Molecular Methods for the Detection of Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 250

system for this virus. Recovery of norovirus is dependent on both the fil-
ter type (electronegative or electropositive) and sample source (tap, bottles,
river water). 129 Huguet et al. 130 recently determined that in situ lysis of viral
particles and recovery of viral RNA was superior to recovery of intact virus
particles. Methods that rely on immunoaffinity with magnetic beads have
shown great promise and can simultaneously help purify the sample. How-
ever, the cost of this technology is expensive and requires the generation of
specific antibodies for each viral variant. 128,131
Concentration of ooscysts and cysts is also necessary to determine the
presence of low numbers of Cryptosporidium and Giardia , respectively, in
environmental samples. Because oocysts and cysts are naturally electro-
negative, this property is used to concentrate the organism by both coag-
ulation and filtration; however, to date no agreement of a standardized
method for concentration of microsporidia from water samples has been
reached. 69
8.3.2. Molecular extraction
Once concentration has been performed, a standardized robust extraction
protocol needs to be adopted to release the target molecules. Many methods
have been shown to be successful for bacterial cell lysis but to date none
have been accepted as a standard procedure, possibly due to the less than
optimal reproducibility of the protocols from laboratory to laboratory. The
challenge of extraction methods is to obtain target molecules free from
humic acids, metals, and other contaminants that may interfere with sub-
sequent enzymatic reactions and to minimize operator variability. Future
automation using microfluidic technology holds the promise of reducing or
eliminating the human error factor.
8.3.3. Viruses
Viruses constitute a unique group of infectious agents that are extremely
small, have a simple acellular structure, are unable to replicate outside of
their host cell, and yet can cause a diverse array of illnesses. There are more
than 140 enteric viruses known to infect humans, many of which are trans-
mitted via the fecal-oral route through infected surface or groundwater.
The most common enteric viruses associated with human waterborne
illnesses are adenovirus, astroviruses, caliciviruses (noroviruses and sapovi-
ruses), enteroviruses (polioviruses, echoviruses, coxsackieviruses), hepatitis A
and E, and rotavirus ( Table 8.1 ). They have been detected in sewage, surface
water, groundwater, and drinking water sources. 162-164