# Method A: Enterococci in Water by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay

April 2010

Note that this method will be updated following it’s validation in marine and fresh ambient waters.

# U.S. Environmental Protection Agency Office of Water (4303T) 1200 Pennsylvania Avenue, NW Washington, DC 20460

EPA-821-R-10-004iii 

## 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 single laboratory validation of this method in fresh and marine waters:

Participant Laboratories

• New York State Department of Health, Environm ental Biology Laboratory: Ellen Braun-Howland and Stacey Chmura

• Mycometrics, LLC: King-Teh Lin and Pi-shiang Laiiv 

## 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 li ability 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 tr ade 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.govv 

## 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 recreationa l water standards based on established relationships between the measured density of enterococci colony forming units (CFU) 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 A describes a quantitative polymerase chain reac tion (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 (lsrRNA, 23S rRNA) from these organisms. The advantage of this method over currently accepted culture methods that require 24-48 hr to obtain results is its relative rapidity. Results can be obtained by this method in 3-4 hr, allowing same-day notifications of recreational water quality. While measurements of en terococci DNA by Method A and enterococci CFU’s by culture methods such as EPA Method 1600 are performed for essentially the same purpose, i.e ., to indicate fecal pollution, the results of these two approaches may not always be correlated with each other due to potential differences in the ratios of viable and non-viable bacteria in different water environments. Never-the-less, more recent epidemiological st udies conducted at freshwater recreational beaches (Reference 17.3) have demonstrated similar or im proved positive correlations between enterococci DNA measurements by this method and swimming-associated GI illness rates compared with those established for enterococci CFU measurements.

In Method A, water samples are filtered to collect en terococci on membranes. Following filtration, total DNA is solubilized from the retentate using a bead beater. Enterococci target DNA sequences present in the clarified homogenate are detected by the real time polymerase chain reaction technique using TaqMan® Universal Master Mix PCR reagent and th e TaqMan® probe system. The TaqMan® system signals the formation of PCR products by a process i nvolving enzymatic hydrolysis of a fluorogenicallylabeled oligonucleotide pr obe when it hybridizes to the target sequence.

Method A uses an arithmetic formula, the comparative cycle threshold (C T ) method, to calculate the ratio of Enterococcus lsrRNA gene target sequences (target sequences) 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. Mean estimates of the absolute quantities of target sequences in the calibrator sample extracts are then used to determin e the absolute quantities of target sequences in the water samples. C T values for sample processing control (SPC ) sequences added in equal quantities to both the water filtrate and calibrator samples before DNA extraction are used to normalize results for potential differences in DNA recovery or to signa l inhibition or fluorescence quenching of the PCR analysis caused by a sample matrix component or possible technical error.vi 

## Table of Contents

1.0 Scope and Application ..................................................................................................... ................1

2.0 Summary of Method ......................................................................................................... ...............1

3.0 Definitions ................................................................................................................ .......................1

4.0 Interferences.............................................................................................................. .......................2

5.0 Safety .................................................................................................................... ...........................3

6.0 Equipment and Supplies ..................................................................................................... .............3

7.0 Reagents an d Standards ..................................................................................................... ..............5

8.0 Sample Collection, Handling, and Storage .................................................................................. ....8

9.0 Quality Control ........................................................................................................... .....................9

10.0 Calibration and Standardization of Method-Related Instruments..................................................11

11.0 Procedure ................................................................................................................ .......................11

12.0 Data Analysis and Calculations ........................................................................................... ..........20

13.0 Sample Spiking Procedure.................................................................................................. ...........23

14.0 Method Performance........................................................................................................ ..............23

15.0 Pollution Prevention ..................................................................................................... .................24

16.0 Waste Management......................................................................................................... ...............24

17.0 References................................................................................................................ .....................25


### 18.0 Acronymsvii 

## List of Appendices

Appendix A: ABI 7900 and ABI 7500 Sequence Detector Operation

Appendix B: Cepheid Smart Cycler® Operation1 

## Method A: Enterococci in Water by Quantitative Polymerase Chain Reaction (qPCR) Assay

April 2010
1.0 Scope and Application

1.1 Method A describes a quantitative polymerase ch ain reaction (qPCR) procedure for the measurement of large subunit ribosomal RNA (lsrRNA, 23S rRNA) gene sequences (target sequences) from all known species of enterococci bact eria 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 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 Enterococci are commonly found in the feces of humans and other warm-blooded animals. Although DNA from these organisms can be persistent in the environment, its presence in water is an indication of fecal pollution and the possible presence of enteric pathogens.

1.3 The Method A test is recommended as a measure of ambient fresh and marine recreational water quality. Epidemiological studies have been conducted at freshwater and marine water beaches has been conducted that may lead to the potential development of criteria that can be used to promulgate recreational water standards based on established relationships between health effects and water quality measurements by this method. The significance of finding Enterococcus DNA target sequences in recreational water samples stems from the direct relationship between the density of these sequences and the risk of gastrointestinal illness associated with swimming in the water that have been observed thus far in th e epidemiological studies (Reference and 17.9).

1.4 This method assumes the use of an ABI Sequence De tector 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 shoul d thoroughly read the method in its entirety before preparation of reagents and commencement of the method to identify differences in protocols for different platforms. 

## 2.0 Summary of Method

The method is initiated by filtering a water sample through a membrane filter. Following filtration, the membrane containing the bacteria l cells and DNA is placed in a microcentrifuge tube with glass beads and buffer, and then ag itated to extract the DNA into solution. The supernatant is used for PCR amplification and detection of target sequences using the TaqMan® Universal Master Mix PCR reagent and probe system.



## 3.0 Definitions

3.1 Enterococci: all species of the genus Enterococcus for which lsrRNA gene nucleotide sequences were reported in the GenBank database ( http://www.ncbi.nlm.nih/gov/Genbank) at the time of method development.Method A

2 3.2 Target sequence: A segment of the Enterococcus lsrRNA 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, containing nucleotide sequences that are homologous to the primers and probe used in the SPC qPCR assay. SPC seque nces are added as part of a total salmon DNA solution in equal quantities to all water sample f iltrate and calibrator samples prior to extracting DNA from the samples.

3.4 DNA standard: A purified, RNA-free and spectr ophotometrically quantified and characterized E. faecalis strain ATCC #29212 genomic DNA preparation. 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 9.6. Also used for quantifying target se quences in calibrator sample extracts as described in Section 12.2.

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 ΔΔ C T comparative cycle threshold calculation method as described in Section 12.4. Analysis r esults of these samples provide corrections for potential daily or weekly me thod-related variations in Enterococcus cell lysis, target sequence recovery and PCR efficiency. QPCR analyses for SPC sequences from these samples are also used to correct for variations in total DNA recove ry in the extracts of water sample filtrates that can be caused by contaminants in these filtrates as described in Section 12 .4 and/or to signal potentially significant PCR inhibition caused by th ese contaminants as described in Section 9.8.

3.6 ΔΔ C T comparative cycle threshold calculation method ( ΔΔ C T method): A calculation method derived by Applied Biosystems (Reference 17.1) fo r 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 C T values for a reference (SPC) sequence that is initially present in e qual quantities prior to DNA extraction.

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 comparative cycle threshold calculation methods. 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 standa rd curve as described in Section 12.2.



