Session 6 Harwood

7/30/2019 Session 6 Harwood

1/22

Performance Criteria anPerformance Criteria anMethods for HumanMethods for Human--

Source Tracking MarkSource Tracking MarkBacteria in Fresh anBacteria in Fresh an

alerie J. Ha

Katrina V. G

ChristopDepartment of Integrative Biol

EPA BeachesEPA Beaches

d Field Testing of qPCRd Field Testing of qPCRAssociated MicrobialAssociated Microbialers and Fecal Indicatorers and Fecal Indicator

Salt Surface WatersSalt Surface Waters

wood, Ph.D.

ordon, Ph.D.

er Staleygy, University of South Florida

onference 03/16/11onference 03/16/11


2/22

Study O

3 Year Study:

Evaluate the performancmethods for human sewa

Evaluate the performancquan y . co an en er

Compare qPCR methodswith culture (membrane

Determine correlation befecal indicator bacteria

Inter-laboratory compari

transferability

jectives

of two qPCR-based MSTe contamination

of qPCR methods tococc

forE. coliand enterococciiltration) methods

ween human markers and

on of method


3/22

Literature Revi

Select Two HumCriteria included:

Specificity

Sensitivity

uantit in wastewat

Use in multiple labs

Use across geographic

Detection correlatedbacteria (FIB), pathooutcomes

The picks: humanBacte

human polyomavirus

w Conducted to

an MST Markers

r limit of detection

regions

ith fecal indicatorens, human health

roides HF183 and

s (HPyVs)


4/22

Other qPOther qP

Fecal IndicFecal Indic Enterococcus faeciu

Ludwig & Schleifer

E. coli uidA gene (L

GeneralBacteroidal

al., 2008)

R TargetsR Targets

tor Bacteriator Bacteria 23S rRNA (EPA;

2000)

e 2005)

es (EPA; Siefring et


5/22

E. coliE. coli qPCRqPCR

106 copies

ssayssay -- PlasmidPlasmid

101 copies


6/22

E. coliE. coli StanStan

2 = 0.9995

fficiency = 98.6

tandard deviation (CT)

0.063 for 106 copies0.325 for 101 copies

dard Curvedard Curve


7/22

MeMe

Sensitivity ability to de

contamination from spec

Limit of detection qua

sensitivity, i.e. how little

Specificity PCR marke

when sewage from speci

ricsrics

ect PCR marker when

ific source is present

titative assessment of

an we reliably detect?

r should not be detected

ic source is absent


8/22

MethoMetho

Standards made from s

Standard curve run in t

96-well plate amp es an contro s ru

Sensitivity (+/-) and met

(quantitative) on sewage Specificity non-targets

Internal amplification c

with generalBacteroidal

DetailsDetails

nthesized sequences (IDT)

iplicate reactions for each

n n tr p cate react ons

od detection limit

samplesinclude dogs, gulls, cattle

ntrol (IAC) multiplexed

es assay


9/22

SpecSpec

Tested against dog, cow,

The HPyVs marker was

Human Bac HF183 was 8

HF183 cross-reacted wit

chicken and duck feces

*detectable, but not quan

dog feces

ficityficity

ird feces

00%

1% specific

dog*,

tifiable in


10/22

Detection LimiDetection Limi

AmbienAmbien Sewage spike 5 ml into

buffer filter & extr

series qPCR epea proce ure sp

river, tannic, estuarin

Compare sample dete

ambient waters Internal amplification

inhibition

t for Sewage int for Sewage in

WatersWaters500 ml phosphatect DNA dilution

ng sewage n o a e,, marine waters

tion limit in buffer vs.

control checks for


11/22

Ambient WAmbient W

LakeLake

CarrollCarroll

Bahia BeachBahia Beach

(Tampa Bay)(Tampa Bay)

FortFort

DeSotoDeSoto

ter Samplester Samples

Riverfront ParkRiverfront Park

Hillsborough RiverHillsborough River


12/22

Effect of DilutiEffect of DilutiAmbient WatersAmbient Waters

Quantification andQuantification and

Site

HPyVs

Limit of QuantificationLimit

DetectiTarget Sewage Sewag

Copies/5la

Dilutionb

Dilutio

Bahia Beach3.23 102 100 10-1

1.06 101 10-1 10-1

Fort DeSoto2.05 101 10-2 10-2

3.07 101 10-1 10-1

Green

Swamp

3.91 102 100 10-1

1.64 102 100 100

Lake Carroll

(site 6)

1.20 102 100 10-1

6.17 101 100 100

Hillsborough

Riverc

5.47 101 10-2 10-2

3.11 101 100 10-2

n of Sewage inn of Sewage inon MST Markeron MST MarkerDetection by qPCRDetection by qPCR

HF183f

on

Limit of QuantificationLimit of

Detectione Target Sewage Sewage

n

Copies/5la

Dilutionb Dilution

8.31 102 10-2 10-4

6.63 101 10-1 10-1

8.81 101 10-3 10-4

9.07 101 10-3 10-3

1.66 102 10-3 10-3

1.31 101 10-3 10-4

1.98 102 10-3 10-4

5.23 102 10-2 10-2

1.71 101 10-4 10-4

4.30 102 10-2 10-3


13/22

PCR Inhibition in AmPCR Inhibition in Am

by Internal Amplby Internal Ampl

Sample SiteSamplin

Bahia Beach 35.

