Sample Processing - Waterborne Pathogens: Detection Methods and Applications - page 64

For all three techniques, the microorganisms need to be eluted from
the filters. Filtration, as well as elution, needs to be optimized to give suf-
ficient recovery of a control panel of model organisms representing bacteria,
viruses, and parasites, from treated as well as untreated ground and surface
waters.
Secondary concentration procedures will differ between the king-
doms. Thus it may not be possible to deliver a universal protocol from
this step for both this reason and, as discussed earlier, the fact that differ-
ent detection methods place different requirements on sample processing.
For example, bacteria and parasites can be concentrated by e.g. pelleting
by centrifugation and viruses by polyethylene glycol (PEG) precipita-
tion, flocculation or (ultra)filtration 10,11 by various techniques from the
supernatant.
Significant amounts of organic and inorganic inhibitors are known to
be enriched during concentration of water samples. However, molecular
detection and quantification require purified nucleic acids with the absence
of reverse transcription (RT) polymerase chain reaction (PCR) inhibitors.
Various kits for preparation of RNA/DNA, post-lysis additives, and PCR
facilitators have been investigated, 12 but no universal method has been
presented.
Different types of water (quality, source, natural environment, etc.) can
be expected to contain different types and concentrations of inhibitors.
Further, some organisms, such as bacterial endospores and parasite oocysts/
cysts, are difficult to lyse, and this needs to be considered in the develop-
ment of extraction techniques.
Important factors in the analysis of any sample processing technique
are the degree of concentration achieved, the pathogen recovery rate, the
amount of operator skill required, the number of steps, what types of water
the method is appropriate for, and the specificity of isolation (see Box 4.1
for definitions). Ideally, a good sample processing procedure for water sam-
ples should meet the following criteria (adapted from Ref. 13 ):
1. Consistent recovery rate of all types of organisms analyzed;
2. Removal of inhibitory or cross-reacting substances, which are widely
present in natural water, to avoid false negative and false positive results
when molecular detection is used;
3. Generation of an end product that can be utilized directly for bacte-
rial cultivation, microscopic detection of parasites in viral culture, or
molecular detection;
4. Equivalent recovery for DNA and RNA viruses;