## 4.0 Interferences

Water samples containing colloidal or suspende d particulate materials can clog the membrane filter and prevent filtration. These materials can also interfere with DNA recovery and with the PCR analysis by inhibiting the enzymatic activ ity 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.Method A

3 

## 5.0 Safety

5.1 The analyst / technician must know and observe the normal safety procedures required in a microbiology and/or molecular biology laborator y 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 h as been cleaned with bleach and UV sterilized as specified in section 11.6.1 between reagent and sa mple preparation. Under ideal conditions, two dedicated workstations are used for sample pr eparation: 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 microcentrifuge tubes, 2.0-mL ( e.g. , PGC 506-636 or equivalent)
6.4 Glass beads, acid washed, 212 - 300 μ m ( e.g. , Sigma G-1277 or equivalent)

6.5 Autoclave, capable of achieving and maintaining 121 ° C [15 lb pressure per s quare 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.
6.7 Sterile bottles/containers for sample collection
6.8 Membrane filtration units (filter base and funnel) fo r 47 mm diameter filters, sterile glass, plastic ( e.g. Pall Gelman 4242, or equivalent), stainless steel, or disposable plastic ( e.g., Nalgene CN 130-4045 or CN 145-0045, or equivalent); clean ed 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 th e filter flask and the vacuum source
6.13 Forceps, straight or curved, with smooth tip s to handle filters without damage, 2 pairs
6.14 Polycarbonate membrane filters, sterile, white, 47 mm diameter, with 0.45 F m pore size ( e.g. , GE Osmotics Inc. 04CP04700 or equivalent)Method A

4 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 × 15 mm with loose fitting lids
6.17 Disposable loops, 1 and 10 μ L
6.18 Permanent ink marking pen for labeling tubes
6.19 Visible wavelength spectrophotometer capable of measuring at 595 nm
6.20 Single or 8-place mini bead beater ( e.g. , Biospec Products Inc. # 3110BX or equivalent).
6.21 Microcentrifuge capable of 12,000 × g
6.22 Micropipettors with 10, 20, 200 and 1000 μ L 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).
6.23 Micropipettor tips with aerosol barrier for 10, 20, 200 and 1000 μ L 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 us ed 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 ( e.g. , 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 × 125 mm
6.28 Pipet containers, stainless steel, aluminum or borosilicate glass, for glass pipets
6.29 Pipets, sterile, T.D. bacteriological or Mohr, di sposable glass or plastic, of appropriate volume (disposable pipets preferable)
6.30 Vortex mixer (ideally one for each work station)
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 wavele ngths of 260 and 280 nm using small volume capacity ( e.g., 0.1-mL) cuvettes or NanoDrop® (ND- 1000) spectrophotometer (or equivalent) capable of the same measurements at 2 μ L sample volumes
6.39 ABI 7900 Sequence Detector
6.39.1 Optical 96 well PCR reaction tray ( e.g. , Applied Biosystems N801-0560 or equivalent)
6.39.2 Optical adhesive PCR reaction tray tape ( e.g. , Applied Biosystems 4311971 or equivalent) or MicroAmpTM caps ( e.g. , Applied Biosystems N8010534 or equivalent)Method A

5 6.39.3 ABI 7900 sequence detector
6.40 Cepheid Smart Cycler ® (optional to ABI)
6.40.1 Smart Cycler® 25 μ L PCR reaction tubes ( e.g. , 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
7.3 Sample Processing Control (SPC) DNA (source of SPC control sequences)

• Salmon testes DNA ( e.g. , Sigma D1626 or equivalent)

7.4 Phosphate Buffered Saline (PBS)
7.4.1 Composition:

Monosodium phosphate (NaH 2 PO 4 ) 0.58 g Disodium phosphate (Na 2 HPO 4 ) 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 wa ter in a flask and dispense in appropriate amounts for dilutions in screw cap bottles or cultu re tubes, and/or into containers for use as rinse water. Autoclave after preparation at 121 ° C [15 lb pressure per square inch (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 (Na 2 HPO 4 ) 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 × 125 mm tubes and autoclave at 121 ° C (15 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2.Method A

6 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 × 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 E C for 24 hours). Centrifuge to create pellet. Using a sterile pipet, discard supernatant. Resuspend pellet in 10 mL of fresh sterile BHI broth containing 15% glycerol and dispense in 1.5-mL aliquot s in microcentrifuge tubes. Freeze at -20 ° C (short term storage) or -80 ° C (long term storage). Note : Aliquots of suspension may be plated to determine CFU concentration as described in section 11.1. Note: It is advisable to verify the E. faecalis culture as described, for example, in Section 15 of EPA Method 1600.
7.9 PCR grade water ( e.g. , 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 ( e.g. , Qiagen 19077 or equivalent) (Note: pH 8.0 is acceptable) Composition:

10 mM Tris-Cl 0.5 mM EDTA (Ethylenediaminetetraacetic acid)
7.12 Salmon DNA/extraction buffer

7.12.1 Composition: Stock Salmon testes DNA (10 μ g/mL) (Section 7.3) AE Buffer (Section 7.11)

7.12.2 Preparation of stock Salmon testes DNA: Each bottle of Salmon DNA contains a differing number of units. Note the units. Add an equal volume of PCR grade water to dissolve the Salmon testes DNA, and stir, using a magnetic stir bar at low to medium speed, until dissolved (2-4 hours if necessary). The solution at this point will be equivalent to 50 μ g Salmon testes DNA/mL. Dilute using PCR grade water to a concentration of ~ 10 μ g/mL. Determine concentration of Salmon testes DNA stock by OD 260 reading in a spectrophotometer. An OD 260 of 1 is approximately equal to 50
μ g/mL (one Unit). This is your Salmon testes DNA stock solution. Unused portion may be aliquoted and frozen at

! 20 ° C.

Note : For example, if the bottle contains 250 mg of DNA, using sterilized scissors and sterilized forceps, cut a piece of DNA to weigh approximately 20 mg (approximately 304 Units), and place in a sterile weigh boat. After weighing, place the DNA into a sterile 50 mL tube and add 20 mL PCR grade water. Cap tightly, and resuspend by 2-4 hours of gentle rocking. The concentration should be about 1 mg/mL. Remove three, 10 μ L aliquots and dilute each to 1 mL with PCR grade water. Check absorbance (OD 260 ) and calculate DNA using the assumption 1 Unit DNA is equal to 1 OD 260 , which is thenMethod A

7 equivalent to 50 μ g/mL DNA. Adjust this stock to 10 μ g/mL based on calculated initial concentration of 1 mg/mL by diluting with PCR grade water. Aliquot portions of the adjusted DNA stock and freeze.
7.12.3 Dilute Salmon testes DNA stock with AE buffer to make 0.2 μ g/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 μ L) × total number of samples to be analyzed including controls, water samples, and calibra tor samples. For example, for 18 samples, prepare enough Salmon/DNA extraction buffer for 24 extraction tubes (18÷6 = 3, therefore, 3 extra tubes for water sample filtration blanks (method blanks) and 3 extra tubes for calibrator samples). Note that the number of samples is divided by 6 because you should conduct one method blank for every 6 samples analyzed. Additionally, prepare excess volume to allow for accurate dispensing of 600 μ L per tube, generally 1 extra tube. Thus, in this example, prep are sufficient Salmon/DNA extraction buffer for 24 tubes plus one extra. The total volume needed is 600 μ L × 25 tubes = 15,000 μ L. Dilute the Salmon testes DNA working stock 1:50, for a total volume needed (15,000 μ L) ÷ 50 = 300 μ L of 10 μ g/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 μ L - 300 μ L = 14,700 μ L AE buffer needed.
7.13 Bleach solution: 10% v/v bleach (or other reagen t that hydrolyzes DNA) (used for cleaning work surfaces)
7.14 Sterile water (used as rinse water for work surface after bleaching)
7.15 TaqMan® Universal PCR Master Mix ( e.g. , Applied Biosystems 4304437 or equivalent) Composition: AmpliTaq Gold® DNA Polymerase AmpErase® UNG

dNTPs with dUTP Passive Reference 1 (ROXTM fluorescent dye) Optimized buffer components (KCl, Tris, EDTA, MgCl 2 )
7.16 Bovine serum albumin (BSA), fraction V powder ( e.g. , Sigma B-4287 or equivalent) Dissolve in PCR grade water 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: 5'-FAM-TGGTTCTCTCCGAAATAGCTTTAGGGCTA-TAMRA
7.17.2 Salmon DNA primer and probe set (Reference 17.4): Forward primer: 5'-GGTTTCCGCAGCTGGG Reverse primer (Sketa 22): 5'-CCGAGCCGTCCTGGTCMethod A

8 TaqMan® probe: 5'-FAM-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 μ M primer and 100 μ M 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 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 pr oper O-ring seating after the tube has been closed. Autoclave tubes at 121 ° C (15 PSI) for 15 minutes.
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).
7.20 RNase A ( e.g. , Sigma Chemical #R-6513) or equivalent
7.20.1 Composition:

RNase A

Tris-Cl

NaCl
7.20.2 Dissolve 10 mg/mL pancreatic RNase A in 10 mM Tris-Cl (pH 7.5), 15 mM NaCl. Heat to 100 ° C for 15 min. Allow to cool to room te mperature. Dispense into aliquots and store at - 20 ° C. For working solution, prepar e solution in PCR grade water at concentration of 5 μg/μL.