Fort DeSoto 36.

Green Swamp 40.

Lake Carroll 39.

Hillsborough River 42.

Inhibition best relieved by te

ient Waters Detectedient Waters Detected

ification Controlification Control

CT Value

Date 1 Sampling Date 2

-

1 35.4

4 35.6

1 37.8

0 37.9

4 Undetermined

plate dilution


14/22

Task 2: Persi

Mimic persistence of mark

waters & beach following

Cycles of sand wetting an

(approximating tidal cycl

Measure two MST marke

FIB (enterococci & fecal

membrane filtration in w

stence Study

rs in estuarine

sewage spill

drying

s)

rs by qPCR and

oliforms) by

ter


15/22


16/22

Correlation of DeCorrelation of De

and MSTand MST

EntPearson Correlation

Sig. (2-tailed)

E. coli.

Sig. (2-tailed) .

FCPearson Correlation .

Sig. (2-tailed) .

HPyVsPearson Correlation .

Sig. (2-tailed) .

HF183Pearson Correlation .

Sig. (2-tailed) .

cay Rates of FIBscay Rates of FIBs

MarkersMarkersnt E. coli FC HPyVs HF183

1 .959 .941 .816 .875

.000 .000 .007 .002. . .

00 .000 .004 .002

41 .994 1 .828 .864

00 .000 .006 .003

16 .851 .828 1 .980**

07 .004 .006 .000

75 .884 .864 .980 1

02 .002 .003 .000


17/22

Correlation of qPCorrelation of qP

Filtration MeasuFiltration MeasuAmbienAmbien

R vs. MembraneR vs. Membrane

ements of FIB inements of FIB inWatersWaters


18/22

BenBen

Tools for detecting humTools for detecting hum

with wellwith well--defined perfordefined perfor

Quantitative PCR for feQuantitative PCR for fe

quality issuesquality issues

May eventually be less eMay eventually be less e

methods so better samplmethods so better sampl

obtainedobtained

fitsfits

n source pollutionn source pollution

ance characteristicsance characteristics

al indicator bacteriaal indicator bacteria

pensive than culturepensive than culture

ing coverage can being coverage can be


19/22

BenBen qPCR measuremenqPCR measuremen

correlation with hucorrelation with hu

than current culturthan current cultur

Correlation of MSTCorrelation of MST

human health outcohuman health outco

Management decisiManagement decisi Remediation activitiRemediation activiti

Risk assessmentRisk assessment

fitsfitss may provide betters may provide better

an health outcomesan health outcomes

--de endent methodsde endent methods

measurements withmeasurements with

mes will inform:mes will inform:

ns (beach, land)ns (beach, land)eses


20/22

Question

vharwood@

s?

sf.edu


21/22

Target Primer/Probe

HPyVsSM2 5 P6 5

KGJ3 5

HF183

HF183F 5'

SSHBacR 5

SSHBac-PRB 5

Eco-F 5

Table 2-3. Primers and Prob

E. coli Eco-R 5

Eco-PR 5

Enterococci

Entero1A (ECST748F) 5

EnteroR1 (ENC854R) 5

GPL813TQ 5

General

Bacteroidales

GenBacF3 5

GenBacR4 5'

GenBacP2 5'

IAC UCP1 5'

Sequence

AGT CTT TAG GGT CTT CTA CCT TT 3GGT GCC AAC CTA TGG AAC AG 3

(FAM) TCA TCA CTG GCA AAC AT (MGBNFQ) 3

ATCATGAGTTCACATGTCCG 3

ACCCCGCCTACTATCTAATG 3

(FAM) TTAAAGGTATTTTCCGGTAGACGATGG (TAMRA) 3

TCCAAAGCGGCGATTTG- 3

s Used for qPCR Assays.

AGGCCAGAAGTTCTTTTTCCA 3

(FAM) ACGGCAGAGAAGGTA ( MGB NFQ) 3

AGAAATTCCAAACGAACTTG 3

AGTGCTCTACCTCCATCATT 3

(FAM) TGGTTCTCTCCGAAATAGCTTTAGGGCTA (TAMRA) 3

GGGTTCTGAGAGGAAGGT 3

CCGTCATCCTTCACGCTACT 3

(FAM) CAATATTCCTCACTGCTGCCTCCCGTA (TAMRA) 3

(VIC) CCTGCCGTCTCGTGCTCCTCA (TAMRA) 3


22/22

Anomalous PointAnomalous Point

CurveCurve ShouldShouldOutlOutl

45

50

Standar

0

5

1015

20

25

30

35

40

0 1 2 3

CT

Value

Log10 gene copie

on the Standardon the Standard

We Throw OutWe Throw Outers?ers?

Curve

y = -3.6723x + 42.677

R = 0.9825

4 5 6 7

s per reaction

Session 6 Harwood

Documents

Transcript of Session 6 Harwood