Biology Reference
In-Depth Information
are usually present in minute amounts in environmental samples, and they
always require some form of concentration prior to detection. Many meth-
ods for concentration of various target organisms have been published but
will need to be validated to be used as routine water detection systems.
Concentration of samples through filter membranes (0.2-0.45µm)
for bacterial detection is usually employed as an initial step in detection.
Filter blockage can become a problem if large volumes need to be con-
centrated. Centrifugation of the sample has been also used; however, it can
also concentrate contaminates. Hollow-fiber ultrafiltration has been used,
followed by centrifugation, to recover
E. coli
O157 from large volumes of
surface water (up to 40 L). Methods that reduce exogenous DNA
60
from
the samples are best for reducing false-positive reactions.
Enrichment methods for bacterial pathogens have been applied in
many cases to enhance the concentration of the target organism. However,
enrichment of the target organism often allows uncontrollable growth of
nontarget organisms as well. In such cases, estimation of the precise number
of pathogens prior to enrichment is hard to determine. Centrifugation has
been employed with large volume samples to increase the probability of
detecting rare targets; however, it also concentrates other particulate matter
in the samples, whereby environmental samples can be affected by the con-
centration of substances such as humic acids that interfere with downstream
detection. More recently, filtration has been studied as a possible method
for concentration of samples because it rapidly processes 1-100 L of water
and it may be possible to eliminate exogenous DNA.
125
With filtration,
the enrichment step can be eliminated yet still allow the detection and
quantification of the pathogen.
60
Overall, one of the greatest challenges for the detection of virus in water
and environmental samples is the adequate sensitivity due to low concentra-
tion because as few as 1-10 viral particles per gram of soil or liter of water
may be present.
126
Standardization of units of measurement—PFU, focal
forming units, genome equivalents, PCR units, 50% tissue infectivity—
also needs to be considered to better compare one method with another.
Current research is devoted to improving concentration, purification, and
detection; however, there is still a need to have a priori knowledge of the
target. Enrichment of viruses from environmental samples can be accom-
plished by adsorption/elution protocols, entrapment ultrafiltration, ultra-
centrifugation, or hydroextraction.
46,127
An overview of the pros and cons
of each method are summarized by Bosch et al.
128
Recovery of norovirus
from water samples is especially important because there is no cell culture
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