Estimation of Enterococcus qpcr target sequence recoveries from EPA NEEAR study calibrator samples 2014 UNC Water Micro Conference

Transcription

1 Estimation of Enterococcus qpcr target sequence recoveries from EPA NEEAR study calibrator samples 2014 UNC Water Micro Conference Office of Research and Development May Notice: Although this work was reviewed by EPA and approved for presentation, it may not necessarily reflect official Agency policy

2 RWQC Background EPA recreational water quality criteria are intended to protect designated water body uses including swimming and other water contact activities. Previous 1986 RWQC were based on culturable fecal indicator densities: E. coli (freshwater), enterococci (freshwater & marine) 2012 RWQC Based on previous and newer ( NEEAR) epidemiology studies Updated criteria values for culturable E. coli (freshwater) & enterococci (fresh & marine) associated with 32 and 36 GI illnesses per 1000 beach water recreators Geo Mean, single sample statistical threshold values (STV - 95 th percentile) and single sample beach action values (BAV - 75 th percentile) are provided Corresponding values also provided for enterococci CCE by more rapid qpcr methods (EPA Method 1611 and forthcoming Method 1609) 2

3 Objectives of this presentation The basis of real time PCR quantification Comparative C T calibration models The EPA calibration model TSC (target sequence copies) vs CSE (calibrator target sequence equivalents) vs CCE (calibrator cell equivalents) Standardization of EPA qpcr method results and correspondence with RWQC values Points to remember: TSC are what are detected by qpcr and quantified by qpcr calibration models CCE do not necessarily = enterococci cells (or CFU) in water samples (but they are the basis of current EPA RWQC values which reflect a strong relationship between qpcr results and GI illness rates in the EPA NEEAR studies) 3

4 Real-Time PCR Quantification Target sequence copies increase exponentially (i.e. ~ double) during early cycles of PCR amplification. In real-time PCR, more copies = more fluorescence QPCR is based on this predictable increase in fluorescence during early cycles: i.e. a linear relationship between log10 target sequences in a reaction and number of cycles needed to reach a threshold where fluorescence first becomes detectable above background Quantitative Cycle threshold (C T or C q ) is the number of cycles required to reach the threshold If DNA standards with known target sequence quantities are available, a standard curve of their C T values can be used to estimate absolute quantities of target sequences in unknown test samples 4 * * * * * 10E7 sequences 10E6 sequences 10E5 sequences 10E4 sequences 10E3 sequences No DNA Fluorescence threshold = 30 fluorescence units in this example *Cycle threshold: Cycle # at which growth curve crosses 30

5 Comparative C T Calibration Models Used to determine relative quantities of TSC in different samples (not to determine absolute quantities). Results expressed as ratios or fold-differences in target sequence quantities in test samples compared to a designated calibrator sample. Ability to normalize for differences in total DNA recovery in calibrator and test samples by comparing C T values of a reference target sequence expected to be present in equal amounts prior to sample processing. Comparative C T models are widely used for measuring changes in gene expression. (Applied Biosystems, User Bulletin #2, ts/generaldocuments/cms_ pdf) 5

6 The EPA calibration model The qpcr calibration model used in EPA Method 1611 and soon to be released Method 1609 is a hybrid of absolute and relative quantification approaches: C T results are used to calculate ratios of target sequence copies (TSC) recovered from test samples to TSC from whole cell calibrator samples as in comparative C T models Salmon DNA, added to both samples prior to extraction, is used as the reference for total DNA recovery Ratios are converted to equivalents in the test samples of a known absolute value of the calibrator samples, e.g. the number of cells present (CCE) or the number of TSC present (CSE) To date, cell numbers in the calibrator samples have been used as the absolute values and TSC/cell treated as an unknown constant. But for implementation of the EPA methods, a standardized TSC/cell value has been established (as discussed below) 6

