Investigation of qpcr Inhibition Occurrence across Geographically Distributed Recreational Waters

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1 Investigation of qpcr Inhibition Occurrence across Geographically Distributed Recreational Waters Tamara Anan eva US EPA Office of Water ORISE Research Fellow City of Racine, WI Health Department Laboratory Great Lakes Beach Association Conference September 27, 2011

2 Comparative Evaluation of Molecular and Culture Methods for Fecal Indicator Bacteria for Use in Inland Recreational Waters (PATH7R09) 1 year project funded by WERF (Water Environmental Research Foundation) Julie Kinzelman (PI) City of Racine, WI Health Department Laboratory Rebecca Bushon (co PI) U.S. Geological Survey, Columbus, OH Sam Dorevitch (co PI) University of Illinois at Chicago, School of Public Health Rachel Noble (co PI) University of North Carolina at Chapel Hill, Institute for Marine Sciences

3 WERF PATH7R09 Project Objectives Determine the relationship between culture based and qpcr methods for assessing a variety of aqueous environments: inland recreational waters (flowing and still; impacted by effluent or stormwater) and wastewater through the treatment train Investigation of qpcrinhibition Detection of qpcr inhibition Association of inhibition to water body type: effluentdominated waters, inland lakes, rivers, and Lake Michigan coastal waters

4 What is qpcr Inhibition? Delay in target DNA amplification (increased Ct value=lower qpcr signal or Ct=0 (undetermined)) US EPA Method A Enterococci (molecular, Taqman ) 3 Ct Specimen Processing Control (SPC) amplification delay = Inhibition Caused by various compounds present in the environmental sample matrix Some of the known inhibitors: Humic acid, polysaccharides, DNA binding proteins, high concentration of non target DNA (competition), etc. 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.

5 The Problem qpcr assay inhibition may cause: Target underestimation No detect (false negative)

6 Geographical distribution of surface water sample collection sites Surface water samples were comprised of geographically distributed fresh water samples provided by the USGS Water Science Centers (12 sampling location sites)

7 Characteristics of sampling sites

8 Methods Sampling events occurred June October, 2010 Collected samples per site Collected beach sanitary survey (BSS) data including turbidity, rainfall precipitation, sky conditions, etc. Sample analysis within 6 hours of collection by culture based methods Fresh frozen filters for qpcr were prepared within 6 hours of sample collection o 12 filters per sample to distribute to three analytical laboratories Fresh frozen filters were analyzed by qpcr molecular assays

9 Comparison of Culture to qpcr within and between Target Organisms Enterococci Culture Enterococci/Scorpion UNC Enterococci/Taq Racine Enterococci/Taq UNC Enterococci/Taq USGS E. coli culture E. coli qpcr Bacteroides qpcr Entero culture Entero Scorp UNC Entero Taq Racine Entero Taq UNC Entero Taq USGS E. coli culture E. coli qpcr Bacteroides qpcr

10 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

11 Detection of Inhibition Specimen Processing Control (a known quantity of Salmon Testes DNA) was included in all samples (calibrator and unknown) prior to DNA extraction dct SPC 2.0 INHIBITION Mediation of inhibition: serial dilution of DNA extract DNA purification (Qiagen kit)

12 Occurrence of Inhibition Inland Lakes, Rivers and Streams Dependent on characteristics of the water body Greater inhibition in inland lakes Inhibition was more common at sites impacted by wastewater discharge (POTW), non point source pollution (NPS), and agricultural runoff Coastal Recreational Waters Fresh (Great Lakes) Little inhibition seen Wastewater 5 POTWs with different disinfection processes qpcr showed similar log reductions through the waste water treatment process as viable organisms counts Little inhibition seen High concentration of target allowed for serial dilution

13 Occurrence of qpcr inhibition by water body type 20 Percentage of Inhibited Samples Effluent Lake Michigan River Inland lake Lab A Lab B N/A Lab C

14 Follow up to WERF PATH7R09 Objectives Evaluate ability of beach sanitary surveys (BSS) to identify environmental conditions associated with qpcr inhibition Utilize site specific BSS data to predict qpcr inhibition Identify sites/events that could preclude use of qpcr Reduce sample analysis time

15 Study Design Looked at WERF Phase II (fresh surface waters) BSS data from 7/12 sites that demonstrated inhibition Enterococci/EPA Method A only Determined seasonal mean values for: Air/water temperature hr precipitation Turbidity Cloud cover Stranded/submerged algae Odor Bather density Stream flow (where applicable) Compared mean values to incidence of inhibition

16 Incidents of qpcr inhibition occurrence by site

17 Relationship of Inhibition to Seasonal Mean Site Conditions based on BSS

18 qpcr Inhibition Mitigation Serial dilution of DNA extract + Fast, simple, inexpensive May dilute out the target DNA if initial concentration in the sample is low Use of DNA purification kits ± Effective (sometimes) Expensive, increases time of sample processing, contributes to greater variation of the results Mitigation measures are not always effective

19 Conclusions Evaluation and analysis of BSS data showed that: a. occurrence of inhibition was related to rainfall occurring in the previous hrs and increased turbidity at most, but not all sites; b. there was no common pattern in occurrence of inhibition across all affected sites, but site specific patterns of environmental conditions can be established to predict qpcr assay inhibition

20 Conclusions qpcr inhibition was irresolvable at some sites, but managed at others (through serial dilution, and/or use of DNA purification kit) When successful, mitigation of inhibition increased assay cost and turn around time Unsuccessful mitigation of inhibition may preclude use of qpcr assays at some sites Rapid alternatives exist Predictive modeling EPA Virtual Beach and others Utilizes BSS data to estimate bacteria concentrations in real time

21 THANK YOU!