator samples using an arithmetic formula, referred to as the AACT
method. The AACT relative quantitation method also normalizes these ratios for differences in
total DNA recovery from the test and calibrator samples using qPCR analysis CT values for a
reference sequence provided by the SPC DNA. These ratios are converted to absolute
measurements of total target sequence quantities recovered from the test and calibrator sample
extracts and these ratios are converted to CCEs recovered from the test samples. The complete
procedure for determining target sequence quantities and CCEs in water samples is detailed
below.
12.2 Generation of CT value vs. target sequence number standard curve: Three replicate serial
dilutions of a DNA standard, prepared as described in Section 11.2 should be prepared to give
concentrations of 4 x 103, 4 x 102, lx 102, and 4 x 101 IsrRNA gene sequences per 5 uL (the
standard sample volume added to the PCR reactions) and the replicates of each dilution pooled.
Note: A procedure for the determinations of target sequence concentrations in the DNA standard
is also provided in Section 9.10.
Aliquots of each of these dilutions should be frozen in low retention microcentrifuge tubes.
Thawed aliquots should be stored at 4° C and can be reused for repeated qPCR analyses. qPCR
analyses of these diluted standards using the Enterococcus primer and probe assay should initially
be performed at least six separate times in triplicate. CT values from these composite analyses
should be subjected to regression analysis against the log 10-transformed target sequence numbers
per reaction as described in Section 9.10 with example results illustrated in Figure 1.
35
30
25
20
15
JQ y = -3.41673x +36.71941
R2 =0.998
5
0 - • ,
01234
Log Sequences
Figure 1: Example plot and regression analysis of qPCR analysis CT values vs. log target
sequences per reaction.
AF used for subsequent comparative CT calculations (Section 12.4) can be calculated from the
slope value of this curve by the formula AF = 10A(1 / (-)slope value). An example calculation
using the slope value from the example regression is shown below:
AF= 10A(1 73.41673)= 1.96
12.3 Calculation of average target sequence recovery in calibrator sample extracts: A minimum
of nine calibrator sample extracts should initially be prepared from at least three different freezer-
25
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Method 1611
stored aliquots of each cultured E.faecalis stock suspension that is prepared as described in
Section 11.1. Dilutions of each of these calibrator sample extracts equivalent to the anticipated
dilutions of the test samples used for analysis (e.g., 1-, 5- and/or 25-fold) should be analyzed with
the Enterococcus primer and probe assay. The average CT value from these analyses should be
interpolated on the standard curve generated from the DNA standard (Section 12.2) to determine
the average number of target sequences per 5 uL of extract used in the reactions. An example
calculation using an average calibrator extract CT value of 24.31 and standard curve from Figure
1 is shown below:
Average calibrator target sequences/5 uL extract = 10A[(24.31-36.7194) / -3.4167] = 4285.0744
Note: Four places should be kept from this calculation for the following calculation (i.e.,
4285.0744). Dividing this value by 5 gives the average calibrator target sequences uL extract
which can be multiplied by the total volume of the extract at the applicable dilution level (e.g.,
600 uL of original extract volume x 5 = 3000 uL for a 5-fold diluted sample) to determine the
average total quantity of target sequences recovered in the calibrator sample extracts. An example
of this calculation using the average calibrator target sequences/reaction value determined
immediately above is shown below:
(4285.0744/5) x 3000 = 2571045 target sequences / total extract
12.3.1 Calculation of average target sequence recovery per Enterococcus cell in calibrator
sample extracts (optional): In previous studies, measurements of recreational water
quality by the qPCR method have been reported as Enterococcus calibrator cell
equivalents (Reference 17.4).
12.3.2 A prerequisite for making such comparisons is to determine that the ratio of the
numbers of target sequences recovered in calibrator sample extracts to the numbers of
Enterococcus cells added to these samples is consistent in different studies. Example:
The quantity of lab prepared cells added to each calibrator sample is -100,000.
Dividing the average target sequences recovered per calibrator extract (determined as
described in Section 12.3) by this number provides the ratio of target sequence numbers
to cell numbers. An example of this calculation using the average target sequences/
calibrator extract value determined in Section 12.3 is shown below:
Ratio of target sequence numbers to cell numbers = 2571045/100,000 = 25.71
12.4 Calculation of CCEs in test samples: A minimum of three fresh calibrator samples should be
extracted and analyzed at least on a weekly basis and preferably on a daily basis in association
with each batch of water sample filtrates. QC analysis of the results from these calibrator extracts
should be performed as described in Section 9.11. CT values from the Enterococcus and salmon
DNA SPC qPCR assays for both the calibrator and test samples are used in the AACT method to
determine the ratios of target sequences in the test and calibrator sample extracts and these ratios
are converted to CCEs recovered from the test samples as specified below and illustrated in
Table 5.
12.4.1 Subtract the SPC assay CT value (CT, SPC) from the target assay CT value (CT, target) for
each calibrator sample extract to obtain ACT value and calculate the average ACT value
for these calibrator samples.
12.4.2 Subtract the SPC assay CT value (CT, SPC) from the target assay CT value (CT, target) for
each water sample filtrate extract to obtain ACT values for each of these test samples.
Note: If multiple analyses are performed on these samples, calculate the average ACT
value.
26
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Method 1611
12.4.3 Subtract the average ACT value for the calibrator samples from the ACT value (or average
ACT value) for each of the test samples to obtain AACT values.
12.4.4 Calculate the ratio of the target sequences in the test and calibrator samples using the
formula: AFA("AACT), where AF = amplification factor of the target organism qPCR assay,
determined as described in Section 12.2.
12.4.5 Multiply the ratio of the target sequences in the test and calibrator samples by the
estimated count (e.g., CPU) determined in Section 11.1.7 to determine the CCEs for each
of the test samples. Note: This calculation can be applied without modification to the
analyses of diluted extracts if both the test sample and calibrator extracts are equally
diluted and equal volumes of diluted extracts are analyzed.
Table 5. Example Calculations (AF = 1.96)
Estimated
Cell Count 1
1200000
Sample
Type
Calibrator
Test
Cl.target
25.71
33.74
CT.SPC
34.13
25.94
ACT
-8.42
7.8
MCr
—
7.59
Ratio calib/test
1 96-MCT
0.006
CCEs in Test Sample Extract
(1. 96 -MCTxEst cell count)
—
0.006x1200000 = 7264.74
12.5
1 Estimated cell count is based on plating results taking into account the dilution factor (100) and the volume
of the dilution (0.1 ml_) added to the calibrator sample
12.4.6 The geometric mean (optional) of the CCEs and associated coefficients of variation in
multiple water samples can be determined from individual sample CT values using the
following procedure:
12.4.6.1 Use ACT value for each individual water sample extract and the mean
calibrator ACT value to calculate the CCEs in each water sample extract, as
described above.
12.4.6.2 Calculate the loglO of the CCEs in each water sample (log N)
12.4.6.3 Calculate the mean (M) and standard deviation (S) from the values of log
N obtained in the previous step for all of the water sample extracts.