7 The EPA calibration model Expressions of the model: ratio (test to calibrator) = E^(-ΔΔC T ) CCE = ratio x calibrator cells (Method 1611 calibration model) CSE = ratio x calibrator TSC (modified calibration model) where: Amplification efficiency (E) is determined from the slope of a fitted curve of C T Ent values from analyses of serially diluted samples (e.g. calibrator samples or DNA standards) by the formula: 10^(1/slope) ΔΔC T is determined by subtracting C T Ref from C T Ent for both calibrator and test samples (= ΔC T ) and then subtracting Calibrator ΔC T from Test sample ΔC T C T Ref is from a sample processing control (salmon DNA) PCR assay C T Ent is from the Enterococcus PCR assay 7

8 Challenges for standardization of the EPA models and methods Need for consistent TSC/cell from different sources of calibrator cells in original Method 1611 model Need for accurate DNA standards or other methods to determine TSC in calibrator samples for modified calibration model Need to relate TSC in calibrator samples to NEEAR study calibrator samples in order to relate CCE or CSE values to RWQC values 8

9 Log 10 Cell Equivalent per Filter Variability in CCE estimates using different calibrator cell sources Comparison of Enterococcus CCE estimates obtained on StepOnePlus and Biorad IQ platforms using either BioBall (BB550) or frozen filter (FF550) calibrators From Cao et al., Water Research BB550_iQ5 BB550_StepOne Plus FF550_iQ5 FF550_StepOne Plus 0.0 D G Z S1 S2 S3 S4 S5 S6 S7 S8 S9 Sample 9 Conclusions: Negligible differences seen between instrument results compared to differences caused by using different sources of calibrator cells Difference between cell sources was attributed to differences in TSC/cell Results like these are the impetus for moving to the modified calibration model determining CSE

10 Need for accurate methods to determine TSC in calibrator samples Methods available: MPN estimation Sivaganesan et al., J. Microbiol Meth Digital PCR (new technology) Estimation from standard curves using accurately quantified DNA standards (Preferred Method) MPN or digital analysis can be used to corroborate accuracy of DNA standards 10

11 Need for accurate DNA standards Differences in TSC/calibrator cell from direct MPN estimates and from standard curves using unadjusted and MPN-adjusted standard concentrations in a recent study EPA MPN direct estimate (published*) TSC/calibrator cell Combined lab standard curve estimate (MPNadjusted) Combined lab standard curve estimate (unadjusted) 11 * Sivaganesan et al., J. Microbiol Meth. 2011

12 Determining TSC in NEEAR study calibrator samples MPN-based standard curve corroboration of 2009 NEEAR study standard curve for estimating NEEAR study TSC/calibrator cell Study (Year of Study) Mean Percent Amplification Efficiency (lower, upper 95% confidence limits) Mean Fitted Curve Intercept (lower, upper 95% confidence limits) NEEAR (2007) 1 96 (94, 98) (38.22, 38.48) NEEAR (2009) 1 96 (93, 100) (36.83, 37.32) Sivaganesan (2012) 2 93 (90, 96) (36.55, 37.69) 12 1 TSC in DNA standards estimated from spectrophotometric analysis (EPA Method 1611) 2 TSC in DNA standards estimated from MPN analysis (Sivaganesan, et al. 2011)

13 13 Standardization of EPA Methods results EPA Method 1609 (and later to be updated Method 1611) will require estimation of TSC in calibrator samples by all laboratories Option will be available of obtaining EPA-provided DNA standards with concentrations that have been corroborated by digital PCR analysis for this purpose TSC in calibrator samples will be used to estimate CSE in test samples using the modified Method 1609 calibration model Test sample CSE can be divided by 15 (overall mean TSC/calibrator cell estimate from the NEEAR study) to obtain CCE that are comparable to RWQC values Future provision of qpcr values directly as CSE units (or TSC units based only on DNA standards) in an addendum to RWQC is possible The EPA methods, an Excel worksheet and other resources to help users understand & perform these methods are (or soon will be) available at: EPA will also offer training workshops for these methods (first to be held next week). Advance sign-up for future workshops is possible at: