3.2 Library-Independent Approaches
Library-independent analyses target the presence or concentration of genetic
markers associated with specific fecal sources in a water sample, typically using
qPCR and thereby requiring no reference database [56, 57]. Currently, this
approach is generally considered to be simpler to employ and validate and is widely
recommended as the best approach to begin any project that might include MST.
For obtaining the equipment and setting up a lab to perform qPCR, an initial
capital investment (approximately $100K) is required; the subsequent cost for
supplies totals roughly $50 per sample; and a competent technician can process
more than 40 samples in one day (following DNA extraction). The USEPA is
currently developing standard methods for two human-associated qPCR markers
(HF183 and HumM2), based largely on the protocols developed during the SIPP
method evaluation study [5, 6]. The combined use of the two qPCR-based
approaches to measure FIBs and make a rapid determination for the presence of
human-source pollution should further reduce the analytical time needed and
increase affordability and accuracy of monitoring recreational waters. Because of
the many methodological nuances of qPCR, those desiring to adopt this technology
would be best served by partnering with a university research program for guidance
and training until the needed level of expertise has been reached.
3.3 DNA-Based Markers
At present, many DNA‐based marker assays have been developed for MST, and
application results have been published in the scientific literature [57–60]. New
assays that target human and other waste sources are continuously being developed
and reported [61]. Around a dozen DNA-based markers for detecting human
sources have been developed over the last decade. Additional assays are applicable
for tracing fecal wastes of other sources such as different types of livestock,
wildlife, domestic pets, and birds [57, 59]. Also, advances in PCR technology
have stimulated reevaluation of earlier markers for their sensitivity (detecting the
correct host fecal material when it is present) and specificity (no detection of any
other fecal sources; 62, 63). Thus, it is important to keep up to date with emerging
scientific literature that publicizes new assays and their performance or new
developments on older assays. All the markers recommended in this chapter use
PCR directed towards bacterial targets. In general, this process includes sample
collection, filtering, DNA extraction, amplification, and data analysis. The majority
of markers use qPCR for the amplification step, meaning they provide information
about the relative amount of target material in a particular water sample. Those
assays termed “end-point” or conventional PCR provide only qualitative (presence
or absence) data. When both quantitative and end-point PCR assays exist for a
given assay, the quantitative is always preferred.
Microbial Source Tracking: Advances in Research and a Guide to Application
273
Library-independent analyses target the presence or concentration of genetic
markers associated with specific fecal sources in a water sample, typically using
qPCR and thereby requiring no reference database [56, 57]. Currently, this
approach is generally considered to be simpler to employ and validate and is widely
recommended as the best approach to begin any project that might include MST.
For obtaining the equipment and setting up a lab to perform qPCR, an initial
capital investment (approximately $100K) is required; the subsequent cost for
supplies totals roughly $50 per sample; and a competent technician can process
more than 40 samples in one day (following DNA extraction). The USEPA is
currently developing standard methods for two human-associated qPCR markers
(HF183 and HumM2), based largely on the protocols developed during the SIPP
method evaluation study [5, 6]. The combined use of the two qPCR-based
approaches to measure FIBs and make a rapid determination for the presence of
human-source pollution should further reduce the analytical time needed and
increase affordability and accuracy of monitoring recreational waters. Because of
the many methodological nuances of qPCR, those desiring to adopt this technology
would be best served by partnering with a university research program for guidance
and training until the needed level of expertise has been reached.
3.3 DNA-Based Markers
At present, many DNA‐based marker assays have been developed for MST, and
application results have been published in the scientific literature [57–60]. New
assays that target human and other waste sources are continuously being developed
and reported [61]. Around a dozen DNA-based markers for detecting human
sources have been developed over the last decade. Additional assays are applicable
for tracing fecal wastes of other sources such as different types of livestock,
wildlife, domestic pets, and birds [57, 59]. Also, advances in PCR technology
have stimulated reevaluation of earlier markers for their sensitivity (detecting the
correct host fecal material when it is present) and specificity (no detection of any
other fecal sources; 62, 63). Thus, it is important to keep up to date with emerging
scientific literature that publicizes new assays and their performance or new
developments on older assays. All the markers recommended in this chapter use
PCR directed towards bacterial targets. In general, this process includes sample
collection, filtering, DNA extraction, amplification, and data analysis. The majority
of markers use qPCR for the amplification step, meaning they provide information
about the relative amount of target material in a particular water sample. Those
assays termed “end-point” or conventional PCR provide only qualitative (presence
or absence) data. When both quantitative and end-point PCR assays exist for a
given assay, the quantitative is always preferred.
Microbial Source Tracking: Advances in Research and a Guide to Application
273