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Translating QPCR Research Methods
into SOPs Shannon Briggs
Michigan Department of
Environmental Quality
4 points for today
1. What are the basic principles?
2. What is happening with the current
studies?
3. What problems were encountered when
attempting to perform testing?
4. What is possible from this type of testing?
WHY?
Monitoring Michigan Beaches Statewide
for E. coli with QPCR
(USEPA Draft Method C, June 2015)
Shannon Briggs
Michigan Department of
Environmental Quality
Water Resources
11,000 inland lakes 77,000 river miles 1,200 public beaches 4 Great Lakes 3,288 miles of coast 5.5 million acres of wetlands
Michigan Public Health Code and Public Beaches
• Monitoring is Voluntary for all 45 Health Departments, however
• County Ordinance can require testing
• Health Officer has authority to test and close
• Requires signs at all public beaches
• Requires reporting if beach is tested
Beach Monitoring “typical stats”
• 200 inland lake beaches monitored
• 200 Great Lakes beaches monitored
• 3.6% samples exceed WQS, n =186 samples
• 80% of beaches are open all season, n= 332
• 20% of beaches report action, n= 84 beaches
• Most actions are 1 to 2 days
Monitoring Approach Identify Beach
Conduct sanitary survey and collect monitoring data
Evaluate all data
Evidence of contamination
YES, MAYBE NO
Potential Risk Low Risk
Investigate Continue Monitoring
Remediation Reduce or Composite Samples
Continue using sanitary survey & monitoring data
Re-evaluate
Should I consider QPCR? Do you monitor a beach?
Have you closed a beach?
How often do you close a beach?
How far away is the beach from a lab?
Do you want results in 4 hours?
Do you want to use a method that was more closely tied with reported illnesses?
Do you want lab equipment that can identify sources?
What do I need for QPCR?
Proximity of lab to beach
Lab willing to dedicate staff to perform qPCR method (consistency)
Equipment Costs
$50k if lab is already well equipped
$100K if lab has no equipment
Training, preferably “wet-lab, hands-on”
Monitoring budget
How Does PCR Work?
1. Denaturation – DNA helixes are pulled apart into single strands
2. Annealing – Primers bind to specific locations on the single
strands of DNA
3. Elongation/Extension – Facilitated by enzymes that attach to the primers,
nucleotide bases are added to the free strand, forming a double-stranded DNA
DNA from
live & dead
microbes
vs
live (growing)
microbes
Path to qPCR for Beach Testing
QPCR Methods Year Beaches
Kary B. Mullis invents PCR
1985
1986 Criteria
2000 BEACH Act
Path to qPCR for Beach Testing
QPCR Methods Year Beaches
Dr. Joan Rose at Michigan State
University
2003 Monitor Beaches with local, state &
federal funds
Water Fellows Lectures & Discussion
2005 Identify Impaired Beaches
Microbial Source Tracking (MST)
2007 Beach Sanitary Survey Tool
Path to qPCR for Beach Testing
Year QPCR & Beaches
2010 MST at Impaired Beaches
2011 Training Manual & Video for Beach Testing with QPCR
2012 Public Meeting for MST Results
U.S. EPA Rec Water Quality Criteria Includes Enterococci QCPR values
U.S. EPA Rec Water Quality Criteria
2012 criteria
based on
“epi” studies
2013 QPCR Lab at Lake St. Clair Metropark Beach
$100,000 for equipment
Park renovated office to lab ($50,000)
$500,000 for 10 New Labs in 2014
• State of Michigan provided $500,000 to DEQ for rapid beach testing equipment
• Only health departments have authority to test beaches
• DEQ sent letters of invitation to 45 health departments responsible for 83 counties
• 13 responses and description of lab capacity
Questions and Details
• Commitment & Expectations in Memorandum of Understanding between DEQ and HDs
• 10 Health Departments signed MOUs
• MOU included equipment list with 50+ items for each HD
• $58,000 for Training and Support from MSU
10 New qPCR Labs 6 Existing qPCR Labs
20
Path to qPCR for Beach Testing
Year QPCR & Beaches
2014 10 New QPCR Labs (15 total) U.S. EPA draft Method C
(QPCR - E. coli)
2015 Equipment Ordered & Delivered Samples filtered & frozen Train the Trainer for QPCR
Path to qPCR for Beach Testing
Year QPCR & Beaches
2016 4-day Training on Draft Method C Stockholm Water Prize awarded to Dr. Joan Rose
4 Teams
Pre-training Survey
42.9%
0.0%
57.1%
0.0% 0.0%
Not at allexperienced
Slightlyexperienced
Somewhatexperienced
Moderatelyexperienced
Extremelyexperienced
How would you rate your experience with qPCR?
How would you rate your confidence in your skills for each step of qPCR?
0
1
2
3
4
5
6
7
Pipetting PreparingReagents
Filtration DNAExtraction
RunningStepOne Plus
DNA Analysis
Before
After
Avera
ge R
atin
g o
f C
onfidence
Step in qPCR Analysis
Post-training Survey
6.50
6.33
5.50
6.33
I gained usable skillsand will be able toapply them in my
lab.
The trainingimproved my abilityto run qPCR in my
lab.
The trainingimproved my
understanding ofhow to calculate
results.
I am more confidentabout using qPCR.
Neutral
Agree
Somewhat
Agree
Strongly
Agree
How would you rate your overall satisfaction with the training workshop?
0.0% 0.0% 0.0% 0.0% 0.0%
66.7%
33.3%
Completelydissatisfied
Mostlydissatisfied
Somewhatdissatisfied
Neutral Somewhatsatisfied
Mostlysatisfied
Completelysatisfied
Path to qPCR for Beach Testing
Year QPCR & Beaches
2016 Multi-lab Validation Study 2015 & 2016 Samples tested
Review Colilert & QPCR Results
Enterococci Multi-lab Validation Study
Announced Monday, March 14, 2016
Multi-Lab Validation Study for draft Method C (qPCR & E. coli)
Determine if labs and method produce consistent results
• Labs report their Standard Curves
– Demonstrates their ability with Method C
• Test samples sent to each lab
• Labs run samples and report results
• Results evaluated
Monitoring Plan
• Collect water samples
– Need at least 30 “good” and “paired” samples per site
• Analyze with Colilert and draft Method C
• Evaluate the Index of Agreement (IA)
– Sufficient IA > 0.7
• Evaluate R-squared (R2)
– Sufficient R2 > 0.6
Monitoring Plan
• Collect water samples with limited resources
• State requirement to collect 3 samples per beach
– 100 ml from 3 sites at beach
• Composite samples
– Mix three 100 ml samples into one 300 ml sample
• Analyze
– 100 ml to colilert, 100 ml to QPCR, 100 ml potential for enterococci USEPA
Year QPCR & Beaches
2017 Samples tested and reviewed with previous 2 years
2018 Continue sampling Present equivalent QPCR results to
USEPA and Local Health Officers
2019 Beach status determined by QPCR methods
Molecular Source Track Training?
Path to qPCR for Beach Testing
41
Waterborne pathogens
Source: EPA’s National Beach Guidance and Performance Criteria for Grants
http://www.miqe.info/
Clean, safe, fun beaches!
Remember that rapid testing is part of the monitoring plan.
Report Data
Sleeping Bear Dunes Photo by Steve Keighly, Winner of the Instagram Beach Photo Challenge for favorite beach to take a long walk.
QPCR is not the finish line
its just another tool in the tool box
Beach Sanitary Surveys http://www2.epa.gov/beach-tech/beach-sanitary-surveys
48
Beach Sanitary Surveys
Canine Scent Tracking
Environmental Canine Services
Remediation landscaping
redesign slope of beach, groom beach
Marquette in October 5-7, 2016
QPCR USERS