7.21 DNA extraction kit (Gene-Rite #K102-02C-50 DNA-EZ® RW02 or equivalent)



## 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 pr oduction of valid data. Samples not collected according to these procedures should not be analyzed.
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 su rface water. Composite samples should not be collected, since such samples do not display the ra nge 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 storageMethod A

9 temperature. Take care that sample bottles are tightly closed and are not totally 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 A is require d 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 Media sterility check — The laboratory should test media sterility by incubating one unit (tube or plate) from each batch of medium (BHIA/BH IB) as appropriate and observing for growth. Absence of growth indicates media sterility. On an ongoing basis, the laboratory should perform a media sterility check every day that samples are analyzed.
9.3 Method blank (water sample filtration blank) — Filter a 50-mL volume of sterile PBS before beginning the sample filtrations. Remove the funne l from the filtration unit. Using two sterile or flame-sterilized forceps, fold the filter on th e base of the filtration unit and place it in an extraction tube with glass beads as described in S ection 7.20. Extract as in Section 11.5. Absence of fluorescence growth curve during PCR analysis of these samples (reported as “0" on SmartCycler and “undetermined” on ABI model 7900) indicates the absence of contaminant target DNA (see Data Quality Acceptance, below). Prepare one method blank filter for every six samples.
9.4 Positive controls — The laboratory should analyze positive controls to ensure that the method is performing properly. Fluorescence growth curve (PCR amplification trace) with an appropriate cycle threshold (C T ) 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.
9.4.1 Calibrator samples will serve as the positive c ontrol. 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 DNA extracts (Section 11.3) will be used as a positive control for both Enterococcus and SPC qPCR
assays. 9.4.2 If the positive control fails to exhibit the a ppropriate fluorescence growth curve response, check and/or replace the associat ed reagents, and reanalyze. If positive controls still fail to exhibit the appropriate fluorescence growth curve response, prepare new calibrator samples and reanalyze (see Section 9.7).
9.5 No template controls — The laboratory should analyze “No Template Controls” (NTC) to ensure that the Master Mix PCR reagents are not contaminated. On an ongoing basis, the laboratory should perform NTC analyses every day th at samples are analyzed. If greater than one-third of the NTC reactions for a PCR master mix exhibit true positive logarithmic amplification traces with C T values below 45 (not from chemical degradation of probe with linear kinetics that exhibit rising baseline) or if any one NTC reaction has a C T value lower than 35, the analyses should be repeated with new Master Mix working stock preparations.Method A

10 9.6 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 it size, the weight of a single E. faecalis genome can be estimated to be ~3.6 fg and there are four lsrRNA gene copies per genome in this species (see the rRNA operon database at http://rrndb.cme.msu.edu/rrndb). The concentration of lsrRNA gene copies per μ L in the standard E. faecalis genomic DNA preparation can be de termined from this information and from its spectrophotometrically determined total DNA concentration by the formula:

Total DNA concentration (fg/ μ L) 4 lsrRNA gene copies Concentration of lsrRNA gene copies per μ L = 3.6 fg/genome x

genome

A composite standard curve should be generated fro m 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 th at point on, it is recommended that additional standard curves be generated from duplicate analy ses 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 r 2 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 th e “Data Analysis” selection under the “Tools” menu in Excel. Subsequent regressions can be performed by plo tting 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-in tercept values as illustrated in Section 12.2. In the event that the slope valu e from a subsequent standard curve regression is outside of the acceptance range, the diluted standards should be re-analyzed. If this di fference persists, new working stocks of the reagents should be prep ared 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 8, should be modified based on the new slope values. If the slopes are within acceptance range but Y-intercep ts are not within acceptance range of this previous average, new serial dilutions of the DNA standard should be prepared and analyzed as described above.
9.7 Calibrator samples — The cell concentration of each cultured E. faecalis stock suspension used for the preparation of calibrator sample extract s 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 11.2. Dilutions of each of these calibrator sample extracts equivalent to the an ticipated dilutions of th e 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 C T 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 th e 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 C T value from these analyses should not be significantly different fro m the laboratory's average values from analyses of the initial calibrator sample extracts from the same stock cell suspension ( i.e ., not greater thanMethod A

11 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 ge neration of a standard curve as described in Section 9.6.
9.8 Salmon DNA Sample Processing Control (SPC) sequence analyses — 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 simi lar to that described for the Enterococcus DNA standards in Section 9.6. 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 fo r each test sample’s 5 fold dilution should be within 3 C T units of the mean of the 5 fold dilute d calibrator (and/or method blank) sample results. Higher C T 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 C T value greater than 3 C T units higher than the mean of th e 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 sa mple can be accepted if its Salmon DNA assay C T 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 C T 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 C T units of the mean C T result of the 25 fold dilution of the three calibrators.



## 10.0 Calibration and Standardizati on 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 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 least annually 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 sample s (Section 11.3) should be prepared at least weekly.Method A

12 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 phosphate buffered saline (PBS) and vortex.
11.1.3 Use 10 μ L 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 × 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 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 Determination of calibrator sample cell concen trations based on one of the three options 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 ce lls/mL (Y) in undiluted cell suspension using the formula from the standard curv e shown below where X is the average 595 nm spectrophotometer reading. Y = (1 x 10 9 cells / mL × X) / 0.19

• Option 2: Hemocytometer counts – Serially dilute 10 μ L of undiluted cell suspension with PBS to 10 -1 , 10 -2 , and 10 -3 dilutions and determine cell concentration of 10 -2 or 10 -3 dilutions in a hemocytometer or Petroff Hauser counting chamber under microscope.

• Option 3: plating – Note : 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. Note : BHIA plates should be made in advance if this option is chosen. 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 ofMethod A

13 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. 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 te mperature or dried using a laminar-flow hood. G) Each of the following will 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 below for calculation of undiluted cell suspension concentration.

