Molecular Methods for the Detection of Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 223

However, there still exist several disadvantages of the molecular methods
that need to be overcome for the methods to be adopted as viable routine
tests. First, although much research has looked at detection of pathogens by
molecular methods, there exists no standardization of protocols. For instance,
with polymerase chain reaction (PCR)-based protocols, the primer/probes,
conditions, and even the extraction of DNA from the samples vary from
laboratory to laboratory, with varying detection success. 4 Most of the molecular
methods require concentration of the sample so that suitable volumes can be
analyzed. In the case of viruses and protozoa, the infective dose is very small,
requiring large sample volumes to be concentrated to ensure the pathogens
are detected. Secondly, because most molecular methods rely on detection
of a single macromolecule and, in most cases, RNA or DNA, the molecule
of choice needs to be extracted from the sample. The variability within the
samples themselves can create problems with the initial extraction of the
nucleic acid. Contaminants in the samples—for example, humic acids or
metals in environmental samples—can inhibit subsequent amplification and
produce false negatives. Therefore, comparison of gene numbers from differ-
ent samples is problematic. 15 Because the appearance and concentration of
inhibitors are not necessarily known beforehand, it is left to the operator to
determine the best extraction method on a sample to sample basis, which is
time consuming. Lastly, detection of genomic sequences in a sample is not a
definitive test for infectivity because DNA can be found in samples outside
of cells, in dead cells, or in cells in a viable but not culturable state. 12,15
Despite the disadvantages of molecular methods, much research is still
underway to overcome them and identify suitable detection targets in
pathogens. Molecular methods definitely hold much promise as techniques
that can be validated and standardized for use as routine water testing assays
of the future. In general, molecular tools have shifted the paradigm from
detection of indicator organisms on culture media to direct detection and
enumeration of known pathogens, unculturable pathogens, and emerging
pathogens.
8.2. MOLECULAR METHODS
Molecular methods are based on the ability to isolate and detect
specific molecules carried by pathogenic organisms. Several techniques for
the analysis of microbial communities and identification of bacterial spe-
cies have been investigated. The development of many taxonomic tools has
been based on the composition of fatty acids, peptides, or genomic material