Conclusions - Waterborne Pathogens: Detection Methods and Applications - page 370

improvements of several orders of magnitude are required to achieve the lev-
els of sensitivity needed. There are, however, some key exceptions, one being a
photonic crystal virus biosensor and the other piezoelectric excited millime-
ter sized cantilevers for bacteria and protozoa, which are close to demonstrat-
ing single microorganism level sensitivity. Other challenges to be overcome
include the need for sample processing, robustness and long-term operation,
biofouling, and the ability to deliver species and viability based information.
Chapter 8 has given an overview of the types of molecular detection
methods that have been applied to waterborne pathogen monitoring. The
chapter also highlights the key demands placed on sample processing due
to the increased uptake of molecular methods; since environmental con-
taminants can interfere with the amplification and detection of molecu-
lar products, successful detection is reliant upon efficient removal of these
interferents. Molecular methods offer the advantage of efficient speciation
and identification of pathogens, though quantification/sensitivity and via-
bility determination can be more challenging. Targets that have developed
around rRNA genes have had the most success at determining the infectiv-
ity of the pathogen since RNA degrades faster than DNA after the death
of a cell. The lack of RNA detection, however, may still occur in viable but
not culturable cells and initiate incorrect risk assessment decisions. While
many methods have been designed over the past decade, in the case of DNA
based methods, no consensus on a universal primer set for the detection of
common waterborne pathogens has been established nor have the target
genes been agreed on.
Molecular methods have been developed for all the key waterborne
viruses, though at present none of these are practical for routine, large-scale
monitoring. Low copies of adenovirus have been detected from a variety of
water types, indicating the potential of the approach. One particular chal-
lenge is that most of the target viruses are RNA viruses and it is therefore
especially essential to select primers or probes which target highly con-
served regions. In terms of bacteria, many of the primers target the 16S
rRNA gene which is present in all organisms and contains hypervariable
regions that can be targeted down to the genus or species level, and this
approach has been used with polymerase chain reaction (PCR) for E. coli ,
Salmonella , Campylobacter , and Legionella . The best sensitivity demonstrated so
far is 4 cfu mL −1 , with many studies reporting far higher limits of detection.
Detection of protozoa with molecular methods has also been developed in
PCR and microarray formats. Detection limits of 1-5 (oo)cysts have been
reported although this sensitivity required two rounds of amplification.