CFU 1 + CFU 2 + .....+ CFU n CFU/mL undiluted = V 1 + V 2 + ... + V n

Where:

CFU/mL undiluted = E. faecalis CFU/mL in undiluted cell suspension CFU = number of colony forming units from BHIA plates yielding counts within the ideal range of 30 to 300 CFU per plate V = volume of undiluted sample in each BHIA plate yielding counts within the ideal range of 30 to 300 CFU per plate n = number of plates with counts within the ideal rangeMethod A

14 Table 1. Example Calculations of E. faecalis Undiluted Cell Suspension Concentration
CFU / plate (triplicate analyses) from BHIA plates Examples
10 -5 mL plates 10 -6 mL plates 10 -7 mL plates E. faecalis CFU / mL in undiluted suspension a

Example 1 275, 250, 301 30, 10, 5 0, 0, 0 (275+250+30) / (10 -5 +10 -5 +10 -6 ) = 555 / (2.1 x 10 -5 ) = 26,428,571 = 2.6 x 10 7 CFU / mL

Example 2 TNTC b , TNTC, TNTC TNTC, 299, TNTC 12, 109, 32 (299+109+32) / (10 -6 +10 -7 +10 -7 ) = 440 / (1.2 x 10 -6 ) =366,666,667 = 3.7 x 10 8 CFU / mL

a Cell concentration is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plate

b 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 F L) aliquots for calibrator samples and freeze at

! 20 ° C. Note: Cell suspension should be stirred while aliquoting. It is also recommended that sep arate micropipettor tips be used for each 10

F L 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.
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 fo r 60 seconds at the maximum rate (5000 rpm).
11.2.5 Remove the tubes from the mini bead beater and centrifuge at 12,000 × g for one minute to pellet the glass beads and debris.
11.2.6 Using a 200 μ L micropipettor, transfer 350 μ L of supernatants to sterile 1.7-mL microcentrifuge tubes. Recover supernatan ts without disrupting the glass beads at the tube bottom.
11.2.7 Centrifuge crude supernatants at 12,000 × g for 5 minutes and transfer 300 μ L 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 μ L of 5 μ g/ μ L 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 vort exing. Note: In general, a minimum concentration of 5 x 10 8 cells is required for this step.Method A

15 11.2.10 If using the DNA-EZ purification kit, perform the following steps. Insert one DNAsureTM column from the DNA-EZ purification kit into a collection tube (also provided with kit) for ea c h of the two extracts. Transfer each of the extract and binding buffer mixtures from section 11.2.9 to a DNAsureTM column and collection tube assembly and centrifuge for 1 min at 12,000 × g.

11.2.11 Transfer each of the DNAsureTM columns to new collection tubes. Discard previous collection tubes and collected liquid.

11.2.12 Add 500 μ L EZ-Wash Buffer from the DNA-EZ purification kit to each of the DNAsureTM columns and centrifuge at 12,000 × 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 DNAsureTM columns to a clean, labeled 1.7-mL low retention microcentrifuge tube and add 50 μ L of DNA elution buffer to each column. Centrifuge for 30 seconds at 12,000 × g. Rep eat this procedure again to obtain a total DNA eluate volume of ~100 μ L from each column.
11.2.15 Pool the two eluates to make a total volume of approximately 200 μ L.
11.2.16 Transfer 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 minimum volume that can be accurately read by the spectrophotometer (see manufacturer’s recommendation) however, this may reduce th e DNA concentration to a level that can not be accurately read by the spectrophotomete r. If available, readings can be taken of 2 μ L aliquots of the sample with a NanoDropTM Spectrophotometer.

11.2.17 Sample is acceptable as a standard if ratio of OD 260 /OD 280 readings is > or = to 1.75.
11.2.18 Calculate total DNA concentration in sample by formula:

OD 260 reading x 50 ng/ μ L DNA/OD 260 11.2.19 Transfer sample back to labeled 1.7-mL non-retentive microcentrifuge tube and store at -20 ° C.
11.3 Extraction of E. 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 μ L 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 singl e BioBallTM to each of 3, 100-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 extr action tube with glass beads. Prepare one filter for each sample to be extracted in this manner.Method A

1 6 11.3.4 Dispense 590 μ 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 μ L AE buffer (Section 7.11) to the 10 μ L 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 μ L onto the polycarbonate filter in a calibrator sample tube.
11.3.7 Tightly close the tube, making sure that the O-ring is seated properly.
11.3.8 Repeat Sections 11.3.6 and 11.3.7 for the ot her two filters to prepare three calibrator samples with E. faecalis

. 11.3.9 Place the tubes in the mini bead beater a nd shake for 60 seconds at the maximum rate (5000 rpm).
11.3.10 Remove the tubes from the mini bead beat er and centrifuge at 12,000 × g for one minute to pellet the glass beads and debris.
11.3.11 Using a 200 μ L micropipettor, transfer the crude supernatant to the corresponding labeled sterile 1.7-mL microcentrifuge tube. Transfer 400 μ L of supernatant without disrupting the debris pellet or glass beads at the tube bottom. Note : The filter will normally remain intact during the bead b eating and centrifugation process. Generally, 400 μ L of supernatant can be easily collected . Collect an absolute minimum of 100
F L of supernatant.
11.3.12 Centrifuge at 12,000 × 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 1x 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 F L aliquot of each 1x E. faecalis calibrator extracts and dilute each with 200 μ L AE buffer (Section 7.11) to make 5 fold dilutions. In appropriately labeled tubes using a micropipettor, add a 50 F L aliquot of each 5 fold dilution and dilute each with 200 μ L 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. For long term storage, freeze at -20 ° C or -80 ° C.
11.4 Water sample filtration

Note : It is required that one water sample filtrati on blank (method blank) be prepared for every 6 water samples (Section 9.3) analyzed by the same procedure.
11.4.1 Place a polycarbonate filter (Section 6.14) on the filter base, 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 been pulled through the filter. Turn off the vacuum and remove the funnel from the filter base.Method A

1 7 11.4.4 Label an extraction tube with glass beads (S ection 7.20) to identify water sample. Leaving the filter on the filtration unit base, fold into a cylinder with the sample side facing inward, being careful to handle the f ilter only on the edges, where the filter has not been exposed to the sample. Insert th e rolled filter into the labeled extraction tube with glass beads. Prepare one filte r for each sample filtered in this manner.

11.4.5 Cap the extraction tube. Tubes may be frozen at -20 ° C or -80 ° C until analysis.
11.5 DNA extraction of water sample filter extracts and method blanks
11.5.1 Using a 1000 μ L micropipettor, dispense 590 μ L of the Salmon DNA/extraction buffer (Section 7.11) to 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 × g for 1 minute to pellet the glass beads and debris. Note : To further prevent contamination, a new pair of gloves may be donned for steps 11.4.5, 11.4.6, and 11.4.7 below.
11.5.5 Using the 200 μ L micropipettor, transfer 400 μ L of the supernatant to corresponding labeled sterile 1.7-mL microcentrifuge tube, taking care not to pick up glass beads or sample debris (pellet). Note : The filter will normally remain intact during the bead beating and centrifugation process. Generally, 400 μ L of supernatant can easily be collected. Collect an absolute minimum of 100 F L of supernatant. Recover the method blank supernatant last.
11.5.6 Centrifuge crude supernatant from Section 11.4.5 for 5 minutes at 12,000 × g. Transfer 350 μ L of the clarified supernatant to another 1.7-mL tube, taking care not to disturb pellet. Note : pellet may not be visible in water samples. Recover the method blank supernatant last.
11.5.7 Label the tubes from Section 11.4.6 as undilute d or 1x water sample extracts with sample identification. These are the water sample f ilter extracts. Also label tubes for method blanks. Label additional 1.7-mL tubes for 5 and 25 fold dilutions. In appropriately labeled tubes, using a micropipettor, add a 50 F L aliquot of each 1x water sample extract and dilute each with 200 μ L AE buffer (Section 7.11) to make 5 fold dilutions. In appropriately labeled tubes using a micropipettor, add a 50 F L aliquot of each 5 fold dilution and dilute each with 200 μ L of AE buffer to make 25 fold dilutions. Dilute the method blank supernatant last
11.5.8 Store all diluted and undiluted extracts in refrigerator. Note: Use of 5 fold diluted samples for analysis is currently recomme nded 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.
11.5.9 If the extracts are not analyzed immediately, refrigerate. For long term storage, freeze at -20 ° C or -80 ° C.
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 ofMethod A

18 15 minutes prior to rinsing with sterile water. If available, turn on UV light for 15 minutes. After decontamination, discard gloves and replace with new clean pair.
11.6.2 Remove primers and probe stocks from the freezer and verify that they have been diluted to solutions of 500 μ M primer and 100 μ M probe.
11.6.3 Prepare working stocks of Enterococcus , and Salmon DNA (Sketa 22) primer/probe mixes by adding 10 μ L of each Enterococcus or Salmon DNA (Sketa 22) primer stock and 4 μ L of respective probe stock to 676 μ L 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 2

. Table 2. PCR Assay Mix Composition
Reagent Volume/Sample (multiply by # samples to be analyzed per day)

Sterile H 2 O 1.5 μ L

BSA 2.5 μ L
TaqMan® master mix 12.5 μ L
Primer/probe working stock solution 3.5 μ L

Note : This will give a final concentration of 1 μ M 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 nega tive 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 pul se microcentrifuge to coalesce droplets. Return the primer/probe working stocks and other reagents to the refrigerator.