12.4.6.4 Calculate the geometric mean as 10M.
12.4.6.5 The implied coefficient of variation (CV) is calculated, based on the log
normal distribution, as the square root of iov/0434 - 1, where V = S2.
Reporting Results: Where possible, duplicate analyses should be performed on each sample.
Report the results as Enterococcus CCEs per volume of water sample filtered.
13.0 Sample Spiking Procedure
13.1 Method 1611 QC requirements (Section 9) include the preparation and analysis of spiked
reference (PBS) and matrix samples in order to monitor initial and ongoing method performance.
For the IPR (Section 9.3), OPR (Section 9.4), and MS (Section 9.5) analyses it is necessary to
spike samples with either laboratory-prepared spiking suspensions (Section 13.3) or BioBalls
(Section 13.7).
13.2 Preparation of Laboratory-Prepared Undiluted Spiking Suspension. Note: when using laboratory-
prepared cultures, it is recommended to prepare calibrator samples and spike suspensions from
the same culture using the same cell enumeration results (see section 11.1).
27
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Method 1611
13.2.1 Stock Culture (E. faecalis). Prepare a stock culture by inoculating a BHIA slant with E.
faecalis ATCC® #29212™ (e.g., KWIK-STIK™) and incubate at 35°C ± 0.5°C for 21 ±
3 hours. This stock culture may be stored in the dark at room temperature for up to 30
days.
13.2.2 E. faecalis Spiking Suspension (Undiluted). From the stock culture of E. faecalis
ATCC® #29212™, transfer a small loopful of growth to the BHIB and vigorously shake a
minimum of 25 times. Incubate at 35°C ± 0.5°C for 21 ± 3 hours. The resulting spiking
suspension contains approximately 1.0 x 109 enterococci CPU per mL. This is referred to
as the "undiluted spiking suspension."
13.2.3 Proceed to Section 13.3 for sample spiking and enumeration of spiking suspension
(Section 13.2.2).
13.3 Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration
Since the objective of spiking the sample is to establish percent recovery, it is necessary to
determine the concentration of E. faecalis in the laboratory-prepared undiluted spiking suspension
(Section 13.2.2). This section provides instructions for sample spiking (Section 13.4) and spiking
suspension enumeration (Section 13.5). If you are using BioBalls instead of a laboratory
preparation, go to Section 13.7.
13.4 Sample Spiking
Please be sure to vigorously shake the spiking suspensions in the steps below to ensure accurate
sample spiking and spiking suspension enumeration.
13.4.1 Dilute spiking suspension
Perform steps 13.4.1.1 through 13.4.1.5 using the laboratory-prepared undiluted spiking
suspension (Section 13.2.2).
13.4.1.1 Mix the undiluted spiking suspension by vigorously shaking the bottle a
minimum of 25 times. Use a sterile pipette to transfer 1.0 mL of the undiluted
spiking suspension to 99 mL of sterile PBS; cap and mix by vigorously
shaking the bottle a minimum of 25 times. This is spiking suspension dilution
"A." A 1.0 mL volume of dilution "A" is equivalent to a concentration of 1 x
107 CPU per mL (10~2 mL of the original undiluted spiking suspension).
13.4.1.2 Mix spiking suspension "A" (Section 13.4.1.1) by vigorously shaking the
bottle a minimum of 25 times. Use a sterile pipette to transfer 1.0 mL to 99
mL of sterile PBS; cap and mix by vigorously shaking the bottle a minimum
of 25 times. This is spiking suspension dilution "B." A 1.0 mL volume of
dilution "B" is equivalent to a concentration of 1 x 105 CPU per mL (10~4 mL
of the original undiluted spiking suspension).
13.4.1.3 Mix spiking suspension "B" (Section 13.4.1.2) by vigorously shaking the
bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99
mL of sterile PBS; cap and mix by vigorously shaking the bottle a minimum
of 25 times. This is spiking suspension dilution "C." A 1.0 mL volume of
dilution "C" is equivalent to a concentration of 1 x 104 CPU per mL (10~5 mL
of the original undiluted spiking suspension).
13.4.1.4 Mix spiking suspension "C" (Section 13.4.1.3) by vigorously shaking the
bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99
mL of sterile PBS; cap and mix by vigorously shaking the bottle a minimum
of 25 times. This is spiking suspension dilution "D." A 1.0 mL volume of
28
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Method 1611
dilution "D" is equivalent to 1 x 103 CPU per mL (10"6 mL of the original
undiluted spiking suspension).
13.4.1.5 Mix spiking suspension "D" (Section 13.4.1.4) by vigorously shaking the
bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99
mL of sterile PBS; cap and mix by vigorously shaking the bottle a minimum
of 25 times. This is spiking suspension dilution "E." A 1.0 mL volume of
dilution "E" is equivalent to 1 * 102 CPU per mL (10~7 mL of the original
undiluted spiking suspension).
13.4.2 Spike samples
13.4.2.1 550 CPU Spike
To spike the sample, add 5.5 mL of spiking suspension dilution "E" (Section
13.4.1.5) to 100 mL of unspiked sample and mix by vigorously shaking the
bottle a minimum of 25 times. This is the "spiked" sample with a
concentration of 550 CPU per 100 mL. The volume (mL) of undiluted
spiking suspension added is 5.5 x 10"7 mL/100 mL. This is referred to as
Vsplked per 100 mL sample in Section 13.6.2.
13.5 Enumeration of Spiking Suspensions (Prepared in Section 13.3.1)
13.5.1 Prepare BHIA according to manufacturer's instructions; add 12 - 15 mL of BHIA per
100 x 15 mm petri dish and solidify. Ensure the agar surfaces are dry. Note: Agar plates
must be dry and free from condensation prior to use. To ensure the agar surfaces are dry,
plates should be made several days in advance and stored inverted at room temperature or
dried using a laminar-flow hood.
13.5.2 Each of the following should be conducted in triplicate, resulting in the evaluation of nine
spread plates:
• Pipet 0.1 mL of dilution "C" (Section 13.4.1.3) onto surface of pre-dried
BHIA plate (10~6 mL of the original spiking suspension).
• Pipet 0.1 mL of dilution "D" (Section 13.4.1.4) onto surface of pre-dried
BHIA plate (10~7 mL of the original spiking suspension).
• Pipet 0.1 mL of dilution "E" (Section 13.4.1.5) onto surface of pre-dried
BHIA plate (10~8 mL of the original spiking suspension).
13.5.3 After pipetting the 3 spread plates for each dilution, use a sterile bent glass rod or
spreader to distribute inoculum over the surface of the medium by rotating the dish by
hand or on a turntable. Repeat for next 2 dilutions. Note: Please ensure that inoculum is
evenly distributed over entire surface of the plate.
13.5.4 Allow inoculum to absorb into the medium completely.
13.5.5 Invert plates and incubate at 35°C ± 0.5°C for 21 ± 3 hours.
13.5.6 Count and record number of colonies per plate. Refer to Section 13.6 for calculation of
spiking suspension concentration.
