Quantitative Polymerase Chain Reaction (qPCR)

A More Rapid Test for the Enumeration of Bacteria in Water Outline

• What is qPCR? • qPCR terminology • Pre‐analytical considerations • qPCR methods for surface waters – E. coli and enterococci • Sample collection/processing • Results • Inhibition • Examples What is qPCR?

• an enzymatic reaction that makes many copies of target DNA fragments from a template (e.g. E. coli isolated from surface water). • Using PCR, specific sequences within a template (e.g. E.coli DNA) can be copied (amplified), to millions of copies. • With PCR; 2n copies of DNA can be amplified from a template where “n” is the number of cycles. • So, if there are 40 cycles of PCR, the yield would be 240 copies of DNA. What is qPCR (con.)?

• Quantitative Polymerase Chain Reaction (qPCR) combines the working principle of PCR with the use of a fluorescent labeled probe – an oligonucleotide (short nucleic‐acid chain usually consisting of up to approximately 20 nucleotides) complementary to the target DNA sequence. – Traditional qPCR uses staining to visualize and quantify a DNA target • http://users.ugent.be/~avierstr/principles/pcr. html Components of qPCR

• Sample DNA (template DNA), containing target DNA sequence that will be amplified (copied) • Heat stable DNA polymerase (i.e. Taq Polymerase) • Forward and reverse primers (short nucleotide sequences complimentary to target DNA) • Deoxynucleoside triphosphates (dNTPs; the building blocks of DNA, used to synthesize the new strand) – a phosphate group, the bases adenine, cytosine, guanine, and thymine, and a pentose sugar • Buffer solution that provides optimal pH conditions for the reaction to proceed • Mg2+ ions (to reduce synthesis error rate) Type of Fluorescent Probes Steps of qPCR

1. Denaturation (92‐98°C): This step occurs at a higher temperature causing a disruption of hydrogen bonding between the template DNA strands and separation of double stranded DNA into single‐stranded DNA. 2. Annealing (50‐65°C): Primers anneal (or bind) to the target DNA sequence, followed by annealing of Taq Polymerase to the target DNA sequence. 3. Elongation/extension (72‐80°C): Taq Polymerase synthesizes a new complimentary strand.

What Does qPCR Measure?

• The intensity of the fluorescence is measured by the thermocycler • It is proportional to the concentration target DNA in the sample. • At the end of the reaction, the amount/intensity of fluorescence is plotted against the number of cycles • This represents the accumulation of DNA product over the duration of the entire PCR reaction. Remember qPCR is not Measuring the Same Thing as a Culture… • qPCR differs from traditional culture‐based assays in that it measures all DNA: – live cells – dead cells – non‐culturable cells – free DNA • Culture assays only measure cells possessing the ability to grow on the selective media you are using • Water body characteristics and environmental conditions may effect • Inhibition • Culture/qPCR relationship • Ability to use qPCR as an analytical method qPCR Terminology

• Precision – Are your results reproducible • Accuracy – False negatives? – False positives? – What is the gold standard? • Variability – Confidence in your result • Reproducibility – Inter‐ and Intra‐analyst variability • Limit of Detection – Lowest quantity that can be distinguished from background levels • Inhibition – Inability to detect or underestimation of your target • Equivalence – How do your results compare to ‘conventional’ fecal indicator bacteria data/historical data  In order to set up qPCR laboratory that works for your application, consideration to all of the above will be necessary Physical Space Requirements

• Segregated work spaces – Sample filtration/DNA extraction – Preparation of master mix – Sample analysis • Washable surfaces – Non‐porous – UV disinfection, bleach • Unidirectional workflow • Dedicated equipment • Dedicated PPE • Proper disposal of DNA waste Choice of Molecular Target

• Site Specific Suitability – E. coli or enterococci for fresh water – Enterococci for marine water • Wet chemistry or beads – Assay time – Precision/accuracy • Sample DNA extract ‐ pre‐treatment? – Crude – Serial Dilution – DNA extraction or purification kits US EPA Method 1611: Enterococci

• Uses probes/primers designed to target 16S DNA of enterococci. [Salmon DNA used as a processing control]. • TaqMan System (Applied Biosystems) – Universal PCR Master Mix : AmpliTaq Gold DNA Polymerase, dNTPs with dUTP, and optimized buffer components. – Add probes/primers, PCR grade water and bovine serum albumin (BSA). • Run time = 1:54 (Total time = 4 hours) • Cost per test = $ 12.00 ‐ $15.00 Cepheid CSR for Enterococci/ E. coli • Assays feature Customer Specific Reagents (CSRs) / Smartbeads™ (Cepheid) • Lyophilized bead technology • QPCR reactions only require tubes and PCR grade water • Omnimix™ beads contain all PCR reagents, Smartbeads™ contain primers, probes, and internal controls. • Reduces pipetting steps and errors, and interanalyst variability. • Run Time = 0: 54 (Total time = 3 hours) • Cost per test = $30 QA/QC • What QC samples should be run? – Method Blank (contamination during sample filtration) – Filter Blank (contamination in extraction process) – NTC (reagent contamination) – Calibrator (target quantification check) – SPC control (extraction and inhibition control) • Number/Frequency? • Definition of inhibition – >3 cycle threshold difference b/w cal‐SPC and unknown‐SPC (EPA definition) QA/QC