11.7 ABI 7900 & ABI 7500 (non-Fast) qPCR assay preparation (Reference 17.1)

Transfer 20 μ L of mastermix containing Enterococcus primers and probe to wells of a 96-well PCR reaction tray equal to number of samples to be analyze 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 it doesn’t make contact with anything else). Example: For the analysis of 18 recreational water sa mples, 51 wells will require the addition of assay mix with Enterococcus primers and probe as follows: 18 samples, two replicates each (36), 3 method blanks, two replicates each (6), 3 no te mplate controls, one replicate each (3), and 3 calibrators, 2 replicates each (6) = 51 wells.
11.7.1 Transfer 20 μ L of mastermix containing Enterococcus primers and probe to wells of a 96-well PCR reaction tray equal to number of sa mples to be analyzed including calibrator and negative control samples. Pipette into the center of the wells, taking care to not touch the well walls with the pipette tip. (Note: Th e 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).Method A

19 11.7.2 When all wells are loaded, cover tray loosel y with aluminum foil or plastic wrap and transfer to refrigerator or directly to th e 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 Transfer 5 μ L each of the diluted (or undiluted) DNA extracts of method blanks and water samples (Section 11.5.7), and then 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.
11.7.4 Transfer 5 μ L each of the diluted (or undiluted) DNA extracts of method blanks and water samples (Section 11.5.7), and corres ponding 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.
11.7.5 Transfer 5 μ L aliquots of AE buffer to wells of PCR reaction tray containing Enterococcus master mix that are designated as no-te mplate controls. Record positions of these samples.
11.7.6 Tightly cap wells of PCR reaction tray containing samples or cover tray and seal tightly with optical adhesive PCR reaction tray.
11.7.7 Run reactions in ABI 7900 & ABI 7500 (non-Fast) sequence detector. For platformspecific operation see Appendix A.

11.8 Smart Cycler® qPCR assay preparation
11.8.1 Label 25 μ L Smart Cycler® tubes with sample identifiers and assay mix type (see Section 11.7.8 for examples) or order tubes in rack by sample number and label rack with assay mix type. It is recommended that the unloaded open SmartCycler® tubes be irradiated under ultraviolet light in a PCR cabinet for 15 minutes. Using a micropipettor, add 20 μ L of the Enterococcus assay mix (section 11.6.5) 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 μ L of AE buffer to no-template control tubes and close tubes tightly.
11.8.3 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.
11.8.4 Transfer 5 μ L each of the diluted (or undiluted) DNA extracts of method blanks and water samples (Section 11.5.7), and then corresponding dilutions of calibrator samples (Section 11.3.13) to tubes containing Enterococcus and Salmon DNA (Sketa 22) mixes. Close each tube tightly after adding sample. Load the method blank PCR assays last . Label the tube tops as appropriate.
11.8.5 When all Smart Cycler® tubes have been loaded, place them in a Smart Cycler® centrifuge, and spin for 2-4 seconds.
11.8.6 Inspect each tube to verify th at 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 chambe r has not been properly filled, carefully open and reclose the tube, and re-centrifuge). Transfer the tubes to the thermocycler.Method A

20 11.8.7 For platform-specific operation see Appendix B.
11.8.8 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 3 and 4 , below.

Table 3. Calibrator PCR Run - 14 Samples
Sample Description* Quantity PCR Assay Master Mix
3 Calibrators × 2 replicates (1, 5, or 25 fold dilution ) 6 Enterococcus
3 Calibrators × 2 replicates (1, 5, or 25 fold dilution ) 6 Salmon DNA
No template controls (reagent blanks) 2 Enterococcus * Diluted equivalently to the water samples

Table 4. Water Sample PCR Run - 14 Samples
Sample Description* Quantity PCR Assay Master Mix
Water samples, (1, 5, or 25 fold dilution ) 6 Enterococcus
Method blank, (1, 5, or 25 fold dilution ) 1 Enterococcus
Water samples, (1, 5, or 25 fold dilution ) 6 Salmon DNA
Method blank, (1, 5, or 25 fold dilution ) 1 Salmon DNA * Use of five-fold diluted samples for analysis is curr ently 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.



## 12.0 Data Analysis and Calculations

12.1 Overview: This section describes a method for determ ining 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 ΔΔ C T comparative cycle threshold calculation method. The ΔΔ C T relative quantitation method also normalizes these ratios for differences in total DNA recovery from the test and calibrator samples using qPCR analysis C T values for a reference sequence provided by the SPC DNA. These ratios are converted to absolute measurements of tota l target sequence quantities recovered from the test samples by multiplying them by the average total number of target sequences that are normally recovered from a constant number of target organisms that are added to all calibrator samples. The complete procedure for determining target sequence quantities in water samples is detailed below.

12.2 Generation of C T value vs. target sequence number standard curve: Three replicate serial dilutions of a DNA standard, prepared as described in section 9.6 should be prepared to give concentrations of 4 x 10 4 , 4 x 10 3 , 4 x 10 2 , 2 x 10 2 and 1 x 10 2 lsrRNA gene sequences per 5 μ L (the standard sample volume added to the PCR reactions) and the replicates of each dilution pooled. Note: a procedure for the determinations of ta rget sequence concentrations in the DNA standard is also provided in Section 9.6.Method A

21 Aliquots of each of these dilutions should be stored at 4 o C in low retention microcentrifuge tubes and can be reused for repeated qPCR analyses. QPCR analyses of these diluted standards using the Enterococcus primer and probe assay should be performed at least three separate times in duplicate. C T values from these composite analyses should be subjected to regression analysis against the log10-transformed target sequence numbers per reaction as described in Section 9.6 with example results illustrated in Figure 1

.

y = -3.4777x + 38.422
R 2 = 0.9951

20 22 24 26 28 30 32 34

12345 Log target sequences per reactionCycle Threshold (CT)

Figure 1: Example plot and regression analysis of qP CR analysis cycle threshold values vs. log target sequences per reaction.