13.6 Calculation of Spiked E. faecalis Percent Recovery (Laboratory-Prepared Undiluted
Spiking Suspension)
The spiked E. faecalis percent recovery will be calculated in three steps as indicated in Sections
13.6.1 through 13.6.3 below. Note: The calculated numbers in the table examples provided below
have been rounded at the end of each step. If your laboratory recalculates the examples using a
29
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Method 1611
spreadsheet and rounds only after the final calculation (Section 13.6.3), the percent recovery may
be slightly different.
13.6.1 Calculate Concentration of E. faecalis (CFU/mL) in Undiluted Spiking Suspensions
13.6.1.1 The number of E. faecalis (CFU/mL) in the undiluted spiking suspensions will
be calculated using all BHIA plates from Section 13.5.2 yielding counts
within the ideal range of 30 to 300 CPU per plate.
13.6.1.2 If the number of colonies exceeds the upper range (i.e., >300) or ifthe
colonies are not discrete, results should be recorded as "too numerous to
count" (TNTC).
13.6.1.3 Calculate the concentration of E. faecalis (CFU/mL) in the undiluted spiking
suspensions according to the following equation. An example calculation is
provided in Table 6.
E. faeCaliS undiluted spike
Where,
tL. JaeCallS undiluted spike
CPU
V
= (CFUj + CFU2
CFUn)
V2
-Vn)
= E. faecalis (CFU/mL) in undiluted spiking suspension
= Number of colony forming units from BHIA plates yielding
counts within the ideal range of 30 to 300 CPU per plate
= Volume of undiluted sample on each BHIA plate yielding
counts within the ideal range of 30 to 300 CPU per plate
= Number of plates with counts within the ideal range
Table 6. Example Calculation of E. faecalis Spiking Suspension Concentration
CPU / plate (triplicate analyses) from
BHIA plates
10-6 ml plates
TNTC, TNTC, TNTC
10'7 ml plates
94,106,89
10-8 ml plates
9,11,28
£ faecalis CPU / ml in undiluted
spiking suspension
(£. faecalis undiluted spike)*
(94+1 06+89) /(10-7+10-7+10-7) =
289 / (3.0x1 0-7) =
9.6 x 108 CFU/mL
*£. faeca//s undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CPU per plate
13.6.2 Calculate "True" Spiked E. faecalis (CFU/100 mL)
Calculate the true concentration of spiked E. faecalis (CFU/100 mL) according to the
following equation. Example calculation is provided in Table 7.
Tspiked K faecalis ~ (E. fc
Where,
A Spiked E. faecalis
fL. JaeCallS undiluted spike
» spiked per 100 mL sample
ndiluted spike) X (V splked per 1 00 mL sample)
Number of spiked E. faecalis (CFU/100 mL)
E. faecalis (CFU/mL) in undiluted spiking suspension
mL of undiluted spiking suspension per 100 mL sample
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Method 1611
Table 7. Example Calculation of Spiked E. faecalis
E. faecalis undiluted spike
9.6x108CFU/mL
V spiked per ml sample
5.5 x 1Q-7 mL/100 ml of sample
Tspiked £ faecalis
(9.6 x 108 CFU/mL) x (5.5 x KHmL/lOO ml) = 528 CPU/ 100 ml
13.6.3 Calculate Percent Recovery
13.6.3.1 Calculate percent recovery (R) using the following equation.
R
100
(NS-NU)
Where,
R
Ns
Nu
A Spiked R faecalis
= Percent recovery
= E. faecalis (CPU/100 mL) in the spiked sample
= E. faecalis (CPU/100 mL) in the unspiked sample
= True spiked E. faecalis (CPU/100 mL) in spiked sample
Example percent recovery calculation is provided in Table 8.
Table 8. Example Percent Recovery Calculation
Ns (CPU /100mL)
521
Nu (CPU /100mL)
3a
T Spiked E faecalis
(CPU /100ml)
528
Percent recovery (R)
100x(521-3)/528 = 98%
The result of 3 CPU is acceptable if the sample was a recreational water; for PBS Nu should be <1 CPU/100 mL.
13.7 BioBall® Sample Spiking and Enumeration
This section provides instructions for sample spiking (Section 13.7.1) and spiking suspension
enumeration (Section 13.7.2) using BioBalls instead of a laboratory preparation of' E. faecalis.
Upon receipt, BioBalls should be stored at -20°C. Preparation of BioBalls prior to spiking is not
necessary, as they can be spiked directly into the sample once the vial is opened.
13.7.1 Sample Spiking
13.7.1.1 Open BioBall® vial by removing the cap. Aseptically add one 550 CPU E.
faecalis BioBall® to 100 mL of sample and mix by vigorously shaking the
bottles a minimum of 25 times.
13.7.1.2 This is the "spiked" sample with a concentration of-550 CPU per 100 mL
Note: BTF provides mean and standard deviation of CPU concentrations in
their certificates of analysis for each lot of BioBalls. These values can be
accessed athttp://www.btfbio.com/cofa.php?nav=BioBall.
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Method 1611
13.7.2 Enumeration of BioBall®
13.7.2.1 Prepare BHIA according to manufacturer's instructions. Add 12 - 15 mL of
BHIA per 100 x 15 mm petri dish and let solidify. Ensure the agar surfaces
are dry. Note: Agar plates must be dry and free from condensation prior to
use. To ensure agar surfaces are dry, plates should be made several days in
advance and stored inverted at room temperature or dried using a laminar-
flow hood.
13.7.2.2 The following should be conducted in triplicate, resulting in the evaluation of
three spread plates.
• Open BioBall® vial by removing the cap and aseptically add one
BioBall® to 1.0 mL of sterile PBS. Allow BioBall® to dissolve
completely.
• Mix suspension to homogenize. Pipet 0.1 mL of the BioBall
suspension onto a 100 x 15 mm BHIA plate.
13.7.2.3 For each spread plate, use a sterile bent glass rod or spreader to distribute
BioBall® inoculum over the medium surface by rotating the dish by hand or
on a turntable. Note: Please ensure that inoculum is evenly distributed over
entire surface of plate.
13.7.2.4 Allow inoculum to absorb into medium completely.
13.7.2.5 Invert plates and incubate at 35°C ± 0.5°C for 21 ± 3 hours.
13.7.2.6 For each dilution, count and record the sum of the number of colonies (CPU)
on the triplicate plates.
13.8 Calculation of BioBall® Spike Percent Recovery
Spiked BioBall® percent recovery will be calculated using the lot mean value provided by
manufacturer according to Section 13.8.2. Recovery may also be calculated following the two
steps as indicated below in Sections 13.8.1 and 13.8.2. Note: The calculated numbers in the
example provided in the tables below have been rounded at the end of each step. If your
laboratory recalculates the examples using a spreadsheet and rounds only after the final
calculation (Step 2), the percent recovery may be slightly different.
13.8.1 Calculate Mean E. faecalis per BioBall® and "True" Spiked E. faecalis (CFU/100
mL)
The mean concentration of E. faecalis (CPU) in the BioBalls will be calculated using all
three BHIA plates (from Section 13.7.2.6). Since one BioBall® is spiked per 100 mL
sample, use the mean number of E. faecalis per BioBall® as the "true" spiked E. faecalis.