• Field replicates vs. sample replicates – Sample replicates = 2 sub‐samples from 1 DNA extraction • Detection in the negative controls – >35 CT in more than a single NTC – <45 CT in a third of NTC rxns for a single MM • Non‐agreement/non‐consensus – Replicate analyses should agree within 1 CT – When is it ok to average replicate CT values? – How do you know what the right answer is? • Reporting out “non‐detects” – Less than half the reciprocal of the dilution factor – What does that mean? Controls

• Calibrators and Controls – Lab‐prepared vs. commercial products – Precision (cal curves, replicate sample agreement) – Accuracy (calibrators, SPC) – Reproducibility (b/w run calibrator agreement) • Inter‐laboratory validation studies Creating a Standard Curve

• Standard Curve is created from analyzing known quantities of target, i.e. E. coli or enterococci • Unknown samples values are interpolated from standard curve

E.coli DNA Standard Curve (Fresh Cells, 2007)

6.00 5.00 4.00 3.00 y = -0.2668x + 12.185 FAM CT FAM 2.00 R2 = 1.0 1.00 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Log Cells Measured Sample Handling & Storage

• Storing supplies – Calibrators, Lab prepared cells @ ‐80 °C – Controls, SPC @ 4 –8 °C – DNA Reagents, product dependent – How long? • Stock salmon DNA was good for 5 years @ 4 °C • Lab prepared cell suspensions were good for 5 years @ ‐80 °C • Storing samples – Fresh, analyzed w/i 4 hours of collection – Filters, freeze @ ‐80 °C – DNA extracts, good for 7 days @ 4 °C (up to 1% loss/day) • Mailing samples and/or reagents – Degradation can occur if temp is not maintained Sample Collection

• Fixed monitoring stations • Fixed sampling frequency • Sampled at same time each day • Collected in a consistent manner • Keep good sampling records Sample Collection (con.)

• Carefully move to the first sampling location while wading slowly in the water, try to avoid mixing bottom sediment at the sampling site. • Wade out until you reach an approximate depth of 1 m. The sampling depth should approximately 6 to 12 inches below the surface of the water. • Open a sampling bottle and grasp it at the base with one hand and plunge the bottle mouth downward into the water to avoid introducing surface suspended material. • Position the mouth of the bottle into the current away from your hand. – Do not touch the inside of the sample container. – Do not put caps on the ground while sampling. • Remove the bottle from the water body and pour out a small portion to allow an air space of 2 cm for proper mixing of the sample before analyses. • Tightly close the cap and label the bottle. • Store sample in a cooler filled with ice or suitable cold packs immediately. • Note time, date, and location of sample collection, current weather conditions, water temperature, clarity, wave height and any abnormal environmental conditions. Steps in the Analytical Process

 Sample Processing 1. Sample handling 2. Membrane filtration 3. DNA extraction a. Bead beating (mechanical cell lysis) b. Kit extraction c. DNA purification  Reagent Preparation 1. Master mix preparation 2. Preparation of cuvettes/plates  QPCR analysis 1. Assay parameters 2. Setting the baseline

3. Quantification (converting CT values into a “result”)

Membrane Filtration DNA Extraction: Bead Beating

• Add filter to 2 ml tube containing 1mm beads, Buffer AE, and SPC • “Bead beat” for 2 minutes • Pellet cellular debris and beads • Collect supernatant material and make dilutions Watch the Video

• http://www.cws.msu.edu/videos/videos.php Threshold: The threshold (baseline) should be set in Results the region associated with an exponential growth of PCR product . It is assigned for each run to calculate the Ct value for each amplification.

CT (threshold cycle): Reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number. The Ct value assigned to a particular reaction thus reflects the point during the reaction at which a sufficient number of amplicons have accumulated. Results

• Units of measure = CCE (calibrator cell equivalents) • Relative difference calculated between CT value of calibrator and paired specimen processing control with that of the unknown sample and its processing control • Best done in an excel spreadsheet • See Method 1611 • The lower the CT higher the concentration of target DNA Results What do your QPCR results mean?

• Are the values the same as what you got with the culture‐based assay? – Should they be? – How do you interpret lack of correlation? – Is there a way to predict inhibition? • Would I make the same beach management decision using these values? – Same regulatory action? – Can I still use old techniques, i.e. composite samples? • What happens if I use enterococci instead of E. coli? • What about measuring water quality in aqueous environments other than coastal waters? What is qPCR Inhibition?

• Delay in target DNA amplification – increased Ct value=lower qPCR signal – Ct=0 (undetermined)

• Inhibitors may interfere with the qPCR by: – binding to and/or degrading target DNA; – interacting with Taq Polymerase and inhibiting its enzymatic activity; – preventing annealing of the probe and primers to the target DNA template

T. Anan’eva, GLBA 2011 Inhibition or Underestimation

• Chemical or inorganic constituents in the sample which prevent reaction from occurring – Humic substances – Non‐point source pollutants • Competition for target with bacteria‐rich samples • Other environmental conditions which result in no detection in the absence of inhibition – i.e. some algal blooms Frequency of Inhibition (Lab & Water Body Type)

20

15 Samples

10 Inhibited of

5 Percentage

0 Effluent Lake Michigan River Inland lake Lab A 0.99 0 9.17 17.14 Lab B 1.16 4 8.06 7.69 Lab C 0N/A 10.2 18.75

T. Anan’eva, GLBA 2011 Enterococci vs. Enterolert (2011)

Method 1600 qPCR Method 1600 vs. qPCR LOG10 Method 1600 LOG10 1 4.5 0.5615192 qPCR (Sk22) LOG10 43 1 4

3.5 ml 3 CCE/100 2.5 LOG10 2

1.5 Enterococci, y = 0.4775x + 1.8633 R² = 0.3153 1

0.5

0 0 0.5 1 1.5 2 2.5 3 3.5 Enterococci, LOG10 cfu/100 ml E. coli, Colilert‐18 vs. qPCR (2011) 1000

900 C‐18, PM qPCR, PM 800 C‐18, PM 1 qPCR, PM 0.903933 1 700 CCE) 600 or

(MPN

500 ml

C‐18, PM 100

per qPCR, PM 400 Units

300

200

100

0 Jul Jul Jul Jul Jul Jul Jul Jul Jul Jul Jul Jul Jul Jul ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ Sep Aug Aug Aug Aug Aug Aug Aug Aug Aug Aug Aug Aug Aug Aug Aug ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ 5 7 9 1 2 4 6 8 13 15 17 19 21 23 25 27 29 31 11 10 12 14 16 18 20 22 24 26 28 30 Date Enterococci, Bacteroides, and E. coli (2011) 6.0

5.0 ml

4.0 CCE/100

3.0 Enterococci LOG10

Bacteroides 2.0 E. coli

Concentration, 1.0

0.0 70111 70711 71211 71511 72011 72511 72811 80211 80511 81011 81511 81811 82311 82611 83111 90611 90911 91411 91911 92211 92711 93011

The use of enterococci would have resulted in significantly more exceedances.

For culture‐based assays, the use of enterococci would have resulted in 7 (IDEXX Colilert‐18 vs. Enterolert) to 13 (USEPA Method 1603 vs. 1600) more advisories based on proposed SVT values of 235 and 61 for E. coli and enterococci respectively.

For qPCR, the use of enterococci would have resulted in 10 additional exceedances based on a an SVT of 235 CCE/100 ml for E. coli (use of same criteria but alternative analytic method as per p. 55) and 1000 CCE/100 ml for enterococci. E. coli CCE/100 mL vs. Time of Sample (2011) 9

8

7

6

5 E. coli CCE/100 mL (Log Normalized) NB-AM qPCR Avg. Value 4 NB-PM qPCR Avg. Value 3

2

1

0 5 6 7 11 13 14 18 19 20 21 25 26 27 28 1 2 3 4 8 9 10 11 Date of Sample (July 5, 2011 - August 11) E. coli CCE/100 mL vs. Date of Sample (2011) 9 8 7 6 E. coli CCE/100 mL 5 (Log Normalized) 4 NB-AM qPCR Avg. Value 3 NB-PM qPCR Avg. Value 2 1 0 5 7 13 18 20 25 27 1 3 8 10 Date of Sample (July 5, 2011 - August 11) Implementation ‐ 2012 • Regulatory monitoring 5 days/week – Received approval as an ATP indicator/method combination in May 2012 – 2 beaches – E. coli by qPCR (BioGx Smartbeads™: E. coli and Sketa) – Compare to culture 4 days/week – Compare to model 5 days/week – Compare E. coli/enterococci by culture (qPCR to follow) • Archived filters (Fall 2012) Murphy’s Law…sort of

SITE Numerical Regulatory # Advisories #Advisories Correlation (r) Agreement E. coli ‐ Cx Alternative North Beach Colilert vs. 0.94 88% 5 9 Enterolert Colilert vs. 0.59 89% 6 12 qPCR Zoo Beach Colilert vs. 0.97 96% 3 5 Enterolert Colilert vs. 0.46 98% 6 7 qPCR Data Handling Reality Check • Replicate analyses do not always agree – Open, advisory, closed, coin toss (?) • Culture does not always agree with qPCR • Results do not always make sense based on environmental conditions • Inhibition • False negatives w/o inhibition (algae blooms?) • Filter blanks or NTC are contaminated • Contamination as a result of visitors/observers not wearing PPE Thank You!