Molecular Methods for the Detection of Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 256

beads are used to bind to antigens present on the surface of the target virus.
A magnet is then used to attract the beads to one side of the tube, thereby
concentrating the virus. This method can be combined with PCR to pro-
vide comparable results to plaque assays.Viruses such as enteroviruses, hepa-
titis A, rotavirus, and Norwalk virus have been successfully detected from
sewage and artificially contaminated environmental water samples using this
technology. 170-172 Drawbacks of this method are the availability of an anti-
body that can target all strains of a particular virus, the cost of the magnetic
beads depending on the reagents coupled to them, and the conditions of the
environmental sample because pH and colloidal particles may interfere with
the initial binding step between the antibody and antigen. 173,174
Molecular methods based on PCR have been developed for the key
enteric viruses. With the exception of adenoviruses, which contain dsDNA
as their nucleic acid, the rest are RNA viruses; all of them contain ssRNA,
with the exception of rotaviruses, which possess a dsRNA genome. RNA
viruses represent a major challenge in the development of molecular meth-
ods because their genomes are replicated with error-prone polymerases that
promote high mutation rates and recombination events. Targets for PCR
primers or microarray probes need to be carefully selected to ensure they
target highly conserved regions.
The advantage of PCR-based methods is that amplification of the target
sequence can increase the sensitivity of the assay and eliminate the need
to re-concentrate samples. Direct PCR, nested PCR and RT-qPCR were
able to provide rapid sensitive detection of adenovirus in wastewater, drink-
ing water, recreational waters, and rivers, 46,175 with qPCR providing some
measure of the concentration of the virus. Jothikumar et al. 176 designed a
broadly reactive TaqMan assay for the detection of all adenovirus and a spe-
cifically reactive assay, using FRET probes, to successfully detect the adeno-
virus fiber gene in AdV40 and AdV41. They were able to detect as few as 5-8
copies of AdV40/41 with TaqMan and 3-5 copies with FRET-PCR. Melting
curve analysis allowed them to differentiate between AdV40 and AdV41. 176
Like immunological techniques, PCR methods can also be inhibited by
environmental contaminants, causing false negatives in this case. 131
RT-qPCR has also been successfully applied to the detection of hepa-
titis A, 177 hepatitis E, 136 Norwalk-like virus 178 and norovirus. 179 Recently,
Jothikumar et al. 44 developed an RNAX buffer that allows improved isola-
tion of RNA from environmental sample. Using the buffer, they found that
contaminants in water were not as much of a problem when applied in an
RT-qPCR assay.