For example, if the triplicate results from Section 13.7.2.6. are 57, 52, and 49, then the
triplicate plate counts for the 550 CPU BioBall® concentration are as follows:
TSplked E. faecalis (CFU/100 mL) = [(54 + 52 + 49) x 10] / 3 = 517 per BioBall®
Where,
Tspiked K faecalis = True spiked E. faecalis (CFU/100 mL) in spiked sample
Thus, 517 is the "true" spiked E. faecalis (CFU/100 mL)
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Method 1611
13.8.2 Calculate Percent Recovery
Calculate percent recovery (R) using the following equation:
R = 100 x (N.-1SU
T
Where,
R = Percent recovery
Ns = E. faecalis (CPU/100 mL) in the spiked sample (or CPU per BioBall®)
Nu = E. faecalis (CPU/100 mL) in the unspiked sample
T spiked R faecalis = True spiked E. faecalis or CPU/100 mL in spiked sample
Percent recovery example calculation is provided in Table 9.
Table 9. Percent Recovery Example Calculation
Concentration
550
Ns (CPU /100mL)
505
Nu (CPU /100mL)
10
T (CPU /100ml)
517
Percent recovery (R)
100x(505-10)/517 = 96%
14.0 Method Performance
14.1 Multi-laboratory Validation (MLV) of Method 1611 in Marine and Fresh Water
14.1.1 Marine Water
14.1.1.1 Eleven volunteer laboratories participated in the U.S. Environmental
Protection Agency's (EPA's) MLV study of EPA Method 1611. The
purposes of the study were to characterize method performance across
multiple laboratories and multiple marine water matrices, assess method
reproducibility, and to develop QC acceptance criteria. A detailed
description of the study and results are provided in the validation study report
(Reference 17.9). Results submitted by laboratories were validated using a
standardized data review process to confirm that results were generated in
accordance with study-specific instructions and the April 2010 draft version
of EPA Method A (1611).
14.1.1.2 Recovery — Method 1611 was characterized by mean laboratory-specific
recoveries, of enterococci from marine water samples spiked with BioBalls
ranging from 68% to 841% (StepOnePlus™) and 89% to 632% (ABI 7500),
with an overall mean recovery of 401% (StepOnePlus™) and 274% (ABI
7500). Mean laboratory-specific recoveries of enterococci from PBS
samples spiked with BioBalls ranged from 37% to 606% (StepOnePlus™)
and 89% to 627% (ABI 7500), with an overall mean recovery of 326%
(StepOnePlus™) and 297% (ABI 7500).
14.1.1.3 Precision — Method 1611 was characterized by laboratory-specific relative
standard deviations (RSDs) from marine water samples spiked with BioBalls
ranging from 35% to 91% (StepOnePlus™) and 43% to 71% (ABI 7500),
with an overall pooled, within-laboratory RSD of 67% (StepOnePlus™) and
33
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Method 1611
58% (ABI 7500). For PBS samples spiked with BioBalls, laboratory-specific
RSDs ranged from 29% to 1 14% (StepOnePlus™) and 48% to 74% (ABI
7500), with an overall pooled, within-laboratory RSD of 81%
(StepOnePlus™) and 58% (ABI 7500).
14.1.1.4 Reproducibility — Each laboratory analyzed a total of five matrix samples
received during a single week. Each sample was split into eight replicates (4
unspiked and 4 spiked). Samples were spiked with E. faecalis (550 CPU)
BioBalls. Overall mean recoveries were 127%, 133%, 82%, 111%, and
228% for matrices 1-5, respectively.
14.1.2 FreshWater
14.1 .2.1 Ten volunteer laboratories participated in the U.S. Environmental Protection
Agency's (EPA's) MLV study of EPA Method 1611. The purposes of the
study were to characterize method performance across multiple laboratories
and multiple fresh water matrices, assess method reproducibility, and to
develop quantitative QC acceptance criteria. A detailed description of the
study and results are provided in the validation study report (Reference 17.9).
Results submitted by laboratories were validated using a standardized data
review process to confirm that results were generated in accordance with
study-specific instructions and the April 2010 draft version of EPA Method
14.1.2.2 Recovery — Method 1611 was characterized by mean laboratory-specific
recoveries, of enterococci from fresh water samples spiked with BioBalls
ranging from 7% to 156%, with an overall mean recovery of 83%. Mean
laboratory-specific recoveries of enterococci from PBS samples spiked with
BioBalls ranged from 35% to 103%, with an overall mean recovery of 69%.
1 4. 1 .2.3 Precision — Method 1611 was characterized by laboratory-specific relative
standard deviations (RSDs) from fresh water samples spiked with BioBalls
ranging from 35% to 1554%, with an overall pooled, within-laboratory RSD
of 554%. For PBS samples spiked with BioBalls, laboratory-specific RSDs
ranged from 20% to 104%, with an overall pooled, within-laboratory RSD of
58%.
14.1 .2.4 Reproducibility — Each laboratory analyzed a total of five matrix samples
received during a single week. Each sample was split into eight replicates (4
unspiked and 4 spiked). Samples were spiked with E. faecalis (550 CPU)
BioBalls. Overall mean recoveries were 13%, 151%, 87%, 35%, and -97%
for matrices 1-5, respectively. It should be noted that for Matrix #5 the
mean background enterococci concentration was 37,329 CCEs/100 mL;
approximately 68 times greater than the spike level of 550 cells added to
each sample.
15.0 Pollution Prevention
15.1 The solutions and reagents used in this method pose little threat to the environment when
recycled and managed properly.
15.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
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Method 1611
16.0 Waste Management
16.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations
governing waste management, particularly the biohazard and hazardous waste identification rules
and land disposal restrictions, and to protect the air, water, and land by minimizing and
controlling all releases from fume hoods and bench operations. Compliance with all sewage
discharge permits and regulations is also required.
16.2 Samples, reference materials, and equipment known or suspected to have viable enterococci
attached or contained must be sterilized prior to disposal.
16.3 For further information on waste management, consult "The Waste Management Manual for
Laboratory Personnel" and "Less Is Better: Laboratory Chemical Management for Waste
Reduction," both available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.
17.0 References
17.1 Anonymous. 1997. User Bulletin #2. ABI Prism 7700 Sequence Detection System. Foster City,
CA, Applied Biosystems.
17.2 Bordner, R, J.A. Winter, and P.V. Scarpino (eds.). 1978. Microbiological Methods for
Monitoring the Environment: Water and Wastes, EPA-600/8-78-017
17.3 Cabelli, V. J., A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W. Haberman. 1979.
Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches. Amer. Jour.
Public Health. 69:690-696.
17.4 Haugland, R.A., S.C. Siefring, L.J. Wymer, K.P. Brenner, and A. P. Dufour. 2005. Comparison
of Enterococcus Density Measurements by Quantitative Polymerase Chain Reaction and
Membrane Filter Analysis at Two Freshwater Recreational Beaches. Water Research 39:559-
568.