Amplification factors (AF) used for subsequent comparative cycle threshold calculations (Section 12.4) can be calculated from the slope value of th is curve by the formula AF = 10^(1 / (-)slope value). An example calculation using the sl ope value from the example regression is shown below: AF = 10^(1 / 3.4777) = 1.94
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 freezerstored aliquots of each cultured E. faecalis stock suspension that is prepared as described in Section 11.1. Dilutions of each of these calibrato r 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 C T 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 μ L of extract used in the reactions. An example calculation using an average calibrator extract C T value of 25.21 is shown below: Average calibrator target sequences/5 μ L extract = 10^((25.21-38.44) / -3.477) = 6382

Note : Four places should be kept from this calculation for the following calculation ( i.e
., 6382.6983). Dividing this value by 5 gives the average calibrator target sequences/ μ L extract which can be multiplied by the total volume of th e extract at the applicable dilution level ( e.g., 600 μ L of original extract volume x 5 = 3000 μ L 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 calibrato r target sequences/reaction value determined immediately above is shown below:Method A

22 Average target sequences = 6382 target sequences x 3000 μ L total extract volume

Calibrator extract 5 μ L extract

= 3,829,619 12.3.1 Calculation of average target sequence rec overy 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 (References 17.4). Calculations performed to obtain this reporting unit are identical to those described in Section 12.4 except that the ratios of target sequences obtained as described in Sections 12.4.1 - 12.4.4 are multiplied by the estimated quantities of Enterococcus cells added to the calibrator samples rather than by the average target sequences recovered per calibra tor extract as described in Section 12.4.5. While the use of this reporting unit is no longer recommended because of the false impression it creates concerning the cell concentration detection limit of the qPCR method , it still may be of value for comparing previous results with those of future studies. 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. For the purpose of determining this ratio, it is reco mmended that the cell concentrations of the cultured E. faecalis stock suspension used for the preparation of calibrator samples in each laboratory be determined by at least two of the three alternative methods described in Section 11.1 to establish the degree of agreement between these enumeration methods. The recommended quantity of cells that are a dded to each calibrator sample is 100,000. Dividing the average target sequences recove red 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 calcu lation using the average target sequences/ calibrator extract value determined in Section 12.3 is shown below: Ratio of target sequence numbers to cell numbers = 3,829,619 / 100,000 = 38.29

12.4 Calculation of target sequence quantities in test samples: A minimum of three fresh calibrator samples should be extracted and analyzed at l east on a weekly basis and preferably on a daily basis in association with each batch of water sample filtrates. QC analysis of the analysis results from these calibrator extracts shoul d be performed as described in Section 9.7. CT values from the Enterococcus target sequence and salm on DNA Sample Processing Control (SPC) qPCR assays for both the calibrator and test samples are used in the ΔΔ C T comparative cycle threshold calculation method to determine the ratios of target sequences in the test and calibrator sample extracts and these ratios are converted to abso lute measurements of total target sequence quantities recovered from the test samples as specified below and illustrated in Table 5

. 12.4.1 Subtract the SPC assay C T value (C T,SPC ) from the target assay C T value (C T,target ) for each calibrator sample extract to obtain Δ C T value and calculate the average Δ C T value for these calibrator samples.

12.4.2 Subtract the SPC assay C T value (C T,SPC ) from the target assay C T value (C T,target ) for each water sample filtrate extract to obtain Δ C T values for each of these test samples. Note : if multiple analyses are performed on th ese samples, calculate the average Δ C T value.
12.4.3 Subtract the average Δ C T value for the calibrator samples from the Δ C T value (or average Δ C T value) for each of the test samples to obtain ΔΔ C T values.Method A

23 12.4.4 Calculate the ratio of the target sequences in the test and calibrator samples using the formula: AF^ (- ΔΔ CT ), where AF = amplification factor of the target organism PCR 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 average target sequences/calibrator extract, determined as described in Section 12.2, to determine absolute numbers of total target sequences/ extract 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 (Amplification factor = 1.94)

Target sequences in Sample Sample Type C T,target C T,SPC Δ C T ΔΔ C T Measured Target Sequences in Test Sample Extract (1.94 - ΔΔ CT × avg. target sequences/calibrator)

3,829,619 Calibrator 25.21 30.45 -5.24

---- ----
Unknown Test 32.53 30.65 1.88 7.12 0.0089 x 3,829,619 = 34,198

12.4.6 The geometric mean of the measured target sequences and associated coefficients of variation in multiple water samples can be determined from individual sample C T values using the following procedure:
12.4.6.1 Use Δ C T value for each individual water sample extract and the mean calibrator Δ C T value to calculate the measured target sequence numbers in each water sample extract, as described in Section 12.4.
12.4.6.2 Calculate the log 10 of the measured target sequence numbers 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 10 M

. 12.4.6.5 The implied coefficient of variation (CV) is calculated, based on the log normal distribution, as the square root of 10 V/ 0.434 - 1, where V = S 2

. 12.5 Reporting Results : Where possible, duplicate analyses should be performed on each sample. Report the results as Enterococcus (large subunit ribosomal RNA gene) target sequences per volume of water sample filtered.



## 13.0 Sample Spiking Procedure [This section will be updated after validation study]



## 14.0 Method Performance

14.1 Accuracy (Bias) The lsrRNA gene (23 S rRNA) of Enterococci, which contains the target sequence amplified and detected by the primers and probe of the Enterococcus qPCR assay, is present in multiple copies in the genome of the different Enterococcus species. Enterococcus faecalis and E. faecium each contain 4 and 6 copies of the gene, respectively. The number of lsrRNA gene copies per genomeMethod A

24 has not been ascertained for all of the Enterococcus species which the Enterococcus qPCR assay can amplify and detect. Hence, the use of E. faecalis cells as a calibrator for relative quantitation purposes and E. faecalis DNA as a standard for absolute quantitation purposes creates an inherent bias potentially affecting the accuracy of the quantitation depending on the species composition of the Enterococcus present in a water sample.

The Enterococcus qPCR method makes the assumption that the Enterococcus cells present in the water sample contain the same number of genomes and lsrRNA gene copies as the E. faecalis calibrator cells which have been grown in culture media to a late-log or stationary phase in batch culture. This assumption has not been validated if untrue may bias the accuracy of the results in a systematic manner. Bacterial cells contain mo re than one complete genome during growth and cell division phases of their life cycle. The num ber of genomic copies de pends on their growth rate and cell division time. More than one ce ll division cycle is often required to complete replication of the genome during rapid log-phase gr owth and cell division. The lsrRNA genes are replicated early in the cell cycle maximizing th e number of lsrRNA gene copies present in cells during log phase growth. This facilitates the e nhanced ribosome production needed for the high level of protein translation needed during rapid cell growth and division. [This section will be updated after validation study]



## 15.0 Pollution Prevention

15.1 The solutions and reagents used in this me thod pose little threat to the environment when recycled and managed properly.
15.2 Solutions and reagents should be prepared in vol umes consistent with laboratory use to minimize the volume of expired materials to be disposed.



## 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 benc h operations. Compliance with all sewage discharge permits and regula tions is also required.
16.2 Samples, reference materials, and equipment know n 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 Ch emical Society’s Department of Government Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.Method A

25 

## 17.0 References

17.1 Anonymous. 1997. User Bulletin #2. ABI Pris m 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 quan titative is quantitative PCR with respect to cell counts? System Appl. Microbiol. 23: 556-562.
17.6 USEPA. 1983. Health Effects Criteria for Ma rine Recreational Waters. EPA-600/1-80-031.
17.7 USEPA. 1984. Health Effects Criteria for Fr esh Recreational Waters. EPA-600/1-84-004.
17.8 USEPA . 1986. Ambient water quality criteri a for bacteria - 1986. EPA 440/5-84-002.
17.9 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. Environmenta l Health Perspectives 114: 24-28.Method A

2 6 

## 18.0 Acronyms

ΔΔ C T comparative cycle threshold calculation method AF amplification factor BHIA brain heart infusion agar BHIB brain heart infusion broth BSA bovine serum albumin CFU colony forming units DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid lsrRNA large subunit ribosomal RNA NTC no template control PBS phosphate buffered saline PCR polymerase chain reaction qPCR quantitative polymerase chain reaction SPC sample processing control TNTC too numerous to count UV ultraviolet (light)Method A

27 

## Appendix A: ABI 7900 and ABI 7500 Sequence Detector OperationMethod A

A -1 

## Appendix A - ABI 7900 and ABI 7500 (non-Fast) Sequence Detector Operation

1.0 ABI 7900 Sequence Detector Operation

1.0.1 Turn on the ABI Model 7900 sequencer 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.
1.0.2 Under File menu, select New

