Validating
MST Methods
(Stoeckel et al.) Chapter 2
Authors:
Don Stoeckel, John Griffith and Asja Korajkic
Background- What do we
mean by method validation?
- Validation
needs depend on study objectives
- Resolution
of sources (categories; human/nonhuman)
- To
quantify or to categorize?
- Validation
includes three levels – source identifier, measurement protocol, and
interpretation paradigm
- Summary of
source identifiers
- Technique
used to develop the method (e.g. metagenomics, subtractive hybridization,
etc)
- Target
characteristic (e.g. 16S or other genes)
- Target
host (General fecal/ruminant/bird or species-level association)
- Summary of
validation literature
- Lessons
learned from FC/FS ratio
- Mention
observed changes in application from library-dependent to
library-independent methods following USGS and SCCWRP validation attempts
- Existing
compiled sensitivity and specificity information for markers
- Stoeckel
and Harwood
- Series by
Shanks
- SCCWRP
Study – Boehm et al., Layton et al.
- Validation
of the Source Identifier
- Theoretical
tests
- If
genetic, check sequences in GenBank
for specificity and evenness within host population
- Controlled
tests for sensitivity and specificity
- For
sensitivity, serial dilutions of the “real-life” intended target (i.e.
sewage for human markers) (use of aged or partially-treated material
for enhanced relevance)
- For
specificity, test method against non-targets (use of pooled material
for efficiency)
- Protocol
Performance
- Analytical
limit of detection and quantifiable range of the target
- Inter-lab
performance comparisons to validate transferability of methodology
- Interpretation
Paradigm
- Test
method against known levels of target seeded into ambient water
- Field
test at sites known to be contaminated versus “pristine”
(Each topic carries specific examples of how to
obtain the needed information to “validate” the use of a source identifier,
protocol, and interpretation paradigm.)
- Coverage
across “intended” hosts – has variability been fully captured?
- Understanding
of population of source required in order to detect contamination (e.g.,
septic systems vs WWTP losses)
- Understanding
of units needed to measure “fecal contamination” (i.e., targets per gram
fecal material, per unit of pathogen carried, or per unit of fecal
indicator carried depending on the objective of the study)
- Coverage
across “alternate” hosts – has potential for false-positive results been
characterized?
- Limits
of detection and quantifiable ranges – does the protocol deliver data that
meet the goals of the study
- Geographic/temporal
stability – is the source identifier and protocol used to measure the
source identifier verified in the study area/study lab?
- Effect
of aging on marker stability and relationship to regulated FIB
- Quantitation
– have all sources of loss been mathematically corrected?
- Recovery
efficiency
- Extraction
efficiency
- Volumetric
losses during processing
- Matrix
inhibition/detection efficiency
- Degradation
during storage
- Standard
curve
- Reporting
– are the results reported in relevant units?
- Targets
per liter of water
- Targets
per nanogram DNA extracted
- Targets
per E. coli or enterococci
cells (to allocate fecal contamination and meet regulatory criteria)
- Effect
of aging on marker stability and relationship to regulated FIB
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