17.5 Ludwig, W. and K.-H. Schleifer. 2000. How quantitative is quantitative PCR with respect to cell
counts? System Appl. Microbiol. 23:556-562.
17.6 USEPA. 1983. Health Effects Criteria for Marine Recreational Waters. EPA-600/1-80-031.
17.7 USEPA. 1984. Health Effects Criteria for Fresh Recreational Waters. EPA-600/1-84-004.
17.8 USEPA. 1986. Ambient Water Quality Criteria for Bacteria - 1986. EPA 440/5-84-002.
17.9 USEPA. 2011. Results of the Multi-Laboratory Validation of EPA Method A for Enterococci in
Marine and Fresh Waters and Method B for Bacteroidales in Marine Waters by TaqMan®
Quantitative Polymerase Chain Reaction (qPCR) Assay. EPA 820-R-12-010.
17.10 Wade, T.J., Calderon, R.L., Sams, E., Beach, M., Brenner, K.P. & Dufour, A.P. 2006. Rapidly
measured indicators of recreational water quality are predictive of swimming associated
gastrointestinal illness. Environmental Health Perspectives 114: 24-28.
35
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Method 1611
18.0 Acronyms
AACT comparative cycle threshold calculation method
AF amplification factor
BHIA brain heart infusion agar
BHIB brain heart infusion broth
BSA bovine serum albumin
CCE calibrator cell equivalent
CPU colony forming units
CT cycle threshold
CV coefficient of variation
DNA deoxyribonucleic acid
EDTA ethylenediaminetetraacetic acid
GI gastrointestinal illness
IPR initial precision and recovery
IsrRNA large subunit ribosomal RNA (i.e., 23S rRNA)
MS matrix spike
MLV multi-laboratory validation
NTC no template control
OD optical density
OPR ongoing precision and recovery
PBS phosphate buffered saline
PCR polymerase chain reaction
QA quality assurance
QC quality control
qPCR quantitative polymerase chain reaction
RNA ribonucleic acid
RPD relative percent difference
RSD relative standard deviation
SLV single-laboratory validation
SPC sample processing control
TNTC too numerous to count
TSC target sequence concentration
UV ultraviolet (light)
36
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Method 1611
Appendix A:
ABI StepOnePlus™, ABI 7900, and ABI 7500 Real-Time PCR System
Operation
37
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Method 1611
StepOnePlus™, ABI 7900, and ABI 7500 Real-Time PCR System
Operation
1.0 ABI StepOnePlus™
1.0.1 Turn on the StepOnePlus™ and then the computer. Launch the StepOnePlus™ software
program by double clicking on its icon on the computer desktop or from the Computer
Programs menu.
1.0.2 On the StepOnePlus™ home screen select Advanced Set Up.
1.0.3 On the right side of the main screen click in the box Experiment Name, enter identifying
information for the experiment (Name/Date etc such that experiment can be identified).
Then go through the following fields:
• Which instrument is going to be used to run the experiment: StepOnePlus™
Instrument (96 wells) is default (highlighted).
• What experiment do you want to set up: Quantitation-Standard Curve is default
(highlighted).
• Which reagents will be used to detect the target: TaqMan® Reagents is default
(highlighted).
• Which ramp speed do you want to use in the instrument run: Click on Standard-2
hours (Not Default)
1.0.4 Click on Plate Set Up from the navigational pane of the present screen to define the
targets, and then assign them to wells in the reaction plate (Step 1.0.8).
1.0.5 Define target - You can add a new target or use a saved target. By clicking on Add Saved
Target the window with the target library will open.
1.0.6 Select the target(s) for your assay(s) and click on Add Selected Targets. All of the
targets may be selected simultaneously by holding the Ctrl key and highlighting the
desired targets (e.g., Entero, Sketa). The selected targets will then be added on to the
define target and sample screen.
1.0.7 Optional: If a new target is to be added click the Enter Target Name cell and type the
name. From the Reporter dropdown menu, select FAM (default). From the Quencher
dropdown menu, select Tamra (NFQ-MGB) (default). Leave the default in the color field.
1.0.8 Click on the tab Assign Targets and Samples to see the screen view of the plate layout
with 96 wells.
• Select the wells, based on the plate set up, by highlighting, one target at a time.
• Select the wells for the first target by checking the box for the desired target in Assign
Target to the selected wells and it will automatically populate the wells for that target.
If more than one target is being used, repeat the process above for each target.
• The wells can be selected individually or by rows by clicking in the left corner of the
row. In addition, the whole plate may be selected by clicking in the left corner of the
plate.
• To deselect a row or well press Ctrl & click the selected portion one more time and it
will deselect the row or well. Selected wells/rows will be highlighted grey, while
unselected wells/rows will remain white.
A-l
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Method 1611
1.0.9 Run the Method
• On the run method screen, review the reaction volume and the thermal profile for the
default run. If needed: The default run method can be edited or replaced with one
from the run method library. Click either the Graphical view (default) or Tabular
View tab.
• Make sure the reaction volume per well field displays 25 \\L, this is not the default
setting.
• Set the thermal profile to the following holding and cycling stages: Holding Stage 1:
50.0°C for 2:00 minutes, Holding Stage 2: 95.0°C for 10:00 minutes, Cycling Stage:
95.0°C for 0:15 seconds. The second step of the Cycling Stage is defaulted at 60.0°C
for 1 minute.
• Note: When using a run from the library click on the tab Open Run Method in the
graphical view. Select Run Method and click ok. It will replace the default run with
the saved run.
1.0.10 Load the plate into the instrument.
1.0.11 Click on the Plate Set Up tab and start the run by clicking the green button in the upper
right hand corner of the screen.
• Save Experiment dialogue box - Click Save to accept default file name and location
(the name assigned when setting up the experiment). The experiment is saved by
default to the :\ applied Biosystems\\experiment folder.
• Run progress can be viewed from the touch screen of the instrument. At the
beginning of a run do not leave the instrument or computer until you verify that the
run has started.
1.0.12 Once the run is completed remove the reaction plate and discard.
1.0.13 Analyze the run
• Click on the tab in the top right corner of screen to Analyze the Run.
• Highlight all of the sample wells and click on Analysis Settings to get to CT settings
(default CT settings).
• Highlight all the targets running in the plate; uncheck the default setting for automatic
threshold and set the threshold to 0.03. Keep the Automatic Baseline. Click on
Apply Analysis Settings to save changes. The new settings can be confirmed by
viewing the threshold line on the amplification plot.
1.0.14 Press the Export tab on the left corner of the view plate layout screen. Click on the
browse button to find the correct place to export the data and click Start Exporting. After
the export is done close export tool.
1.1 ABI 7900 Real-Time PCR System Operation
1.1.1 Turn on the ABI Model 7900 and then the computer. Launch the SDS 2.2.2 software
program by double clicking on its icon on the computer desktop or from the Computer
Programs menu. The computer will establish communication with the 7900 instrument
and if the connection is successful, the software will display the Connected icon in the
status bar when a plate document is opened.
A-2
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Method 1611
1.1.2 Under File menu, select New.
1.1.3 In resulting New Document window that appears, change container selection from 384
well clear plate to 96 well clear plate using drop down menu. Accept default selections
of Absolute Quantification and Blank Template. Click OK to display a new plate
document.
1.1.4 Click, hold and drag mouse over all PCR reaction tray wells containing samples in upper
left window. Selected wells will be outlined with a bold line and their position numbers
should appear in the results table in the lower left window. To unselect wells, repeat
above process while holding down control key.
1.1.5 Above right hand window, click on Setup tab.
1.1.6 Click on Add Detector button at the bottom of the setup screen. Note: Before any
analyses are performed, a specific detector for the method must be created. To do this, go
to step 1.1.7. Once the detector has been created, go to step 1.1.8.
1.1.7 Click on New in the pop-up window that appears. Another pop-up window will appears.
Under Name, type in a name for the detector that will be used by this method (e.g.,
"Method 1611"). Under Group select Default. Under Reporter select FAM. Under
Quencher select TAMRA. Click on OK to close second pop-up window. This step only
needs to be performed before the initial analysis run of the method. The detector that is
named is selected in all subsequent analysis runs as indicated in step 1.1.8)
1.1.8 In pop-up window that was opened in step 1.1.6, select the desired detector under Names
menu (e.g., Method 1611) and click on Copy to Plate Document button. Click on Done
button to return to setup screen.
1.1.9 Click on Use box next to FAM detector in right hand window. This box should become
marked with an X. Name and color code for FAM detector should appear in each of the
selected well positions in the upper left window and a data column for this detector should
be created for each of the selected well positions in the results table in the lower left
window.
1.1.10 Click on Instrument tab right hand window.
1.1.11 In instrument screen, change sample volume to 25 uL and choose 9600 emulation.
1.1.12 Still in instrument screen, click on Connect, then click on Open/Close button in lower
right hand "Real Time" window to open PCR reaction tray holder door on instrument.
1.1.13 Insert PCR reaction tray with prepared reactions in holder.
1.1.14 Click on Open/Close button to close PCR reaction tray holder door on instrument.
1.1.15 Click on Start button in lower right hand "Real Time" window to start thermal cycling in
instrument.
1.1.16 Name run file at prompt.
1.1.17 At termination of the run, instrument-calculated cycle threshold values should
automatically appear for each well position and detector entry in the lower left hand
results table window.
1.1.18 At termination of the run success complete, choose Analysis Settings from the toolbar. In
that box enter a value for the Manual Ct Threshold (see 1.1.19). Click on OK. Click on
Analyze from the toolbar. You should see CT values in the Results Table.
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Method 1611
1.1.19 The instrument-selected threshold line is indicated by the bold red line on the plot and the
value is listed below the window. This can be manually adjusted by either click-hold and
dragging the line up or down or entering new values below. Note: Based on results thus
far with the instrument, the threshold value can be adjusted from the default value of 0.2 to
0.03, however, this should be done only if the threshold line remains above the
background values (seen before the growth curves) for all of the samples.
1.1.20 Calculated CT values for each of the sample tray positions in the lower left hand "Results
Table" will automatically be updated following adjustments of the threshold line. Once
the threshold is adjusted to the desired level, select "Print Report" under the "File" menu.
Check or uncheck desired report items by clicking on their associated boxes and the click
on "Print" button. Note: Minimum report should have both detector boxes, i.e., "FAM"
checked which will show CT values for all selected tray positions for this detector.
1.1.21 Export data by clicking on File from the toolbar. From the drop down menu choose
Export. In the box you will see Look in: and here you choose a directory to send the
exported file too. Click on Export. Save changes to document? will appear, click on
Yes. Click OK.
1.2 ABI 7500 (non-Fast) Sequence Detector Operation
1.2.1 Turn on the ABI Model 7500 sequencer and then the computer. Launch the software
program by double clicking on its icon on the computer desktop or from the Computer
Programs menu. The computer will establish communication with the 7500 instrument.
See "How to Set Up a New Experiment Using the ABI 7500" for screen shots.
1.2.2 Click on either the New Experiment or Advanced Setup button to create a new
experiment, which will pull up the Experiment Menu.
1.2.3 From the Setup menu, select Experimental Properties to select the experiment type and
give the experiment a name. Enter the experiment name in the asterisked box.
• Click on 7500 (96 Wells) to select the instrument type. Note: This protocol is not
designed for the 7500 Fast.
• Scroll down to access more experiment properties options. Click on Quantitation -
Standard Curve to select the experiment type
• Click on TaqMan* Reagents to select the reagents used.
1.2.4 From the Setup menu, select Plate Setup. Click on the Define Targets and Samples tab
to define the reporter-quencher dye for each target and also to enter the sample
identifications (e.g., Ent 5X).
• Click on Add New Target and enter the name of the target. Under the Reporter
heading, click on the drop down menu to select the reporter dye FAM. Under the
Quencher heading, click on the drop down menu to select TAMRA as the quencher.
• Repeat the process above to add more than one target, e.g., Sketa 5x.
• In the Define Samples section, click on Add New Sample and enter the sample
name in the Sample 1 box.
• Click on the Assign Targets and Sample tab to assign the Target, sample ID and
sample type to the wells.
o Drag the mouse over the desired cells to assign the Target, Task and
Sample.
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Method 1611
o While the cells are highlighted, in the Assign target(s) to the selected
wells section, check the box under Assign for the appropriate Target, then
under Task, select U for unknown, S for standard or N for negative
control.
o In the Assign sample(s) to the selected wells section, check the box
under Assign to label the highlighted cells as Sample 1.
• Repeat the process above, by dragging the mouse over the appropriate cells, and
then assigning them as Unknowns, Standards or Negative Controls, as appropriate.
Note: For Standards, in the Assign target(s) to the selected wells section, if there
are 4 different values, each cell will need to be highlighted separately, and a quantity
(e.g. 40000) typed in the Quantity box.
• Click on Print Report at the top of the screen to print a plate layout for loading
master mix and sample extracts.
o Select Plate Layout option by clicking on the box next to it.
o Click Print Report to print the plate layout.
1.2.5 From the Setup menu, select Run Method to set up the thermo cycling profile.
• In the Graphical View tab, change the Reaction Volume Per Well from the default
50 uL to 25 uL by typing in 25.
• Under Cycling Stage section, change the Number of Cycles to 45.
• Check that the default settings of Temperature and Time for the two Holding Stages
and the Cycling Stage are correct. Specifically, these should be: Holding Stage 1:
50.0°C for 2:00 minutes, Holding Stage 2: 95.0°C for 10:00 minutes, and Cycling
Stage: 95.0°C for 0:15 seconds. Note: The purpose of Holding Stage 1 is to
eliminate potential PCR carryover products in the reactions using the Amp-Erase®
UNG enzyme provided in the TaqMan® Universal PCR master mix. The purpose of
Holding Stage 2 is to inactivate the Amp-Erase® UNG enzyme.
• The second step of the Cycling Stage is defaulted at 60.0°C for 1 minute - change
the time to 2:00 minutes. Note: This sets the detection of the fluorescence signal to
occur at the end of the second step in each cycle.
