prove superior to Gull2SYBR and LeeSeaGull, and will require a comparable
evaluation before adoption for widespread use [80].
At present, several dog-associated qPCR and end-point PCR marker assays have
been published. Two assays were included and evaluated in the SIPP study,
DogBact [72] and BacCan-UCD [59]; both performed equally well when dogs
were a potential fecal source [81]. Though highly sensitive and specific, both
markers occasionally cross-react with other species such as humans and cattle,
and such potential cross-reactions should be evaluated with local fecal material in
whatever region these two recommended dog markers are desired to be used.
Horse-associated markers are relatively uncommon to date, and only two have
been published. Only one PCR-based horse marker, conventional HoF597 (1), was
included in the SIPP study, and it was adequately sensitive and specific. Its use is
recommended when horses are present in the watershed and all other potential
sources have been already ruled out. The horse marker does not show as much
sensitivity as most other host-associated assays. Since another quantitative horse
assay has not yet been thoroughly evaluated for inclusion in the SIPP study, the
conventional HoF597 assay remains the recommended horse marker for now.
Multiple cow- and ruminant-associated DNA marker assays have been developed in the past decade, and several evaluation studies have been conducted to
validate their performance. Three qPCR assays have consistently performed well in
locales around the world and were included in the SIPP validation: BacR, CowM2,
and Rum2Bac. Although the performances of all three markers were good [82],
CowM2 [73] is the recommended cattle marker, and it is expected to become the
USEPA-approved method in the near future. When a non-bovine ruminant fecal
source such as deer or goat is suspected, Rum2Bac [74] should be used instead of
CowM2. When both cattle and other ruminants live in the watershed, both CowM2
and Rum2Bac markers are recommended. Rum2Bac occasionally demonstrates
false-positive results with septage, so in watersheds containing both septage and
ruminant sources, septage presence should be conclusively ruled out before
employing Rum2Bac.
Relatively few pig-specific markers have been developed to date and two pig
markers, one based on conventional PCR (PF163) (1) and one quantitative
(Pig2Bac) [74], were included in the SIPP study. Due to its demonstrated high
sensitivity, Pig2Bac performs better than PF163 and is the recommended method
for detection of pig feces. However, Pig2Bac sometimes cross-reacts with human/
septage and dogs so these would either need to be ruled out first or accounted for
when employing Pig2Bac.
4 Microbial Community Analysis Approach
In spite of the considerable progress that has been achieved employing DNA-based
MST markers described in Sect. 3, they share one disadvantage with traditional FIB
measurements in that they are still based on a single marker. This is limiting
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B. Badgley and C. Hagedorn
evaluation before adoption for widespread use [80].
At present, several dog-associated qPCR and end-point PCR marker assays have
been published. Two assays were included and evaluated in the SIPP study,
DogBact [72] and BacCan-UCD [59]; both performed equally well when dogs
were a potential fecal source [81]. Though highly sensitive and specific, both
markers occasionally cross-react with other species such as humans and cattle,
and such potential cross-reactions should be evaluated with local fecal material in
whatever region these two recommended dog markers are desired to be used.
Horse-associated markers are relatively uncommon to date, and only two have
been published. Only one PCR-based horse marker, conventional HoF597 (1), was
included in the SIPP study, and it was adequately sensitive and specific. Its use is
recommended when horses are present in the watershed and all other potential
sources have been already ruled out. The horse marker does not show as much
sensitivity as most other host-associated assays. Since another quantitative horse
assay has not yet been thoroughly evaluated for inclusion in the SIPP study, the
conventional HoF597 assay remains the recommended horse marker for now.
Multiple cow- and ruminant-associated DNA marker assays have been developed in the past decade, and several evaluation studies have been conducted to
validate their performance. Three qPCR assays have consistently performed well in
locales around the world and were included in the SIPP validation: BacR, CowM2,
and Rum2Bac. Although the performances of all three markers were good [82],
CowM2 [73] is the recommended cattle marker, and it is expected to become the
USEPA-approved method in the near future. When a non-bovine ruminant fecal
source such as deer or goat is suspected, Rum2Bac [74] should be used instead of
CowM2. When both cattle and other ruminants live in the watershed, both CowM2
and Rum2Bac markers are recommended. Rum2Bac occasionally demonstrates
false-positive results with septage, so in watersheds containing both septage and
ruminant sources, septage presence should be conclusively ruled out before
employing Rum2Bac.
Relatively few pig-specific markers have been developed to date and two pig
markers, one based on conventional PCR (PF163) (1) and one quantitative
(Pig2Bac) [74], were included in the SIPP study. Due to its demonstrated high
sensitivity, Pig2Bac performs better than PF163 and is the recommended method
for detection of pig feces. However, Pig2Bac sometimes cross-reacts with human/
septage and dogs so these would either need to be ruled out first or accounted for
when employing Pig2Bac.
4 Microbial Community Analysis Approach
In spite of the considerable progress that has been achieved employing DNA-based
MST markers described in Sect. 3, they share one disadvantage with traditional FIB
measurements in that they are still based on a single marker. This is limiting
276
B. Badgley and C. Hagedorn