Validation of qpcr Rapid Bacterial Quantification through Viable E. coli cell count in the Saginaw Bay Watershed

Validation of qpcr Rapid Bacterial Quantification through Viable E. coli cell count in the Saginaw Bay Watershed TYLER LEFEVRE ADVISOR: DR. TAMI SIVY 1

Overview Introduction to fecal coliforms Current detection methods qpcr rapid testing EPA Method C validation study The qpcr quantification issue Viability testing Future research 2

Fecal Indicator Bacteria Coliforms Indicator of water quality Characteristics: Rod shaped Gram-negative Non-spore forming Fecal Coliforms Indicator of fecal contamination Typically Escherichia coli Snyder, K. Genomes of Two Popular Research Strains of E. coli Sequenced https://www.bnl.gov/newsroom/news.php?a=11019 (accessed April 2, 2017). 3

Overview Introduction to fecal coliforms Current detection methods qpcr rapid testing EPA Method C validation study The qpcr quantification issue Viability testing Future research 4

Current Methods Idexx Colilert-18 Enzymatic, colorimetric Measures coliforms and fecal coliforms after overnight incubation period (18 hours) Faster methods being researched/validated qpcr rapid testing (3-4 hours) Michigan beach allowable threshold: 300 Colony Forming Units (CFU)/100 ml water Set by the Michigan DEQ 4-Methylumbelliferyl β-d-glucuronide (MUGlcU) idexx.com 5

Polymerase Chain Reaction (PCR) Annealing Extension http://www.mun.ca/biology/scarr/gr12-26.html 6

Polymerase Chain Reaction (PCR) Ethidium bromide staining Presence/absence (qualitative) http://www.madsci.org/posts/archives/1999-02/919869466.mb.r.html www.researchgate.net 7

Overview Introduction to fecal coliforms Current detection methods qpcr rapid testing EPA Method C validation study The qpcr quantification issue Viability testing Future research 8

Quantitative PCR (qpcr) Utilizes fluorescent probes to quantitate DNA Fluorescence monitored in real time Probe is hybridized to fit between primers http://www.sinobiological.com/real-time-pcr-service-gene-expression-analysis-by-qpcr-cro-service.html 9

Controls Blanks Known levels of Salmon DNA Standard curve used to calculate the number of 1S rrna sequence in calibrator Non Template Controls (NTCs) Duplicates Background dye (ROX) 10

qpcr Run Protocol Run time approximately 90 minutes Combined extension and annealing steps 11

qpcr Raw Data Cycle Threshold (C T ) Fluorescence α 1/ C T α [DNA] [DNA] Genome Equivalents (GE) 7 12

Overview Introduction to fecal coliforms Current detection methods qpcr rapid testing EPA Method C validation study The qpcr quantification issue Viability testing Results Future research 13

EPA Validation Study of Method C: Escherichia coli in Water by TaqMan Quantitative Polymerase Chain Reaction (qpcr). 24 L water collected from various sites Filtered at SVSU Distributed to multiple labs Tested: Colilert-18 qpcr Goal: to find a reliable and reproducible conversion between CFU from Colilert and GE from qpcr Determine an allowable level of GE Log (Target Sequence Copies) 14

Filtration Vacuum filtration Polycarbonate filter membranes Rod-shaped bacteria 0.5 µm 0.4 µm pore size, 47mm diameter 100 ml of sample 100 ml Phosphate-buffered saline (PBS) rinse PBS Blanks E. coli calibrators 90% ethanol sterilization 15

DNA Extraction (EPA Method C) Bead beat with acid-washed glass beads in ph 8.0 Tris-EDTA buffer Salmon testes internal control Purify through several centrifugation steps 16

qpcr setup Contamination controls Aerosol barrier micropipette tips Biosafety cabinet Primer/Probe Mix Forward Primer Reverse Primer Probe PCR-grade water Master Mix PCR-grade water Bovine serum albumin (BSA) Taqman Environmental Master Mix 2.0 Primer/probe mix http://www.pocdscientific.com.au/eppendorf_eptips_dualfilter_barrier_tips.php 17

Colilert log CFU/100 ml qpcr log GE/100 ml SVSU Validation Study Results 5 4.5 4 3.5 3 2.5 2 1.5 1 qpcr 0.5 Allowable CFU Colilert 0 Saginaw Bay (7/19) Saginaw Bay (7/20) Saginaw Bay (7/21) Saginaw River (7/27) Saginaw River (7/28) Saginaw River (7/29) Sampling Locations (Dates) 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 18

Colilert (log CFU/100 ml) SVSU Validation Study Results Colilert vs. Method C 4 3.5 3 y = 0.7314x + 1.3421 R² = 0.914 2.5 2 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 3 3.5 E. coli Genome Equivalents (log GE) Pearson Correlation Coefficient: 0.96 19

Overview Introduction to fecal coliforms Current detection methods qpcr rapid testing EPA Method C validation study The qpcr quantification issue Viability testing Future research 20

Colilert log CFU/100 ml qpcr log GE/100 ml SVSU Validation Study Results 5 4.5 4 3.5 3 2.5 2 1.5 1 qpcr 0.5 Allowable CFU Colilert 0 Saginaw Bay (7/19) Saginaw Bay (7/20) Saginaw Bay (7/21) Saginaw River (7/27) Saginaw River (7/28) Saginaw River (7/29) Sampling Locations (Dates) 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 21