• Click on Save at the top of the screen to save the run before actually running the
experiment.
• The Save screen will open so that you may select the folder to save the Run Data
File. Type in the Experiment Name (if you did not already do so at the beginning of
the Experiment Setup) and click on Save. If you had previously typed in an
Experiment Name and there is no change, click on Save. Note: You must save to
the hard drive (rather than a flash drive).
1.2.6 From the Experiment menu, select Run to monitor the run in real time.
• The Run Status screen will open - click on Start Run. When the run has
successfully started, the Start Run button will turn change from green to red.
1.2.7 When the run has completed, from the Setup menu, select Analysis to export your results.
See "How to Export Results from the ABI 7500 Software" for screen shots.
• The Analysis Settings for Experiment name screen will open.
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Method 1611
• Under the CT Settings tab, click on the Target desired to be set from Default to
Manual Threshold setting (generally, this would be the Ent).
• In the CT Settings for ENT section, uncheck the boxes next to Use Default
Settings, and Automatic Threshold. Enter the desired Threshold (e.g., 0.025). If
more than one target is present, highlight the next target (e.g., Sketa) and repeat this
step.
• Click on Apply Analysis Settings to save the changes.
1.2.8 Click on Export at the top of the screen to save the experiment results data and to save the
selected plots required in the report. The threshold value will have changed on the
Amplification plot curve graphic.
• The Export Data screen will open. In the Select data to export section, check all of
the boxes (i.e., Sample Setup, Raw Data, Amplification Data, Results, and
Multicomponent Data).
• For Select one file or separate files, select One file to export all the data into one
file with multiple tabs.
• For Export File Name, enter the name of the Experimental data file to be exported,
and for File Type, choose *.xls.
• For Export File Location, click on Browse to select the folder in which you wish
to export your file. Always save your file to the hard drive.
• Click on Start Export. When this is completed, an Export Completed screen will
open - click on Close Export Tool to complete this task.
1.2.9 How to interpret your results. See "How to Interpret Results from the ABI 7500" for
screen shots.
• After exporting your data, from the Experiment menu, select Analysis.
o From the Analysis menu, select Amplification Plot to view the results of
the selected samples at different plot settings.
o Drag the mouse over the desired cells to select the samples to be plotted in
the amplification plot.
o In the Amplification Plot section, in the Plot Settings tab, select the Plot
Type (from the drop down menu) and the Graph Type (from the drop
down menu).
o If the plot requires the display of the Threshold and Baseline, in the
Options tab, check the boxes next to Threshold and Baseline. If you
want to see the results in tabular format, click on View Well Table, and
scroll down or sideways for more samples and analysis parameters.
o The toolbar above the graph can be used to make hard copy prints, saving
the file as *.jpg, zooming in and out, and changing the plot properties.
• For the Standard Curve, select Standard Curve from the Analysis menu to view
the results of the same sample (assuming they have the same target) and standards in
the plot.
o In the Plot Settings tab, click the drop down menu arrow beside Target
to select a different target (if the standards were analyzed in more than
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Method 1611
one target), and click the drop down menu arrow beside Plot Color to
change the plot color.
o The standard curve parameter display below the plot (i.e. Target, Slope,
Y-Intercept, R2, and Eff% (percent efficiency) is useful for data analysis.
• For Multicomponent plots, click on Multicomponent Plot from the Analysis menu
to view the variation in the fluorescence of the dyes used in the experiment over the
ascending repeat of the 45 thermo cycles based on well, target, or dye.
o Click on the drop down menu arrow next to Plot Color in the Plot
Settings tab to select the parameter for the basis of the graph (e.g., well,
target, or dye).
• For raw data plots, select Raw Data Plot from the Analysis menu to view the
variation in the emission of fluorescence of the dyes in different filters present over
the ascending repeat of the 45 thermo cycles.
o In the Options tab, drag the pointer on the cycle scale to the desired cycle
number to view the fluorescence variation through filters of the samples at
that cycle.
• For quality controls, click on QC Summary from the Analysis menu to view the
analysis summary of the samples.
• To view all the plots, click on Multiple Plots View from the Analysis menu to see
all of the plots for the samples in a single window. Each plot will have its own drop
down menu as in Section 1.2.9.5.
1.2.10 To print the report, click on Print Report at the top of the screen.
• Check all of the boxes (i.e., Experimental Summary, Results Summary, Plate
Layout, Amplification Plot (3 boxes), Standard Curves, Results Table, and QC
Summary).
• Click on Print Report at the bottom of the screen.
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Appendix B
Cepheid Smart Cycler® Operation
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Method 1611
Appendix B - Cepheid Smart Cycler® Operation
1.0 Smart Cycler® Operation
1.1 This protocol is intended to provide only information about critical instrument settings required to
perform EPA Method 1611. Further details concerning the operation of the instrument can be
obtained from the Smart Cycler® Operation Manual, Cepheid Part #D0190, Rev. D.
1.2 Turn on the Smart Cycler®; then the computer.
1.3 Double-click on the Smart Cycler® icon on the computer desktop.
1.4 The following steps for defining a protocol are only required before the initial run of the
instrument. The protocol that is defined in these steps is used in all subsequent runs of the
instrument. See "How to Set Up a New Experiment Using the Smart Cycler® (Software Version
2.0" for screen shots.
1.4.1 Click on the Define Protocols icon to go to Define Protocols screen.
1.4.2 Click on the New Protocol button to open the Protocol Name? dialog. Enter "EPA
Enterococcus Method 1611" for the new protocol name, and click OK. The protocol
stages are defined in the series of boxes at the bottom of the Define Protocol screen.
Make sure the new protocol is highlighted and begin to choose state settings. To define
Stage 1, click on its drop-down box to display the menu of stage types; then select Hold.
In the Temp column, enter 50.0, and in Sees column, enter 120, leaving the Optics setting
as the default Off setting. (Note: This stage is performed to eliminate potential PCR
carryover products in the reactions using the Amp-Erase® UNG enzyme provided in the
TaqMan® Universal PCR master mix. The optical reading cannot be programmed during
a Hold stage.).
1.4.3 To define Stage 2, click on its drop-down box to display the menu of stage types, and
again select Hold. In the Temp column, enter 95.0, and in Sees column enter 600. Again,
leave the Optics setting on Off. (Note: This stage is performed to inactivate the Amp-
Erase® UNG enzyme).
1.4.4 To define Stage 3, select 2-Temperature Cycle from its drop down menu. For the first
step, enter 95.0 in the first row of the Temp column and 15 in the Sees column, Optics
column Off. For the second step, enter 60.0 in the second row of the Temp column, 120
in the Sees column, and click on the Optics cell to select On from the drop-down menu.
(Note: This sets the detection of the fluorescence signal to occur at the end of the second
step in each cycle.) Enter 45 in the Repeat field at the top of the Stage 3 box to specify
that it should be repeated for 45 cycles. Click the Save Protocol button.
1.4.5 To display primary curve graphs, click Define Graphs. Highlight FAM in Graph
column. Check the box for Automatically add to new runs. Under Graph Type choose
Optics from the pull down menu. Under Channels check the box for Ch 1 (FAM).