. 1.0.3 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.
1.0.4 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.
1.0.5 Above right hand window, click on Setup tab.
1.0.6 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 1.6.1. Once the detector has been created, go to step 1.7.
1.0.7 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 1600"). 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 1.7)
1.0.8 In pop-up window that was opened in step 1.6, select the desired detector under Names menu ( e.g., Method 1600) and click on Copy to Plate Document button. Click on Done button to return to setup screen.
1.0.9 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 a nd 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.
1.0.10 Click on Instrument tab right han d window.
1.0.11 In instrument screen, change sample volume to 25 μ L and choose 9600 emulation

. 1.0.12 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.
1.0.13 Insert PCR reaction tray with prepared reactions in holder.
1.0.14 Click on Open/Close button to close PCR reaction tray holder door on instrument.
1.0.15 Click on Start button in lower right hand “Real Time ” window to start thermal cycling in instrument.Method A

A -2 1.0.16 Name run file at prompt.
1.0.17 At termination of the run, instrument-calculated cycle threshold values should automatically appear for each well position a nd detector entry in the lower left hand results table window.
1.0.18 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 1.18). Click on OK . Click on Analyze from the toolbar. You should see Ct values in the Results Table.
1.0.19 The instrument-selected threshold line is indicat ed 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.
1.0.20 Calculated C T 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 clic king 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 C T values for all selected tray positions for this detector.
1.0.21 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

. 1.1 ABI 7500 (non-Fast) Sequence Detector Operation
1.1.1 Turn on the ABI Model 7500 sequencer and th en 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.

1.1.2 Click on either the New Experiment or Advanced Setup button to create a new experiment, which will pull up the Experiment Menu.
1.1.3 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.
1.1.3.1 Click on 7500 (96 Wells) to select the instrument type. Note : This protocol is not designed for the 7500 Fast.
1.1.3.2 Scroll down to access more experiment properties options. Click on Quantitation – Standard Curve to select the experiment type
1.1.3.3 Click on TaqMan® Reagents to select the reagents used.
1.1.4 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).
1.1.4.1 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.Method A

A -3 1.1.4.2 Repeat the process in Section 1.1.4.1 to add more than one target, e.g. , Sketa.
1.1.4.3 In the Define Samples section, click on Add New Sample and enter the sample name in the Sample 1 box.
1.1.4.4 Click on the Assign Targets and Sample tab to assign the Target, sample ID and sample type to the wells.
1.1.4.5 Drag the mouse over the desired cells to assign the Target, Task and Sample.
1.1.4.6 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.
1.1.4.7 In the Assign sample(s) to the selected wells section, check the box under Assign to label the highlighted cells as Sample 1 ( e.g. ).
1.1.4.8 Repeat the process in Section 1.1.4.4, dragging the mouse over the appropriate cells, and then assigning them as U nknowns, S tandards or N egative Controls, as appropriate. Note that for the 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. 40000.0) typed in the Quantity box.
1.1.4.9 Click on Print Report at the top of the screen to print a plate layout for loading master mix and sample extracts.
1.1.4.9.1 Select Plate Layout option by clicking on the box next to it.
1.1.4.9.2 Click Print Report to print the plate layout.
1.1.5 From the Setup menu, select Run Method to set up the thermo cycling profile.
1.1.5.1 In the Graphical View tab, change the Reaction Volume Per Well from the default 50 μ L to 25 μ L by typing in 25.
1.1.5.2 Under Cycling Stage section, change the Number of Cycles to 45.
1.1.5.3 Check that the default setti ngs of Tem p erature 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.
1.1.5.4 The second step of the Cycling Stage is defaulted at 60.0°C for 1 minute – change the time to 2:00 minutes.
1.1.5.5 Click on Save at the top of the screen to save the run before actually running the experiment.
1.1.5.6 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

. 1.1.6 From the Experiment menu, select Run to monitor the run in real time.
1.1.6.1 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.Method A

A -4 1.1.7 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.
1.1.7.1 The Analysis Settings for Experiment name screen will open.
1.1.7.2 Under the C T Settings tab, click on the Target desired to be set from Default to Manual Threshold setting (generally, this would be the Ent).
1.1.7.3 In the C T 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.
1.1.7.4 Click on Apply Analysis Settings to save the changes.
1.1.8 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. Note th at the threshold value will have changed on the Amplification curve graphic.
1.1.8.1 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).
1.1.8.2 For Select one file or separate files , select One file to export all the data into one file with multiple tabs.
1.1.8.3 For Export File Name , enter the name of the Experimental data file to be exported, and for File Type , choose *.xls.
1.1.8.4 For Export File Location , click on Browse to select the folder in which you wish to export your file.
1.1.8.5 Click on Start Export . When this is completed, an Export Completed screen will open – click on Close Export Tool to complete this task.
1.1.9 How to interpret your results. See “How to Interpret Results from the ABI 7500” for screen shots.
1.1.9.1 After exporting your data, from the Experiment menu, select Analysis.
1.1.9.1.1 From the Analysis menu, select Amplification Plot to view the results of the selected samples at different plot settings..
1.1.9.2 Drag the mouse over the desired cells to select the samples to be plotted in the amplification plot.
1.1.9.3 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).
1.1.9.4 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.
1.1.9.5 For the Standard Curve, select Standard Curve fro m the Analysis menu to view the results of the same sample (assuming they have the same target) and standards in the plot.Method A

A -5 1.1.9.6 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 one target), and click the drop down menu arrow beside Plot Color to change the plot color.
1.1.9.7 The parameter display below the plot ( i.e. Target, Slope, YInter(cept), R 2 , and Eff% (percent efficiency) is useful for data analysis.
1.1.9.8 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 the wells, target, or dye.
1.1.9.9 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).
1.1.9.10 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.
1.1.9.11 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.
1.1.9.12 For quality controls, click on QC Summary from the Analysis menu to view the analysis summary of the samples.
1.1.9.13 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 Sections 1.1.12-1.1.15.
1.1.10 To print the report, click on Print Report at the top of the screen.
1.1.10.1 Check all of the boxes ( i.e. , Experimental Summary, Results Summary, Plate Layout, Amplification Plot (3 boxes), Standard Curves, Results Table, and QC Summary).
1.1.10.2 Click on Print Report at the bottom of the screen.Click either button tocreate new experimentHow to Set Up a New Experiment Using the ABI 75002. Enter theExperiment Name1. Click “Experiment Properties” toselect the experiment type and givean experiment nameClick the type of instrumentused - in this case it is the7500 (96 wells) plateScroll down for moreexperiment propertiesoptionsClick to selectexperiment typeClick on TaqMan® Reagents toselect the reagents used2. Click “Define Targets and Samples” tab to definethe reporter-quencher dye for each target and alsoenter sample IDs.1. Click on“Plate Setup”1. Click to AddNew Target2. Enter theTarget Name3. Select the Reporterdye FAM from thedrop down menu4. Select the QuencherTAMRA from the dropdown menuYou can add more than one target- usuallyper target DNA (ENT, SKETA)Select to Add a New SampleEnter the name of thesampleClick to designate the target, sampleID and sample type to the wellsDrag the mouse over thedesired cells to assign theTarget, Task and Sample IDWhile the cells are highlighted,check the desired Target anddesignate whether the sample is anU (Unknown), S (Standard) or N(Negative Control)Assign the sample thatis to be analyzed in thehighlighted cell(s)To designate standards,highlight the desired cells, addthe target and select the “S” forStandardType in a Quantity for eachstandard - if there are 4different values, highlighteach cell independently andfill in the quantity2. Click on “Print Report” to print a platelayout for loading master mix and sampleextracts1. Select “Plate Layout” option byclicking in the box next to it2. Click “Print Report”to print the platelayout.Select “Run Method” to setup the thermo cycling profile1. Change the Reaction Volume PerWell from the default of 50uL to 25uL2. Change the Number of Cycles to 454. In the data collection step,change the timeframe fromthe default 1 minute to 2minutes3. Confirm that the default Temperatureand Time settings in both HoldingStages and in step 1 of the CyclingStage are as displayed – if not, resetthem1. Click to save the runbefore starting the run2. Select the folder forsaving the Run Data file3. Enter the experiment name if not entered at thebeginning of the experiment setup and click “Save”or if already have the appropriate experiment name,without making any changes click “Save”Click “Run” to monitor the run in real-timeClick to start the run. Thegreen box will turn red whenthe run has been startedsuccessfullyClick on the “Analysis” tab in the Experiment Menuto access the “Analysis Settings”How to Export Results from the ABI 7500 Software1. To change the threshold setting fromAutomatic to Manual, click on “AnalysisSettings”2. Under the “CTSettings” tab,click on the Target desired tobe set from Default to ManualThreshold setting3. Uncheck the box next to“Use Default Settings”Uncheck the box next to“Automatic Threshold” toenter the desired thresholdEnter the desired Threshold.*If more than one target is present, highlight the nexttarget and repeat this processSelect “Apply Analysis Settings” to savethe changesClick on “Export” to save experiment resultsdata and to save the selected plots requiredin the reportNote that the threshold value has changed on the Amplification PlotCheck the USE boxes next to theCheck the boxes nextto the desired results toexportFrom the drop-down menu select“One File” to export all data into asingle file with multiple tabsEnter the name of theexperiment data file to beexportedSelect *.xls (Excel) from the “File Type”drop-down menuClick on “Browse” to select thefolder / location to export the FileClick on “Start Export”to export the data to thedetermined locationA pop-up window “Export Completed” appearsonce the exporting of the file is completed.Click on “Close Export Tool” toclose the export window oncedata file exporting is completed.Select “Amplification Plot” to view the results ofthe selected samples at different plot settingsHow to Interpret Results From the ABI 75002. Select the“Plot Type”from the dropdown menu3. From the “Graph Type”drop down menu select eitherLog or Linear1. Drag the mouse over thedesired cells to select thesamples to be plotted in theamplification plotAnalysis Menu- Amplification PlotIf the plot requiresthe display of thethreshold andbaseline select bychecking the “USE”boxes in “Show”next to Thresholdand BaselineSelect “View Well Table” to viewthe results in a tabular formatScroll down or sidewaysfor more samples andanalysis parametersAnalysis Menu- Amplification PlotSelect “Standard Curve” to view the results ofthe samples (if same target) and standards in theplotAnalysis Menu- Standard CurveStandard curveparameters maybe useful in dataanalysisClick here to select a different Target (if thestandards were analyzed in more than one target)or change the Plot ColorAnalysis Menu – Standard CurveSelect “Multicomponent Plot” to view the variation in thefluorescence of the wells, targets or dyes used over theascending repeat of the 45 thermo cyclesClick on the “Plot Color” drop down menu to select theparameter based on which the graph required to be plottedAnalysis Menu – Multicomponent PlotSelect “Raw Data Plot” to view the variation in the emission offluorescence of the dyes in different filters present over theascending repeat of the 45 thermo cyclesDrag the pointer on thecycle scale to the desiredcycle number to view thevariation in the fluorescencethrough filters of thesamples at that cycleAnalysis Menu – Raw Data PlotSelect “QC Summary” to view theanalysis summary of the samplesin the experimentAnalysis Menu – QC SummarySelect “Multiple Plot View” to view all the plots for thesamples in one windowEach plot has a drop downmenu located on the top toaccess various parametersrequired to displayed andalso has the toolbar featurewith various options ofprinting, saving, and otherparametersAnalysis Menu – Multiple Plots ViewClick on “Print Report” to make a hardcopy of the results with required plotsAnalysis – To Print a Data Report1. Select the required data andplots to be printed by checkingthe boxes next to the data andplot types in which results can beprinted2. Click “Print Report” toprint the required dataMethod A



## Appendix B : Cepheid Smart Cycler® OperationMethod A

B -1 

## Appendix B - Cepheid Smart Cycler® Operation

1.0 Smart Cycler® Operation

1.1 This protocol is intended to provide only informa tion about critical instrument settings required to perform EPA Method A. Further details concer ning the operation of the instrument can be obtained from the Smart Cycler® Operati on 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 th ese 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 TaqMan Method" 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 Secs 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 r eactions using the Amp-Erase® UNG enzyme provided in the TaqMan® Universal PCR Master Mix. Note that 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 Secs column enter 600. Again, leave the Optics setting on Off. ( Note : This stage is performed to inactivate the AmpErase® 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 Secs column, Optics column Off

. For the second step, enter 60.0 in the second row of the Temp colum n, 120 in the Secs column, and click on the Optics cell to select On from the drop-down menu. 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 μ L reaction).Method A

B -2 1.7 Click the Add/Remove Sites button. The Select Protocols and Sites... dialog will appear. Highlight (click on) the “EPA Method A" 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 us ing 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 m e nu.
1.14 At the end of the run, it is recommended to ch eck the cycle threshold values calculated by the instrument for each sa mple 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 si ngle horizontal red line and the cycle thresholds for each site are shown as vertical red lines. To vi ew 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 visibl e 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 adju sted 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 cha nges to the Results Table or Analysis Settings, and immediately print or export the data, then close th e run without saving the changes. In this case,Method A

B -3 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. Analysts will late save the file as an XLS file.

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 a line 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 analy s is (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 TaqMan Method 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.1. Click on “New Protocol”2. This screen will appear - type in the name of the new protocolHow to Set Up a New Experiment Using the Smart Cycler(Software Version 2.0)3. Click on OK when done1. New Protocol name will appear in listMake sure new protocol is highlightedand begin to choose stage settings2. Set the Temperature in the“HOLD” stage to 50.0 degrees and120 seconds.NOTE opticalreadings cannot be programmedduring a HOLD stageIn Stage 2, set the temperature to 95.0 degreesand 600 seconds1. In Stage 3, set the drop downmenu at “2-Temperature Cycle”to repeat 45 times (cycles).2. For the first temp cycleset the temp at 95degrees for 15 secondswith optics OFF.3. In the second Tempcycle, set the temp for 60degrees for 120 secondswith the optics ON4. Click Save Protocolwhen finishedTo set up automatic export of Raw Datainto computer, click on “SETUP”, then“System Defaults” then “Export Settings”How To Set Up Automatic Export of Raw Data1. Check Results Table and Analysis settings box3. Be sure that Automatic export on run completion is checked2. Choose browse toselect a folder wherethe raw data will exportto4. Click OK when finishedHow To Manually Export Raw DataOnce run is finished, click ExportCheck the Export Results Table and Analysis Settings boxthen click “Export”Choose the appropriate file folder to save the Raw Data toand click SAVE. NOTE: All raw data will export in the form ofa CSV file. Analysts will later save file as an “XLS” file.Right click on graph area and select “Print” then“Print Graph with Results Table”How to Print Run DataThis sheetwill pop up.Click“Print” toprint fileTo print the generated report, click on “Report”Click “Print”to print outthe generatedrun datareportClick “Create Run”How to Set Up a New RunClick “Add/Remove” Sites1. Be sure the correctprotocol is highlightedbefore selecting sites2. Highlight the amount ofsites needed for assay (A1-A16)Click “right” arrow tomove sites to the“Selections” columnSites have been moved toselections column. Doublecheck that the correct protocolis selected and click on “OK”The Sample IDs and run name can be entered at thispointUnder the “Usage” column, select “Unused” from thedrop down menu for Cy3, TxR and Cy5. Leave the“Assay” on the FAM line for this particular protocol1. Set the Manual ThresholdFluorescence Units to 8.0 on theFAM line2. When smart cycler tubes have been put in toappropriate sites, click “Start Run” - a red lightshould appear on sites that are in use