Fluorescence Peak Area The qpcr Quantification Issue qpcr is DNA-based DNA doesn t need a living host to exist Obtained results can be falsely high How can this be studied? Compare viable (live) cell DNA to non-viable (dead) cell DNA Fluorescent viability dyes Study effects of UV-sterilization 5000 4500 4000 3500 3000 2500 2000 1500 1000 Live Dead 500 0 Pre- or Post-UV Samples 22

Ultraviolet Sterilization 100 400 nm Relatively high energy Causes thymine dimers in DNA Renders DNA unusable for transcription/translation and replication Cell death/non-viability 23

SYTO 9 PI Cell Viability Dyes SYTO 9 nucleic acid dye Live and dead cells Green Fluoresces at 510-540 nm Propidium iodide Dead cells Red Fluoresces at 620-650 nm https://bio-ggs.blogspot.com/2010_05_01_archive.html 24

Cell Staining 3 ml samples 9 μl dye mix Equal volumes of 3.34 mm SYTO 9 in dimethyl sulfoxide (DMSO) 20 mm propidium iodide in DMSO Incubated in the dark at room temperature for 15 mins 25

Fluorescence Spectrophotometry https://micro.magnet.fsu.edu/primer/java/fluorescence/exciteemit/ https://www.researchgate.net/figure/23236997_fig1_figure-1-101- Fluorescence-fundamentals-Jablonski-diagram-displaying-the-energy-states 26

Fluorescence Spectrophotometry Excitation: 470 nm Slit width 10.0 nm Live Dead Emission: 490-700 nm Slit width 10.0 nm Live and dead fluorescence regions Live: 510-540 nm Dead: 620-650 nm Integrate peak areas 27

% Living E. coli Results Fluorescence Spectrophotometry % Living E. coli Pre- and Post-UV Decrease in live cells after UV treatment in 2/3 sampling dates Expected More dead cells than live cells in all samples 20 15 10 5 Pre-UV Post-UV 0 2/15/2017 3/13/2017 3/21/2017 Sampling Date 28

qpcr DNA Extraction (Pre/Post-UV) Bead beat with acid-washed glass beads in ph 8.0 Tris-EDTA buffer Salmon testes internal control Centrifugation FastDNA Spin Kit for Soil Protein precitipation solution (PPS) Binding matrix Centrifuge with spin filters SEWS-M Washes impurities away from bound DNA DNA elution solution (DES) 29

E. coli Genome Equivalents (log GE)/100 ml Results qpcr 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 2/15/17 3/13/17 3/21/17 Sampling Date Pre-UV Post-UV E. coli GE per 100 ml water Log scale Amount of detected GE decreases from pre- to post-uv in 2/3 sampling dates Not the same two as in fluorescence data For true comparison, quantification data from fluorescence spectrophotometry is required 30

Live/Dead Area Ratio Results Quantification Via Fluorescence Known concentration of cells Create standards with known ratios of live to dead cells Test their fluorescence emission Compare intensity of standards to intensity of samples at live and dead wavelength ranges 2.00 1.50 1.00 0.50 Fluorescence Standard Curve y = 0.0184x + 0.006 R² = 0.7532 0.00 0 10 20 30 40 50 60 70 80 90 100 Percent Live Cells 31

Fluorescence Intensity Fuorescence Intensity Results Quantification Via Fluorescence 35000 Live Fluorescence Standard Curve (510-540 nm) 16000 Dead Fluorescence Standard Curve (620-650 nm) 30000 y = 284.62x - 807.95 R² = 0.7631 15000 25000 14000 y = -38.748x + 15421 R² = 0.8564 20000 13000 15000 10000 12000 5000 11000 0 0 10 20 30 40 50 60 70 80 90 100 10000 0 20 40 60 80 100 120 Percent Live Cells Percent Dead Cells 32

Future Research Test additional samples with Colilert and qpcr to add to the Method C data set Continue viability testing and compare with qpcr results Explore methods to quantify E. coli using fluorescence spectrophotometry Investigate use of alternative viability testing technologies, i.e. flow cytometry Perform controlled assays of UV light exposure on contaminated samples 33

Acknowledgements Dr. Tami Sivy The SVSU Honors Program The SVSU Undergraduate Research Program for funding The Saginaw Bay Environmental Science Institute (SBESI) at SVSU for providing facilities and equipment Carol Injasoulian and the Bay City Wastewater Treatment Plant The MWEA 34

References EPA Method C: Escherichia coli in Water by TaqMan Quantitative Polymerase Chain Reaction (qpcr). Thermo Fisher Scientific. TaqMan Real-Time PCR (qpcr). Thermofisher.com (accessed April 2, 2017). Thermo Fisher Scientific. Invitrogen LIVE/DEAD BacLight Bacterial Viability Kits. Thermofisher.com (accessed April 4, 2017). MP Biomedicals. FastDNA SPIN Kit for Soil. mpbio.com (accessed April 4, 2017). 35

Thank You! 36

Questions? 37