Molecular Methods for the Detection of Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 266

of inhibition due to contaminates. This is particularly true with samples
needing to be monitored for viruses and protozoa because samples of up to
100 L may need to be concentrated to ensure detection because infectivity
of the pathogen can be as low as one particle.
For viral sample concentration methods, environmental virology has
focused on filtration methods that can maximize viral trapping within the
filter during filtration and maximize viral detachment from the filter for
recovery. 14 The recent increase in more robust and reliable concentration
methods will increase the likelihood of detecting of all types of waterborne
pathogens.
Once the sample is prepared, isolation of the macromolecules requires
specialized protocols for both the matrix in question and the pathogens to
be detected. There has been much success with extraction methods, but still
one has not been universally accepted as the method to follow as a rou-
tine testing protocol. High-quality extraction of the macromolecules from
samples is highly variable and dependent of the composition (organic mat-
ter, humic acids, detergents, pesticides, herbicides, pharmaceuticals, etc.) of
target water samples. Validation of the method for reproducibility between
operators and laboratories also requires cooperation between research and
testing laboratories.
Low pathogen numbers generate low molecule concentrations that can
cause random fluctuations in PCR efficiency of detection. Because sam-
ples may contain a high variety of pathogens (virus, bacteria, protozoa),
competition for nucleic acid templates may interfere with single pathogen
detection and confirmatory results may require multiple analysis.
One direction that current research has taken to improve concentration,
purification, and detection of waterborne pathogens is to use IMS. Although
it still requires a priori knowledge of the target, it can bind specifically and
concentrate the pathogen while leaving the contaminates behind.
Many methods have been designed and developed for detection of
pathogens targets over the last decade. However, in the case of DNA-based
methods, no consensus on a universal primer set for the detection of com-
mon waterborne pathogens has been established, nor have the target genes
been agreed upon. In the end, the primer sets will need to consistently
amplify targets with high reproducibility, sensitivity, and specificity. One
concern is the ability to establish the physiological condition of the tar-
get organism—dead or alive, infectious or noninfectious. Targets that have
developed around rRNA genes have had the most success at determining
the infectivity of the pathogen because RNA degrades faster than DNA