validation of qpcr rapid bacterial quantification through ... · epa validation study of method c:...

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).

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qPCR rapid testing



Viability testing

Future research

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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

idexx.com 4-Methylumbelliferyl β-D-glucuronide (MUGlcU)

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Polymerase Chain Reaction (PCR)

http://www.mun.ca/biology/scarr/Gr12-26.html 6

Annealing

Extension

Polymerase Chain Reaction (PCR) Ethidium bromide staining

www.researchgate.net http://www.madsci.org/posts/archives/1999-02/919869466.Mb.r.html

Presence/absence (qualitative)

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qPCR rapid testing



Viability testing

Future research

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Quantitative PCR (qPCR) Utilizes fluorescent probes to quantitate DNA

Fluorescence monitored in real time

Probe is hybridized to fit between primers

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http://www.sinobiological.com/real-time-pcr-service-gene-expression-analysis-by-qpcr-cro-service.html

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)

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qPCR Run Protocol

Run time approximately 90 minutes

Combined extension and annealing steps

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qPCR Raw Data Cycle Threshold (CT)

Fluorescence α 1/ CT α [DNA]

[DNA]

7 Genome Equivalents (GE)

12



qPCR rapid testing



Viability testing

Results

Future research

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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

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Log (Target Sequence Copies)

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

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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

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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

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http://www.pocdscientific.com.au/eppendorf_eptips_dualfilter_barrier_tips.php

SVSU Validation Study Results

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Saginaw Bay (7/19) Saginaw Bay (7/20) Saginaw Bay (7/21) Saginaw River (7/27) Saginaw River (7/28) Saginaw River (7/29)

qP

CR

lo

g G

E/1

00 m

L

Co

lile

rt l

og

CF

U/1

00 m

L

Sampling Locations (Dates)

qPCR

Allowable CFU

Colilert

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y = 0.7314x + 1.3421 R² = 0.914

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Co

liler

t (l

og

CFU

/10

0 m

L)

E. coli Genome Equivalents (log GE)

Colilert vs. Method C

Pearson Correlation Coefficient: 0.96

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qPCR rapid testing



Viability testing

Future research

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Saginaw Bay (7/19) Saginaw Bay (7/20) Saginaw Bay (7/21) Saginaw River (7/27) Saginaw River (7/28) Saginaw River (7/29)

qP

CR

lo

g G

E/1

00 m

L

Co

lile

rt l

og

CF

U/1

00 m

L

Sampling Locations (Dates)

qPCR

Allowable CFU

Colilert

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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

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Flu

ore

sce

nce

Pe

ak A

rea

Pre- or Post-UV Samples

Live

Dead

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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

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Cell Viability Dyes SYTO® 9 nucleic acid dye Live and dead cells

Green

Fluoresces at 510-540 nm

https://bio-ggs.blogspot.com/2010_05_01_archive.html

SYTO 9 PI SYTO 9 PI

Propidium iodide Dead cells

Red

Fluoresces at 620-650 nm

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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

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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

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Fluorescence Spectrophotometry

Live Dead

Excitation: 470 nm Slit width 10.0 nm

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

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Results – Fluorescence Spectrophotometry

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5

10

15

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2/15/2017 3/13/2017 3/21/2017

% L

ivin

g E.

co

li

Sampling Date

Pre-UV

Post-UV

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% 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

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)

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Results – qPCR

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2/15/17 3/13/17 3/21/17

E. c

oli

Ge

no

me

Eq

uiv

ale

nts

(lo

g G

E)/1

00

mL

Sampling Date

Pre-UV

Post-UV

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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

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

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y = 0.0184x + 0.006 R² = 0.7532

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1.00

1.50

2.00

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Live

/Dea

d A

rea

Rat

io

Percent Live Cells

Fluorescence Standard Curve

Results – Quantification Via Fluorescence

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y = 284.62x - 807.95 R² = 0.7631

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10000

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35000

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Flu

ore

scen

ce In

ten

sity

Percent Live Cells

Live Fluorescence Standard Curve (510-540 nm)

y = -38.748x + 15421 R² = 0.8564

10000

11000

12000

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15000

16000

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Fuo

resc

en

ce In

ten

sity

Percent Dead Cells

Dead Fluorescence Standard Curve (620-650 nm)

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

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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

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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).

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Thank You!

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Questions?

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