Under Show check the boxes for Primary Curve, Threshold (Vertical) and Threshold
(Horizontal). Under Axes check the box for Fluorescence vs. Cycle. At the bottom of
the screen click on Save Graph.
1.5 Click on the Create Run icon to open the Create Run screen. For each new run, enter a unique
name in the Run Name field. (Note: The software does not allow duplicate run names).
1.6 Enter any additional information about the run in the Notes field. Click the arrow in the Dye Set
box to display a drop-down menu of the possible selections. Select FTTC25. (Note: This selects
the dye set: FAM, TET, Tex Red, Cy5, and a 25 uL reaction).
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Method 1611
1.7 Click the Add/Remove Sites button. The Select Protocols and Sites... dialog will appear.
Highlight (click on) the "EPA Method 1611" protocol developed prior to the first run (Section
1.4.2) in the Protocols list. In the Sites list, highlight the sites on the instrument to be used with
this protocol in the current run by clicking on them with the shift key held down (Note: Sites refer
to the I-core modules in the Smart Cycler® processing block in which reaction tubes will be
placed; a total of 16 are possible per block. When using multiple blocks, the site numbers will be
preceded by the block letters, e.g., A, B, C); then click the right pointing arrow to transfer the
selected sites and protocol to the Selections table.
1.8 Click on the OK button to save the selections, and return to the Create Run screen.
1.9 Place the loaded Smart Cycler® reaction tubes in the I-core module slots, selected above for
current run. The tubes should snap into place. Either the front or back of the caps can face the
front of the processing block.
1.10 In one of the View menus that is shown, select Analysis Settings. The displayed table includes
one row for each of the four possible dye channels defined in the dye set. Click on the cell in the
FAM row under the Usage column heading, and select Assay from the drop down menu. Set the
Usage cells for all other dyes to Unused in the same manner. (Note: All assays in this protocol
use FAM as the reporter dye). All other cells in this table should be left at default settings (See
Smart Cycler® Operation Manual).
1.11 In the other View menu that is shown, select the Results Table. Enter the sample identification
information for each site in the Sample ID column (additional information can be entered into the
Notes column.). Leave the other columns as default settings (see Smart Cycler® Operation
Manual.)
1.12 Click on the Start Run button. The orange LEDs on the Smart Cycler® processing block should
turn on, and the software will automatically switch to the View Results screen.
1.13 To display the real time temperature profiles for all sites, click Temperature in either of the View
menus. To display real time growth curves for all samples (i.e., the fluorescence signal vs. cycle),
click FAM in the other View menu.
1.14 At the end of the run, it is recommended to check the cycle threshold values calculated by the
instrument for each sample by opening the Results Table window by clicking on this selection in
the upper View menu. It is also recommended to inspect the growth curves in the FAM window
which can be opened in the same manner from the lower View menu. The default threshold
fluorescence value is shown in this window as a single horizontal red line and the cycle thresholds
for each site are shown as vertical red lines. To view the data for individual sites in this window,
click on that site number in the table to the right of the graph. If the default threshold fluorescence
line is well above all of the growth curve lines prior to visible amplification, the threshold
fluorescence value can be changed to a lower value. This is done by reopening the Analysis
Settings window from the upper View menu and entering a new value in the Manual Thresh
Fluor Units cell in the FAM row. Conversely if the default threshold fluorescence line is below
any of the growth curve lines prior to visible amplification, the threshold fluorescence value
should be changed to a higher value in the same manner. Previous studies have indicated that a
threshold value of 8 works well for most analyses. Click on the Update Analysis button to view
the new threshold line in the FAM window. The cycle threshold values will be automatically
updated in the Results Table.
1.15 Once the threshold fluorescence value is adjusted to an optimal value, click the Save Run button.
(Note: The Smart Cycler® Software does not give a prompt to save changes before printing or
exporting. Therefore, it is possible to make changes to the Results Table or Analysis Settings, and
immediately print or export the data, then close the run without saving the changes. In this case,
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Method 1611
the data saved in the Smart Cycler® database will not match the printed or exported data. If no
changes are made in the threshold fluorescence value, the run data is automatically saved as it is
when the program is closed or a new run is created.)
1.16 To set up automatic export of raw data, (see "How To Set Up Automatic Export of Raw Data" for
screen shots) from the main menu, click on Setup, then System Defaults, then Export Settings.
From the Export Settings dialog box, check the box beside Results Table and Analysis Settings.
Click the Browse button to select the folder where you want your raw data saved. Click on the
radio button next to Automatic export on run completion, then click the OK button.
1.17 To manually save the Results Table and Analysis setting containing the instrument-calculated
cycle threshold values for each sample (see "How to Manually Export Raw Data" for screen
shots), click the Export button to display the Export Data dialog box. Check the box next to the
heading Export Results Table and Analysis Settings by clicking on it. Leave all other boxes
unchecked. Click on Export. A box labeled Export Data will appear with the run name in the
file name box. Click on the drop down menu to the right of Save In: at the top left of the screen to
change the directory where you want your data saved. Click on Save. Data are exported as
comma-delimited text (*.cvs) files in MS Excel-compatible files to the Export folder in the Smart
Cycler® folder: C:\Smart Cycler®\Export. Always save to the hard drive.
1.18 To archive a run in the Smart Cycler® program, click on Tools. From the drop down menu click
on Data Management and then Archive Runs. Click Proceed. Select the run to be archived by
clicking on its name in the database list. Click OK and then Proceed. A box labeled Archive Run
will appear on the screen. There will be a line labeled Save in:. Input which directory you would
like the run to be saved in. There will be aline File Name:. Enter a file name. Click Save. Click
OK
1.19 To print run data (see "How to Print Run Data" for screen shots), after the run is completed, right
click on the graph area, and select Print, then Print Graph with Results Table. An Optics Graph
print preview screen will pop up, click Print. The screen will switch back to the post run screen.
To print the generated report, click Report. A Run Report print preview screen will pop up, click
Print
1.20 To set up a new analysis (see "How to Set Up a New Run" for screen shots), from the post run
screen, click Create Run at the top left. The results of your just completed will disappear and a
Add/Remove Sites button will appear in the middle of the screen - click it. A Select Protocols
and Sites screen will pop up, select EPA Enterococcus Method 1611 in the Protocols box, and
highlight the amount of sites needed for the assay (A1-A16) in the Sites box. Click the right arrow
to the right of the sites box to move the sights to the Selections box. After double checking that
the correct protocol is highlighted, click OK. Add the Run Name in the Run Name box. The Site
ID, Protocol, Sample ID, Sample Type, Notes (etc.) box will pop up. Enter the Sample ID for
each site. Under this box, find the Usage column and click on Assay to select Unused for Cy3,
TxR and Cy5 (leave Assay for FAM). In the same box, find the Manual Thresh Fluor Units, and
click on the units for FAM to change it from 30.0 to 8.0 (leave the units at 30.0 for Cy3, TxR and
Cy5). After loading the Smart Cycler with tubes, click Start Run. A red light should appear on
sites that are in use.
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